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\begin{document}

\title{Thesis 3}



\author[1]{Glynis Mattheisen}%
\affil[1]{Smith Lab}%


\vspace{-1em}



  
  \date{May 05, 2024}


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\sloppy




\section{Introduction}

{\label{745272}}

\par\null

\subsection{Importance of Voltage-gated Sodium Channels in the
Cortex}

{\label{120939}}

The action potential is an electrical signal that facilitates
interneuronal communication. Action potentials occur in response to
excitatory input which depolarizes the neuronal membrane and activates
voltage-gated sodium channels (VGSCs). These channels drive neuronal
depolarization, set the threshold for action potential generation,
conduct the action potential, and propagate the signal to sites of
neurotransmitter release~\cite{Hodgkin_1952}. In doing so, VGSCs prove
vital to neuronal excitability. This process takes place over
milliseconds and is shaped by a multitude of factors including both the
intrinsic properties of these channels and extrinsic interactions with
additional signaling molecules. While intrinsic factors are coupled to
neuronal identity, extrinsic factors provide more dynamic systems to
modulate and refine the electrical signal (for review
\cite{Catterall_2012,Ahern_2015}).

\par\null

\subsection{Properties of Voltage-gated Sodium
Channels}

{\label{720813}}

In all excitable cells, VGSCs are closed at negative potentials,
activate in response to a depolarization, and then rapidly inactivate at
positive potentials. Within hundreds of microseconds of activation a
large macroscopic current is produced and the proportion that
inactivates rapidly within 1-2 milliseconds is described as the
transient, fast-inactivating VGSC current ~\cite{Hodgkin_1952,Hodgkin_1952a}. The
proportionately small residual VGSC current following is defined as the
persistent VGSC current~\selectlanguage{ngerman}\cite{Hotson_1979,Llinás1980,Stafstrom1985}. Relief of inactivation
occurs only at strongly negative potentials and primes the channels for
further activation~\cite{Hodgkin_1952,Kuo_1994}. Certain cell types, such as
cerebellar Purkinje neurons, exhibit a resurgent VGSC current produced
when depolarization-inactivated VGSCs reopen in response to
repolarization after strong depolarizing pulses~\cite{Raman_1997}.
Resurgent current exhibits its own voltage-dependence and
kinetics~\cite{Raman1997}. ~Voltage-dependent activation,
voltage-dependent inactivation, persistent current, and resurgent
current of VGSCs all influence the action potential waveform. The innate
kinetics of any of these properties or modulation of them will influence
signal conduction.

\par\null

\subsection{Intrinsic VGSC Properties Influencing the Action
Potential~}

{\label{407892}}

Once the threshold for action potential firing is breached, an action
potential will reliably fire with a set amplitude independent of the
stimulus size \cite{Noble1966}. While action potentials are a digital
all-or-none signal in this regard, the VGSC conductance underlying this
phenomenon is influenced by a host of variables, giving rise to
wide-ranging action potential shapes among neuronal types
\cite{Bean_2007}. The kinetics of sodium currents differ immensely
between different types of neurons and, remarkably, even between
different regions of the same neuron~\cite{Engel_2005}. While the
explicit mechanisms producing variability in both the VGSC signal and
action potential waveform are unknown, some variation can be attributed
to VGSC~subunit composition and distribution~\cite{Bean_1997}.

At the structural level, VGSC properties are isoform-specific and
modulated by auxiliary \selectlanguage{greek}β \selectlanguage{english}subunits. Of the nine VGSC \selectlanguage{greek}α \selectlanguage{english}subunits
(Na\textsubscript{V}1.1-1.9), the mammalian central nervous system is
primarily populated by isoforms Na\textsubscript{V}1.2 and
Na\textsubscript{V} 1.6~\cite{Catterall2012}. Na\textsubscript{V}1.2 is
expressed almost exclusively in unmyelinated axons~\cite{Miyazaki2014},~
Na\textsubscript{V}1.6 in myelinated axons of mature neurons at the
initial segment and nodes of Ranvier~\cite{Boiko2003},
Na\textsubscript{V}1.1 and Na\textsubscript{V}1.3 on neuronal
soma~\cite{Westenbroek1989}, and Na\textsubscript{V}1.1,
Na\textsubscript{V}1.3, and Na\textsubscript{V}1.6 have been identified
in dendrites~\cite{Westenbroek1989,Westenbroek1992,Caldwell2000}. The differential distribution of these
neuronal isoforms affect action potential waveform, initiation,
propagation, frequency, and even action potential back~propagation
toward the postsynaptic terminal~\cite{Araya2007}, changing how
signals are integrated in the cortex. The differences in VGSC properties
between isoforms are diverse and not yet fully quantified. Each isoform
exhibits unique gating properties, voltage-dependence, and current
profiles, with even more nuanced differences shown in use-dependent
potentiation or use-dependent reduction of channel current
\cite{Zhou_2004}.

Adding another level of complexity, VGSCs in the mammalian brain are
heterotrimeric complexes of an \selectlanguage{greek}α \selectlanguage{english}subunit and flanking \selectlanguage{greek}β
\selectlanguage{english}subunits~\cite{Messner1985}\cite{Hartshorne1981,Isom1995}. While the \selectlanguage{greek}α \selectlanguage{english}subunit can
form a functional unit on its own with voltage sensor, sodium
selectivity filter, and pore-forming domains, adjacent \selectlanguage{greek}β \selectlanguage{english}subunits
modulate voltage-sensitivity, gating kinetics, and membrane trafficking
of the \selectlanguage{greek}α \selectlanguage{english}subunit ~\cite{Catterall2000,Kazen-Gillespie2000,Qin2003,Yu2003}. The five~mammalian \selectlanguage{greek}β
\selectlanguage{english}subunits~\cite{O_Malley_2015} can increase peak current
density~\cite{Brackenbury_2011}, shift the voltage range over which
activation and inactivation occur, enhance rates of inactivation and
recovery from inactivation~\cite{Yu_2005,Chen_1995}, and alter persistent and
resurgent VGSC current in a tissue-specific pattern~ (reviewed
in~\cite{Calhoun2014}). In addition, the \selectlanguage{greek}β \selectlanguage{english}subunit can receive
posttranslational modifications such as phosphorylation and
glycosylation that alter channel gating~\cite{Johnson_2004,Calhoun_2014}. \selectlanguage{greek}β\selectlanguage{english}~ subunits
therefore influence many of the key conformational changes that VGSCs
undergo during the action potential~\cite{Brackenbury_2011,Patino_2010}. Specific
combinations of \selectlanguage{greek}α \selectlanguage{english}and \selectlanguage{greek}β \selectlanguage{english}subunits contribute to the particular
physiological characteristics of divergent cell
types~\cite{Winters_2016}.

In addition, the location of VGSC expression can modify action potential
shape to control the spatial and temporal features of the signal and, in
many cases, define the role of that input in neuronal processing (for
review~\cite{Lai2006}). A classic example of this is VGSCs
clustering at the axon initial segment and nodes of Ranvier to insure
saltatory conduction along the axon (reviewed in~\cite{Freeman_2015}).
Further studies of VGSCs have revealed novel physiological roles of
VGSCs at specific~physical locations along excitatory cells. For
example, VGSCs were once though to be absent in dendrites but now data
show that sodium channels at these sites play a role in the back
propagation of action potentials \cite{Losonczy2008,Larkum2007,Gasparini2006,Gasparini2004}. ~Patch-clamp
experiments have demonstrated that axonal and somato-dendritic VGSC
currents differ in their voltage-dependent properties, indicating either
different \selectlanguage{greek}α \selectlanguage{english}and ~\selectlanguage{greek}β \selectlanguage{english}subunit compositions and/or distinct
posttranslational modifications of identical
subunits~\cite{Gasparini2002,Colbert1998}. These findings reveal how action potential
waveform can vary widely along individual neurons and between divergent
cell types.

VGSC subunit identity and composition are intrinsic factors that are
coupled to neuronal identity and known to influence the firing
properties of neurons. ~These in addition to spatial distribution
influence the electrical behavior of neurons at a base level (for review
\cite{Lai2006,Cusdin2008}). We now find that these foundational properties can
be further shaped and refined through protein signaling pathways and
posttranslational modifications to add additional complexity to the
electrical contribution of VGSCs.

\subsection{Extrinsic Voltage-gated Sodium Channel
Modulation}

{\label{614735}}

It was once though that the only point of regulation of the sodium
channel signal was voltage-dependent gating on a millisecond time scale.
Early voltage-clamp studies of VGSCs in neurons indicated that VGSCs are
not subject to regulation via second messenger
pathways~\cite{hille2001}(pgs 201-236). An explanation for this
finding is that the methodology of these studies allowed for dialysis of
the cytoplasm resulting in the loss or dilution of critical signaling
components. These studies led to the widespread idea that VGSC channels
are functionally similar in different excitable cells and that they are
not modulated by cellular regulatory pathways. Now this picture has
evolved with the identification of second messenger signaling cascades
that modulate VGSC activity. A multitude of enzymes, including among
others protein kinases A and C~\cite{Sigel1988,Scheuer2011,Murphy1993},
calcium/calmodulin-dependent protein kinase (CAMK)
II~\cite{Ashpole2012,Thompson2017}, Fyn tyrosine
kinases~\cite{Ahn2007}\cite{Scheuer2011},
calcineurin~\cite{Murphy1993}, induce posttranslational modifications
of VGSCs culminating in short- and long-term alteration of sodium
currents (reviewed in~\cite{Laedermann_2015}). Because the action potential
is the final common pathway for neuronal output, VGSCs are ideally
positioned to serve as an important point of regulation of neuronal
signal transduction. Herein we identify a novel G-protein-dependent
pathway for the dynamic regulation of VGSCs, investigate the components
of this pathway and the kinetics of VGSC modulation, and discuss the
significance of this regulation to neuronal function.

\subsection{The Intersection of G-Proteins and Ion
Channels}

{\label{440996}}

GPCRs are a large protein family of integral membrane
receptors~\cite{Vassilatis_2003} that sense extracellular molecules such as
peptides, lipids, or neurotransmitters and in turn activate
intracellular signaling pathways by regulating the activity of bound
heterotrimeric G-proteins.~Heterotrimeric G-proteins consist of a
G\selectlanguage{greek}\emph{α} \selectlanguage{english}subunit that binds and hydrolyzes GTP into GDP, and \selectlanguage{greek}\emph{β}
\selectlanguage{english}~and~\selectlanguage{greek}\emph{γ} \selectlanguage{english}subunits that form the G\selectlanguage{greek}\emph{βγ~}\selectlanguage{english}
dimer~\cite{Wettschureck_2005}. In the absence of stimulus, the GDP-bound
G\selectlanguage{greek}\emph{α} \selectlanguage{english}~subunit and G\selectlanguage{greek}\emph{βγ~}\selectlanguage{english} dimer are associated with the
receptor~\cite{Sondek1996}. Binding of an extracellular ligand onto
the GPCR induces a conformational change that is transduced to the
G-protein and promotes the exchange of bound GDP for GTP from the
G\selectlanguage{greek}\emph{α} \selectlanguage{english}~subunit. GTP-bound G\selectlanguage{greek}\emph{α}\selectlanguage{english}~ dissociates from the GPCR and
can freely diffuse away from the membrane. Upon activation, the
G\selectlanguage{greek}\emph{βγ}\selectlanguage{english}~dimer also dissociates from the heterotrimer and diffuses
away from the GPCR in a membrane-delimited fashion. Intrinsic hydrolysis
of GTP by the G\selectlanguage{greek}\emph{α} \selectlanguage{english}~subunit allows reassociation of GDP-bound
G\selectlanguage{greek}\emph{α} \selectlanguage{english}~subunit with the G\selectlanguage{greek}\emph{βγ~}\selectlanguage{english}dimer, terminating G-protein
signaling. Both the GTP-bound G\selectlanguage{greek}\emph{α} \selectlanguage{english}~subunit and the G\selectlanguage{greek}\emph{βγ~}\selectlanguage{english}
dimer can mediate intracellular signaling. The activity of GPCRs has
been implicated in a diverse repertoire of processes including
metabolism, development, hormonal homeostasis, and~synaptic plasticity
(reviewed in \cite{Rosenbaum2009,Hoffmann2008}).

G-proteins have been shown to modulate ion channels for dynamic roles in
synaptic transmission, exerting effects on both voltage-gated calcium
(VGCCs) and voltage-gated potassium channels (VGKCs). Data show that
G-protein modulation of these channels is widespread throughout the
cortex. To date,~up to 20 neurotransmitters and corresponding receptors
have been described to modulate VACCs, including
noradrenaline~\cite{Bean1989,McFadzean1989,Lipscombe1989}, GABA~\cite{Deisz1985,Grassi1989,Dolphin1987},
dopamine~\cite{Drolet1997} and acetylcholine~\cite{Bernheim1991,Shapiro1999}.
~Upwards of fifteen GPCRs have also been shown to modulate VAKCs,
including M2-muscarinic~\selectlanguage{ngerman}\cite{Bünemann1995}, A1~adenosine
\cite{Soejima1984}, D2dopamine~\cite{Lacey1988,Saugstad1996}, \selectlanguage{greek}μ-, δ-, \selectlanguage{english}and
\selectlanguage{greek}κ-\selectlanguage{english}opioid~\cite{Grudt1993,North1987}, 5-HT1A serotonin~\cite{Oh1995},
mGluRs~\cite{Saugstad1996}, and GABAB receptors~\cite{Lacey1988}
(reviewed in~\cite{Hille1992,Yamada1998}). GPCR-induced modulation of VGCCs and
VGKCs includes the indirect mechanisms of~phosphorylation, lipid
signaling pathways, and channel trafficking (for
review~\cite{Zamponi2013,Inanobe2014}). Interestingly, both VGCCs and VGKCs
are~subject to direct modulation by the~\selectlanguage{greek}\emph{βγ}\selectlanguage{english}~dimer for reduced
neuronal excitability. VGCC inhibition by G\selectlanguage{greek}\emph{βγ~}\selectlanguage{english}provides negative
feedback over VGCCs and terminates further neurotransmitter discharge
~\cite{Herlitze1996,Ikeda1996} while G\selectlanguage{greek}\emph{βγ~}\selectlanguage{english}activation of\emph{~}
G-protein-coupled inwardly-rectifying potassium channels
(GIRKs)~\selectlanguage{ngerman}\cite{Lüscher1997} reduces the propagation of neuronal inputs
from excitatory synapses as well as the back propagation of action
potentials from the soma to the synapse~\cite{Ponce1996,Morishige1996,Liao1996}.

Given the pervasiveness of G-protein and ion channel interactions, high
structural homology between VGSCs and VGKCs, and the positioning of
VGSCs as critical targets for the modulation of neuronal excitability,
there exists a convincing probability and fortuitous opportunity for
G-protein interaction with these channels. We already know that
G-protein and ion channel interactions modulate the integrative
properties of neuron; it is certain that further research will reveal
additional functional relationships between these two key players in
neuronal excitability and delineate more pathways for the control of
synaptic transmission.

\subsection{G-Proteins and Voltage-gated Sodium
Channels}

{\label{384713}}

The first studies of the G-protein modulation of VGSCs were undertaken
following the identification of several putative phosphorylation sites
on the pore-forming portion of the channel
structure~\cite{Barchi_1988}. This suggested that the channel may be
subject to regulation by second messenger pathways that activate
phosphorylating kinases. Sigel and Bauer, 1988, presented the first
electrophysiological evidence of G-protein modulation of VGSCs by
demonstrating that activators of PKC reduce the amplitude of VGSC
currents in~\emph{Xenopus} oocytes injected with chick brain
mRNA~\cite{Sigel1988}. It was then shown that GTP\selectlanguage{greek}\emph{γ}\selectlanguage{english}S, an
activator of G-protein signaling, inhibited VGSC current
in~\emph{Xenopus} oocytes injected with rat brain
RNA~\cite{Cohen-Armon1989}. Since the initial observations that linked
G-proteins to VGSCs, a complete picture has been painted between GPCRs
at the cellular membrane and changes in VGSC output. Studies now show
that VGSCs in the cortex can be modulated by dopamine, acetycholine,
serotonin and other transmitters acting through GPCRs (ALL THE REFS).
This regulation provides an important form of cellular plasticity, which
controls the excitability of central neurons in response to synaptic
inputs, the threshold for excitability, and the frequency and form of
action potentials.

Evidence for G-protein modulation of VGSCs points to prolific regulation
throughout the cortex as well as a common theme of G-proteins producing
significant reduction in VGSC current. VGSCs have been shown to be
inhibited by activation of dopamine D1-like receptors, metabotropic
glutamate receptor 1 (mGluRs) , muscarinic acetylcholine receptor
(mAChR) M1, and serotonin 5-HT1\textsubscript{a/c} receptor. While some
studies report a decrease in channel conductance without a change to the
voltage-dependent properties of VGSCs~\cite{Cantrell1999,Cantrell1996}, others
indicate G-protein interactions can substantially shift
voltage-dependent inactivation of the channels~\cite{Carlier_2006,Carr2002}. ~The
primary biophysical mechanism of VGSC modulation by GPCRs is
phosphorylation downstream of PKA and PKC activation. This relationship
have been shown to modulate VGSCs in preparations from the hippocampus
(scheuer 1999)(Cantrell ma schemer catterall 1996),
neocortex~\cite{Carlier_2006}, and prefrontal cortex (Maurice summerier
2001)(surmier 2002). These pathways have been proposed to function as
negative feedback loops during periods of intense synaptic transmission.
Preliminary studies has also suggested that VGSCs are also subject to
direct modulation via the G\selectlanguage{greek}\emph{βγ} \selectlanguage{english}of heterotrimeric G-proteins, in
much the same fashion as VGCCS and VGKCS, and that this increases
persistent VGSC current of Na\textsubscript{V}1.1 and
Na\textsubscript{V}1.2~\cite{Mantegazza_2005}, though additional research
is necessary to fully understand the extent and nature of G\selectlanguage{greek}\emph{βγ}
\selectlanguage{english}modulation of VGSCs. Overall, data is amassing for the G-protein
modulation of VGSCs and the varied roles this relationship plays in
synaptic transmission.

\subsection{The Calcium-Sensing
Receptor~}

{\label{435775}}

The calcium-sensing receptor (CaSR) is a GPCR activated by extracellular
calcium expressed in many tissues including the nervous
system~\cite{Leach_2015,Butters1997,Rogers1997}. The CaSR has been studied extensively for
its roles in maintaining calcium homeostasis in the kidney and
parathyroid gland. CaSRs expressed on the parathyroid gland sense serum
calcium levels and modulate parathyroid hormone synthesis and secretion
to maintain serum calcium within a physiological range. For this role,
allosteric modulators of the CaSR provide therapeutic promise for
disorders of calcium homeostasis~\cite{Brauner_Osborne_2007}.

In the cerebellum and cerebral cortex, the CaSR is expressed at nerve
terminals~\cite{Chen_2010,Ruat1995}~where it modulates evoked and spontaneous
synaptic transmission~\cite{Smith_2012,Smith2004}. Previous research in our lab
has shown that presynaptic CaSRs decrease release probability at
excitatory synapses in the neocortex through the regulation of a
nonselective cation channel (NSCC)~\cite{Smith2004}.~This function
forms a feedback loop wherein the CaSR represses a NSCC~current as
extracellular calcium increases and relieves inhibition on the channel
as extracellular calcium falls \cite{Smith2004}. Decreases in
extracellular calcium depress synaptic transmission due to the
sensitivity of neurotransmitter release to extracellular calcium
following its entry via VGCCs~\cite{Heidelberger1994,Schneggenburger2000}. When calcium falls at
the cleft during periods of high synaptic activity CaSR activity is
reduced, leading the relief of suppression of the NSCC conductance. The
addition of the NSCC current depolarizes the terminal to allow for
continued neurotransmitter release during bursts of synaptic
activity.~~In our current theoretical model, increased NSCC activity may
increase action potential duration, prolong calcium entry at the
terminal, and thus increase release probability. Consequently, the CaSR
may be operating as part of a homeostatic pathway to prevent synaptic
failure when extracellular calcium falls (for
review~\cite{Jones2016}).

\subsection{Calcium-Sensing Receptor
Modulators~}

{\label{625573}}

The drugs cinacalcet and calindol serve as positive allosteric
modulators of the CaSR, enhancing its sensitivity to extracellular
calcium. In an antagonistic action, negative allosteric modulators NPS
2143 and calhex decrease the sensitivity of the CaSR to extracellular
calcium. All four drugs exhibit high selectivity for the CaSR and are
known to possess overlapping though not identical binding sites to the
seventh transmembrane domain (TMD) of the receptor (for review
\cite{Ward2012,Conigrave2013}).

Interestingly, cinacalcet, marketed under the name Sensipar, was the
first drug of its class to become available for clinical use and been
shown to treat severe secondary hyperparathyroidism in patients
receiving hemodialysis treatment for chronic kidney disease
\cite{Block2017}. In this role, cinacalcet acts on targets on the
parathyroid to stimulate the CaSR, thereby decreasing circulating
parathyroid hormone levels and producing a concurrent decrease in serum
calcium levels. Cardiovascular disease is the leading cause of death in
patients with chronic kidney disease and a risk factor for secondary
hyperparathyroidism. Cardiovascular disease results from premature
cardiovascular aging characterized by vascular calcification on a
background of hypercalcemia secondary to high levels of parathyroid
hormone. Cinacalcet prevents vascular calcification by reducing
parathyroid hormone and consequentially reduce the risk of
cardiovascular events in patients with chronic kidney disease.~

\par\null

A trial called EVOLVE was designed to evaluate if cinacalcet reduced the
risk of death and nonfatal cardiovascular events among patients with
secondary hyperparathyroidism who were undergoing dialysis. The trial,
published in \emph{The New England Journal of Medicine} in 2012,
concluded that while cinacalcet reduced the levels of parathyroid
hormone effectively, treatment with cinacalcet did not lead to a
statistically significant reduction in the risk of death or
cardiovascular events~\cite{Chertow2012}. The results indicated that
adverse or unknown effects of cinacalcet might be undermining its
effectiveness in the treatment of hyperparathyroidism. A criticism of
the EVOLVE trial was the high nonadherence rate for cinacalcet-medicated
patients due to intolerable side effects~\cite{Moe_2013,Parfrey2016}. Cinacalcet
is lipid-soluble, meaning that it readily crosses the blood brain
barrier. Commonly-reported neurological side effects of cinacalcet
include paresthesia, vertigo, severe hearing loss, and altered mental
status~\cite{Borchhardt_2008}. Despite its ability to readily diffuse into
the nervous system and the results of the EVOLVE trial, the effects
of~cinacalcet in the brain have yet to be studied.

In investigating the role of the CaSR in synaptic transmission, we found
that allosteric CaSR modulators reduced GABAergic transmission between
neocortical neurons. The nature of this inhibition indicated
CaSR-mediated conduction block of the presynaptic action potential and
this hypothesis was validated by the discovery that CaSR allosteric
modulators strongly inhibit VGSCs. This finding was immediately relevant
to the clinical usage of cinacalcet as well as our understanding of the
role of the CaSR in the cortex. Further examination showed that both
allosteric agonists and allosteric antagonists of the CaSR completely
inhibited VGSC current. This inhibition, in all cases, was shown to be
dependent on a G-protein pathway. In Chapter 1, we look at the mechanism
underlying CaSR modulator-regulation of VGSCs, investigating the
kinetics of this effect as well as potential effector proteins within
the pathway.

\subsection{Anandamide and the Calcium-Sensing
Receptor}

{\label{908233}}

Endocannabinoids (eCBs) are a family of lipid molecules that serve as
key players in synaptic plasticity. The two major cannabinoid-signaling
molecules in the central nervous system are anandamide (AEA) and
2-Arachidonoylglycerol (2-AG). Cannabinoid type 1 (CB1) receptors have
been shown to possess the highest sensitivity to AEA, however, data
support the existence of additional receptors that respond to eCBs
(reviewed in~\cite{Castillo_2012,Kano_2009,Katona_2012,Alger_2012}) . We have identified AEA as a positive
allosteric modulator of the CaSR, acting to increase the sensitivity of
the receptor to extracellular calcium in the same fashion as has been
observed for calindol and cinacalcet.

\subsection{Significance of Anandamide in Physiology and
Disease-States}

{\label{149615}}

In the canonical pathway for cannabinoid function, eCBs are produced
on-demand in postsynaptic neurons in response to stimulation then travel
in a retrograde fashion to targets receptors on the presynaptic
component. eCBs function primarily through presynaptically expressed CB1
receptors throughout the cortex on both excitatory and inhibitory
terminals \cite{Herkenham1990}. Activation of presynaptic cannabinoid
receptors can initiate both short- and long- term synaptic depression
(reviewed in \cite{Castillo_2012,Kano_2009,Katona_2012,Alger_2012}). Recent work shows that cannabinoid
function is more diverse than previously believed. In addition to their
role as instigators of synaptic depression, cannabinoids have also been
shown to participate in non-retrograde signaling \selectlanguage{ngerman}\cite{Grueter2010,Chávez2010} and
signaling via astrocytes \cite{Stella2010,Navarrete2008}. Furthermore, studies show
that eCBs act on as-yet unidentified receptors to contribute to these
processes of synaptic plasticity (reviewed in~\cite{Begg2005}).

Importantly, eCBs contribute to a range of physiological processes,
including synaptic plasticity and learning~\cite{Heifets2009},
pain~\cite{Guindon2009}, metabolism and energy
homeostasis~\cite{Viveros2008}, and neural
development~\cite{Fride2008}. Dysregulation of the eCB system has
been implicated in neuropsychiatric conditions, such as depression,
autism, schizophrenia, addiction, stress and
anxiety~\cite{Ruehle2012,Hillard2014,Mechoulam2013,Parsons2015,Volkow2017}. eCBs have also shown therapeutic promise
for Tourette's syndrome~\cite{Müller-Vahl2013}, Huntington's
disease~\cite{Pazos2008}, epilepsy~\cite{Alger2004}, Alzheimer's
disease~\cite{Maroof2013}, depression \cite{Huang2016}, and stroke
\cite{Hillard2008}.~ Advancing our understanding of the many roles of
eCBs in the nervous system will enhance our knowledge of disease states
and synaptic transmission.

\subsection{Allosteric Modulators of the Calcium-Sensing Receptor
Modulate Voltage-gated Sodium Channels via a G-protein
pathway}

{\label{537141}}

In Chapter 1 of this dissertation, we show that synthetic allosteric
modulators of the CaSR substantially reduce VGSC current. ~In a detailed
look at the kinetic properties of VGSCs, it is shown that CaSR
modulators produce a hyperpolarizing shift in steady-state inactivation
of the channels, pushing them into a deeply inactivated state relieved
only by strong, prolonged hyperpolarization. We establish that this
pathway is independent of the CaSR but dependent on G-protein signaling.
~ Cumulatively, these data indicate an important new pathway for
modulating neuronal excitability in the cortex. Following this study, we
sought to identify the VGSC modulatory properties of the endogenous
signaling ligand AEA. In Chapter 2 , we show that AEA completely
inhibited VGSC current also via a change in the inactivation properties
of VGSCs. This block of VGSC current was also independent of the CaSR
but required G-protein activation. The VGSC inhibition also appeared
independent of CB1 receptors, indicating a novel site of action of AEA
as well as a new G-protein-dependent pathway for VGSCs regulation in the
cortex.~

\par\null

The findings presented here are exciting in a number of ways. First,
they lend further support to the idea that VGSCs are subject to dynamic
modulation by second messenger pathways. Second, they show AEA has
actions independent of CB1 receptors in the cortex. And third, they
point to the existence of an as-yet unexplored eCB-activated pathway for
synaptic plasticity in the cortex.

\section{}

{\label{120978}}

\section{Discussion}

{\label{120978}}

Voltage-gated sodium channels are integral to a number of physiological
processes in excitable cells: setting the action potential threshold,
action potential conduction, signal integration, firing properties,
and~synaptic transmissions. The regulation of these channels can alter
the transmission of cellular signals in response to local stimuli.
Neurotransmitters such as acetylcholine and dopamine have been shown to
inhibit VGSCs via G-protein pathways to change the firing properties and
input-output relationships of cortical neurons. The data presented in
this dissertation examines new pathways for the G-protein modulation of
VGSCs by allosteric modulators of the CaSR.

The five allosteric modulators of the CaSR tested all exhibited similar
mechanisms and kinetics of VGSC inhibition. Cinacalcet, calhex, NPS
2143, calindol, and AEA reliably produced near-complete VGSC inhibited
with slow kinetics (full inhibition occurring in 100s of seconds),
dependence on G-proteins, and a shift in the steady-state inactivation
of affected VGSCs (shown with cinacalcet, AEA). Inhibition in all cases
was shown to be independent of the CaSR. Furthermore, AEA-induced
inhibition was independent of CB1 receptors, all indicating off target
effects of these ligands. Of the CaSR allosteric modulators tested, AEA
is the only endogenous ligand. The participation of this major cortical
signaling molecule in the G-protein-dependent mechanism for VGSC
inhibition points to a significant physiological role for this pathway
in the process of synaptic transmission.

\subsection{G-Protein Modulation of Voltage-gated Sodium
Channels}

{\label{186440}}

While it was once believed that VGCs were not subject to regulation by
second messenger systems, recent studies have upended prior claims and
accrued evidence for G-protein modulation of VGSCs in the cortex.
G-protein-dependent inhibition of VGSCs has been measured downstream of
dopamine D1-like, mAChR M1, mGluR1, and serotonin
5HT2\textsubscript{a/c} receptors in the cortex (reviewed in~Cantrell \&
Catterall, 2001).

\subsection{Other Pathways and how ours is
different}

{\label{250007}}

The four known pathways for VGSC modulation thus far (mAChR, mGluR1,
D1-like, 5HT2a/c) all rely on the activity of a protein kinases; either
PKA or PKC (Cantrell, Ma, Scheuer and Catterall 1996, Cantrell, Smith,
Goldin, Scheuer and Catterall 1997, Carr, Cooper, Ulrich, Spruston and
Surmeier 2002, Carlier, Sourdet, Boudkkazi,
D\textbackslash{}selectlanguagengermanéglise, Ankri,
Fronzaroli-Molinieres and Debanne 2006). These kinases phosphorylate a
serine residues in the intracellular loop connecting domains I and II of
the VGSC alpha subunit (Cantrell \& Catterall, 2001). This common theme
is one major divergence from the pathway of CaSR allosteric modulators.
Experiments with cinacalcet showed that the G-protein inhibition induced
by this CaSR allosteric modulator was independent of either PKA or PKC.
Interestingly, the phosphorylation of VGSCs by PK\_ downstream of \_\_\_
and \_\_ was shown to shift VGSC inactivation in the hyperpolarizing
direction, much the same as observed with Cinacalcet and AEA. While this
study targeted PKA and PKC with specific inhibitors chelerythryine
chloride, PKI, PKI\_\_\_\_\_, we cannot rule out the involvement of
staurosporine resistant kinases. Phosphorylation of VGSCs downstream of
AEA may still influence this pathway and produce the shift in VGSC
inactivation.

\subsection{Protein Kinases
Involvement}

{\label{462973}}

Here we attempt to identify the full pathway from ligand to GPCR to
effector proteins and, finally, to changes in VGSC modulation. We
approached this with inhibitors of G-protein signaling and protein
kinases. Several pitfalls prevented a more complete exploration; namely,
the absence of selective, reliable tools by which to inhibit proposed
components of this pathway. One mechanism that has been proposed is
direct Gbetagamma modulation of VGSCs. The first evidence of Gbetagamma
modulation of ion channels shown activate cardiac potassium channels
downstream of acetylcholine on muscarinic receptors (Logothetis et all
1987). Since that discovery, it has been well-established that the
Gbetagamma particle serves a role in inhibiting \_\_\_ and activating
\_\_\_.~ Betagamma has been shown to interact with a wide range of
effectors and proteomic methods and yeast two-hybrid screening have
revealed that no readily apparent consensus sequence or structure
mediates binding of these proteins to
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ.
\selectlanguage{english}\textbackslash{}selectlanguageenglishThe binding of Gbetagamma to VGSCs
has been briefly addressed by Ma et al, 1997. The group showed that Gby
increased persistent VGSC current from NAV1.2 and NaV1.1 but not NaV1.4
or NaV 1.5 when coexpressed in \_\_\_\_ system. This study also garnered
evidence that G-protein \textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits interact with that
C-terminal site of NaV1.2 (Ma et al., 1997). Consistent with this idea,
G-protein \textbackslash{}selectlanguagegreek\selectlanguage{greek}β
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits immunoprecipsitate with
sodium channel \textbackslash{}selectlanguagegreek\selectlanguage{greek}α
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits from cortical neuron
preparations (Marin et al., 2001). Persistent VGSC current through
Nav1.1 and Nav1.2 conduct is modulated by
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ,
\selectlanguage{english}\textbackslash{}selectlanguageenglishbut current of Nav1.4 and Nav1.5 is
not. CHECK THESE STUDIES FOR TRANSIENT It has been proposed that because
Gbetagamma signaling is membrane-delimited, modulation by betagamma
occurs in membrane microdomains (Galbiati et al., 2001) that are
strategically positioned to affect action potential generation in the
axon hillock and back-propagation at dendritic branches. Such tightly
delimited modulation might be critically important for integrative
properties of the neuron. Determining if Gbetagamma interaction with
VGSCs is responsible for modulation of VGSCs by CaSR allosteric
modulators requires reliable, specific Gbetagamma signaling blockers for
which controls can be designed within our system. The tools currently
available to test this hypothesis are insufficient and we therefore
cannot verify whether or not Gbetagamma participates in this pathway.

\subsection{Inactivation Kinetics: Slow
inactivation}

{\label{286747}}

Upon depolarization, VGSCs move from several closed (C) resting states
though an open (O) state to one or several inactivated states (I).
Recovery from inactivation does not pass through the O state, except in
the case of resurgent VGSC current~\cite{Afshari_2004}\cite{Raman_2001}.
While there is variation between models, it has been suggested that
VGSCs in the cortex can exhibit up to five closed stated (Kuo and Bean
1994) and multiple forms of inactivation. These inactivation states vary
in time scale, biophysical mechanism, and pharmacological sensitivity.
Fast inactivation takes place over the timescale of a single action
potential. The forms of non-fast inactivation have been termed
``intermediate,'' ``slow,'' and ``ultraslow.'' Since the molecular basis
clearly defining these forms of inactivation are not yet known, they are
often clumped under the general term ``slow inactivation.'' Slow
inactivation is produced by prolonged depolarizations over tens of
seconds or a series of brief, consequential depolarizations.

Sodium channels are capable of fast inactivation (complete within a few
milliseconds), and different forms of slow inactivation (time constants
ranging from ~100 ms to several minutes)~{[}18{]}. The mechanism of fast
inactivation is well-studied and occurs by a `hinged lid' mechanism
wherein an intracellular linker between domains III and IV occludes the
channel pore after the open state is reached. In contrast, the
structural basis for slow inactivation has not been fully resolved. Slow
inactivation is thought to occur by a conformational rearrangement or
`collapse' of the outer pore region~{[}38{]}. Mutational studies also
indicate long distance interactions with other structural regions
including involvement of residues in domains IV S4 (19), II S5-S6(30),
and II S6 (21). It was demonstrated that channels could enter the
slow-inactivated state from the closed, open, and fast-inactivated
states (10). Slow inactivation decrease VGSC availability and serves to
decrease repetitive action potential firing and back propagation ~(Ruff
1988)~ in a way that is dependent on previous history of action
potential firing~(Mickus 1999, Toib 1998).~ Slow-inactivated
state-perferring drugs have been identified including lacosimide, an
antiepileptic ~~{[}19{]},~{[}23{]}. ~However, their specific action as
state-preferring is contentious. Electrophysiological protocols used to
study the slow inactivated state utilize a prolonged depolarization (to
induce slow inactivation), followed by a hyperpolarizing gap (to allow
recovery from fast, but not slow inactivation). Because availability in
such protocols is solely determined by the extent of slow inactivation,
a drug that decreases availability is considered to be slow-inactivated
state-preferring. However, gating rates (the rate of inactivation and
rate of recovery from inactivation) are altered by drug binding. A
fast-inactivated state-preferring drug stabilizes this state by delaying
recovery. A delayed recovery does not necessarily indicate actual
modification of the gating rate. For example if the bound drug prevents
recovery from inactivation, then recovery will appear to be slowed
because the drug needs first to dissociate~{[}24{]},~{[}25{]}. Because
of the altered gating, the rate of recovery from fast inactivation in
the presence of the drug can easily overlap with the rate of recovery
from slow-inactivated state.

The rate of state-dependent association and dissociation of the drug
should also be taken into account. As a result, interpretation of data
obtained with these protocols is not straightforward
(e.g.~{[}9{]},~{[}30{]}).

shifts inactivation voltage dependence

and slows recovery from inactivatiData show that both cinacalcet and AEA
shift the steady-state inactivation of VGSCs toward more hyper polarized
potentials. This produces reduced VGSC current during depolarizations
and severely diminishes neuronal excitabilty. However, the application
of prolonged, hyperpolarizing pulses provided for complete recovery of
VGSC in the presence of AEA and cinacalcet (Figure \_\_\_). This slowing
of recovery from inactivation can result from two processes: 1) slow
rate of dissociation of the ligand or 2) increased stability of a
slow-inactivated state, on the basis of an increased rate of entry into
the slow-inactivated state in the presence of ligand. These are two
fundamentally different mechanisms that have qualitatively similar
effects on excitability.

Slowed rate of dissociation of a ligand can occur when drugs bind with
high affinity to a specific state-dependent conformation of the
channel~(Hille, 1977;~Hondeghem and Katzung, 1977) or drugs may be
trapped by the channel gates during specific gating states (Starmer,
1986). If the blocking effect requires activation it will demonstrate
use-dependence. With regard to the dissociation hypothesis, drugs are
thought to bind with high affinity to a specific state-dependent
conformation of the binding site (``modulated receptor theory'') (Hille,
1977;~Hondeghem and Katzung, 1977) or drugs may be trapped by the
channel gates during specific gating states (``guarded receptor
theory'') (Starmer, 1986). if drug binds preferentially to a slow
inactivated state, then it would accelerate the development of this
state during high frequency stimulation (or series of APs). Cinacalcet
was shown to demonstrate use-dependence (Figure \_\_\_) with inhibition
slowed \_\_\_ BLAH BLAH BLAH EXPERIMENT.

The stabilization hypothesis suggests that drugs bind to native ``slow''
inactivated states that under drug-free condition require a long time to
develop (Khodorov et al., 1976;~Zilberter Yu et al., 1991;~Balser et
al., 1996b;~Kambouris et al., 1998;~Chen et al., 2000). However,
drug-binding to a slow-inactivated state accelerates the time course of
development of this state during a depolarization (``stabilization''),
thereby increasing the fraction of channels recovering from that state
during a subsequent hyperpolarization. Slow drug dissociation from
fast-inactivated states and stabilization of slow-inactivated states
need not be mutually exclusive, but both mechanisms may operate in
concert, as noted by~Nuss et al. (2000).

Slow inactivation of sodium channels may be pivotal in regulating firing
properties of a range of neurons by increasing spike threshold,
curtailing prolonged action potential bursts, and limiting active back
propagation of action potentials into dendritic regions (Jung et al.,
1997;~Maurice et al., 2004). Furthermore, slow-inactivated state
preference has been proposed as a therapeutic
advantage~{[}19{]}--{[}21{]}, ~in such diseases as epilepsy, neuropathic
pain and certain arrhythmias~{[}19{]}--{[}22{]}. Defective slow
inactivation is associated with several nervous system diseases,
including myotonia, periodic paralysis, and long-QT syndrome.

\subsection{How this fits into what we know of AEA in synaptic
transmission}

{\label{110047}}

While the investigation of synthetic allosteric modulators of the CaSR's
role in this pathway gives us mores detailed insight into the change in
kinetic properties of VGSCs downstream of G-proteins and explores
potential effector proteins, the addition of AEA as an activator of this
pathway to VGSC inhibition presents the greatest evidence for the
physiological relevance of this pathway. AEA are manufactured on demand
from membrane phospholipid through an enzymatic pathway triggered by
neuronal activity, large depolarization-induced calcium increases, the
activation of D2-like dopamine receptors (Giuffrida et al., 1999), and
MAYBE mGLURS???. AEA is canonically thought of a retrograde signaling
molecule that decreases the release of neurotransmitters, both
inhibitory and excitatory, via the activation of presynaptic receptors
(Ohno-Shosaku et al., 2001; Wilson \& Nicoll, 2001; Alger, 2002). AEA is
removed by uptake from its site of action and the enzymes that catalyze
the conversion of AEA to its degradation compound FAAH (Cravatt 1996,
Goparaju 1998) so that it's action is tightly spatiotemporally
regulated. This indicates that the action of AEA is diffusible but local
to its point of production and short-lived. Recent studies show that
eCBs can have diverse functions are mediators of both long and short
term synaptic depression.

The concentration of AEA within synaptic clefts of the cortex during
periods of high activity is unknown. Measurements of basal AEA levels
vary widely between studies due to quantification technique, parameters
for lipid extraction and purification as well as post-mortem handling of
tissue prior to extraction. However the basal AEA level is quite high
and may reach 0.8 umoles/kg brain tissue
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC55427/). Cannabinoid
receptors CB1 and CB2 and the CaSR are high-affinity targets for AEA.
CB1 is primarily expressed in CNS (reviewed by Howlett, 2002) and CB2
receptor expression is restricted to the periphery (Galiegue et al.,
1995). The CaSR is known to be ubiquitously expressed throughout the
cortex. Pharmacological experiments in this manuscript ruled out CB1 and
the CaSR as AEA target for the initiation of this pathway. AEA is also
known to act on GPCRs Muscarinic M1 \& M4, Adenosine A3, 5-HT1 \& 5-HT2,
S1P1, GPR18, GPR55, GPR119, plus a large number of ion channels and
nuclear receptors
(https://link.springer.com/chapter/10.1007\%2F978-3-319-20825-1\_1).
Several cannabinoid-like orphan receptors have been identified as AEA
targets, including GPR18, GPR55, and GPR119, with affinities for AEA in
the nM-uM range nM
(https://link.springer.com/chapter/10.1007\%2F978-3-319-20825-1\_1)
(https://onlinelibrary.wiley.com/doi/full/10.1002/mnfr.201500449). A
discussion of those receptors can be found in the Future experiments
section of this discussion. The role of many of these receptors as part
of an endocannabinoid pathway is largely unknown. In our current
understanding of AEA action in the cortex through the activation of CB1
receptors, the inhibition of VGSCs would prove to be synergistic.
Cannabinoid agonists have been shown to reduce GABA-mediated IPSCs and
Glutamate-mediated EPSCs in postsynaptic terminals and this is blocked
by selective cannabinoid antagonists and mimicked by cannabinoid
receptor agonists, suggesting roles limited to CB1 (Levenes et al.,
1998) (Maejima et al., 2001; Kreitzer and Regehr, 2001b)
(https://www.sciencedirect.com/science/article/pii/S0896627301002471).
The inhibition of VGSCs by AEA acting acting independently of CB1
receptors would serve to further reduce presynaptic excitability. VGSC
inhibition in this instance would reduce or fully block conduction of
presynaptic action potentials to response to and postsynaptic retrograde
signal. Therefore, VGSC inhibition by AEA may contribute to eCB-mediated
STD and LTD. How neurotransmitter release is actually inhibited by
cannabinoids is still under investigation. There are several
possibilities, as cannabinoids are known to have actions on ionic
conductances that could, if exerted on ion channels at the nerve
terminal, inhibit release. The classic work of Ikeda (1996) and Herlitze
et al. (1996) clearly established that the
\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ-\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits
of heterotrimeric G-proteins directly interact with and inhibit the high
voltage-activated Ca2+ channels the mediate transmitter release at most
synapses. CB1Rs are pertussis-sensitive G-protein-coupled receptors, and
their activation reduces N-type Ca2+ current (Mackie and Hille, 1992;
Caulfield and Brown, 1992; Caulfield et al., 1992; Pan et al., 1996;
Twitchell et al., 1997). CB1R activation also increases the conductances
of various voltage-gated K channels in neurons (Deadwyler et al., 1993,
1995; Schweitzer, 2000), and activates GIRKs in heterologous expression
systems (Henry and Chavkin, 1995; Mackie et al., 1995). Effects on any
of these ion channels could reduce transmitter release. Direct
inhibition of transmitter release machinery independent of Ca2+ is also
possible.

Wilson and Nicoll (2001) reported that DSI is expressed on
TTX-insensitive mIPSCs in the presence of a high-K+/-Ca2+ solution.
Evidently by directly depolarizing the inhibitory nerve terminals, this
solution promotes spontaneous, Ca2+-dependent vesicular release from
terminals possessing CB1Rs, and permits DSI to occur. Clearly DSI can be
exerted at the level of the nerve terminal, and does not depend entirely
on axonal conduction block for its expression, because it can be
observed in the presence of TTX. It is not yet clear whether DSI of the
Ca2+-dependent, TTX-insensitive mIPSCs represents a Ca2+-dependent, or a
downstream consequence of CB1R activation. Conceivably, DSI-susceptible
nerve terminals produce a very low level of basal, i.e.
Ca2+-insensitive, mIPSCs. Depolarization by high {[}K+{]}o in the
presence of high Ca2+, by initiating mIPSC occurrence, may simply make
cannabinoid-sensitive release evident, i.e. the fact that these mIPSCs
are Ca2+-dependent does not mean that their inhibition is also
Ca2+-dependent. DSI of the mIPSCs also seems to be a more variable and
less robust phenomenon than DSI of eIPSCs or of sIPSCs in the absence of
TTX. Moreover, the decrease in mIPSCs has not been shown to account
quantitatively for the decrease in eIPSCs. Hence, the data do not rule
out other mechanisms for DSI expression (see Section 2.7.4).

\par\null

AEA are manufactured on demand from membrane phospholipid through an
enzymatic pathway triggered by neuronal activity, large
depolarization-induced calcium increases. It is also reported that
anandamide is released in the striatum by activation of D2-like dopamine
receptors (Giuffrida et al., 1999). In the canonical AEA pathway, AEA
travels postsynaptically to nerve terminals where it decreases the
release of neurotransmitters, such as GABA or glutamate, via the
activation of presynaptic receptors (Ohno-Shosaku et al., 2001; Wilson
\& Nicoll, 2001; Alger, 2002).AEA is removed by uptake from its site of
action and the enzymes that catalyze the conversion of AEA to its
degradation compound FAAH (Cravatt 1996, Goparaju 1998). This indicates
that the action of AEA is diffusible but local to its point of
production and short-lived. Recent studies show that eCBs can have
diverse functions are mediators of both long and short term synaptic
depression. eCBs are implicated in a range of physiological processes,
on a cellular level: only synaptic plasticity and learning (Heifets and
Castillo, 2009), pain (Guindon and Hohmann, 2009), metabolism and energy
homeostasis (Viveros et al., 2008), and neural development (Fride,
2008). They are also implicated in behavior experiments in motivation
and reward (Solinas et al., 2008), emotional state (Lutz, 2009), the
aetiology of drug addiction (Lopez-Moreno et al., 2008; Parolaro and
Rubino, 2008), as well as numerous models of neurodegenerative disease.
In many instances, the evidence for an eCB mechanism is indirect and
pathways not fully delineated. Accordingly, there has been substantial
interest in the quantification of eCB content in a variety of biological
matrices.

https://www.sciencedirect.com/science/article/pii/S0301008202000801?via\%3Dihub\#BIB160

\par\null

synaptic cleft concentrations of the endocannabinoids at times of
activity are unknown. However the basal

Regional brain tissue eCb levels in rats 20 pmol/g in cortex
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931546/)

Measurements vary with brain tissues and between studies due to the
parameters for lipid extraction and purification can have a large impact
of eCB recovery, as can the post-mortem handling of tissue prior to
extraction

\par\null

ECs are released from neurons in a nonvesicular manner in response to
large depolarization-induced Ca2+~elevation (Cadas~\emph{et~al} .,
1996;~Stella~\emph{et~al} ., 1997).

Recent studies have revealed the essential role of ECs in retrograde
modulation of synaptic transmission in the central nervous system:
strong depolarization of postsynaptic neurons releases ECs, which act
backwards onto presynaptic CB1 receptors and decrease the release of
other neurotransmitters, such as
\textbackslash{}selectlanguagegreek\selectlanguage{greek}γ\selectlanguage{english}\textbackslash{}selectlanguageenglish-\textbackslash{}selectlanguageenglishaminobutyric
acid (GABA) or glutamate, from presynaptic terminals
(Ohno-Shosaku~\emph{et~al} ., 2001;~Wilson \& Nicoll, 2001;~Alger,
2002).

;Wilson
\&a\textbackslash{}selectlanguagepolish\selectlanguage{polish}Ė\selectlanguage{english}\textbackslash{}selectlanguageenglishrdJ

DSI shows a substantial delay, several hundred milliseconds to a second
or more, suggesting the involvement of second messengers
(https://ac.els-cdn.com/0896627394904308/1-s2.0-0896627394904308-main.pdf?\_tid=85e10993-1659-4d30-876b-04afbe6625d9\&acdnat=1521588031\_11fb139ed11b6c05be0e412c226a6499)

Measurements vary with brain tissues and between studies due to the
parameters for lipid extraction and purification can have a large impact
of eCB recovery, as can the post-mortem handling of tissue prior to
extraction

ECs are released from neurons in a nonvesicular manner in response to
large depolarization-induced Ca2+~elevation (Cadas~\emph{et~al} .,
1996;~Stella~\emph{et~al} ., 1997).

Recent studies have revealed the essential role of ECs in retrograde
modulation of synaptic transmission in the central nervous system:
strong depolarization of postsynaptic neurons releases ECs, which act
backwards onto presynaptic CB1 receptors and decrease the release of
other neurotransmitters, such as
\textbackslash{}selectlanguagegreek\selectlanguage{greek}γ\selectlanguage{english}\textbackslash{}selectlanguageenglish-\textbackslash{}selectlanguageenglishaminobutyric
acid (GABA) or glutamate, from presynaptic terminals
(Ohno-Shosaku~\emph{et~al} ., 2001;~Wilson \& Nicoll, 2001;~Alger,
2002).

normal'r\_J

\par\null

EXAMPLE WHERE OMISSION OF GTP DOES NOT STOP G PROTEIN PROCESS
https://ac.els-cdn.com/0896627394904308/1-s2.0-0896627394904308-main.pdf?\_tid=85e10993-1659-4d30-876b-04afbe6625d9\&acdnat=1521588031\_11fb139ed11b6c05be0e412c226a6499

GPR55, GPR119, and GPR18 are three cannabinoid-like orphan GPCRS (Mackie
and Stella 2006; Pertwee, 2010). Little is yet known of the
physiological functions of these receptors. All three receptors are
expressed in brain tissue. AEA is identified as a partial agonist of
GPR18, a GPCR expressed in the central nervous system, including the
cortex (Vassilatis et al., 2003). GPR18 was only recently deorphanized
and its role in physiological processes consequentially unexplored.
GPR18 serves roles in microglial migration in the central nervous
system. GPR18 is suggested to have a sympathoinhibitory role in neurons
of the rostral ventrolateral medulla
(https://www.ncbi.nlm.nih.gov/pubmed/24431468). However, further
physiological roles for this receptor are as yet unexplored. GPR119 is
also expressed in the cortex (Bonini 2002 Methods of identifying
compounds that bind to SNORF25 receptors.). Research on GPR 119 has
focused on it's role in the modulation of insulin release by pancreatic
\textbackslash{}selectlanguagegreek\selectlanguage{greek}β-\selectlanguage{english}\textbackslash{}selectlanguageenglishcells
and of GLP-1 secretion by gut enteroendocrine cells
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268073/). It's action in
the cortex is not yet known. GPR55 was identified as a cannabinoid
receptor. Activation stimulates RhoA, cdc42, rac1.Expressed in the brain
(M. Sawzdargo, et al.Identification and cloning of three novel human G
protein-coupled receptor genes GPR52, PsiGPR53 and GPR55: GPR55 is
extensively expressed in human brain). These three GPCRs serve as
putative targets for AEA to stimulate the pathway identified in this
research. Exploring the role of these receptors in the pathway will
require the development of knockout mice and/or highly specific receptor
inhibitors. Without these tools, testing for VGSC inhibition via GPR18,
GPR55, or GPR119 will be difficult to conclude.

\par\null

\subsection{What we already know about AEA on
VGSCs}

{\label{909696}}

\subsection{Potential GPRCs}

{\label{817048}}

\par\null

\subsubsection{Binding sites of CaSR
Modulators}

{\label{956437}}

R 568~ cinacalcet is a derivative of

NPS 2143, Calindol, Calhex 231 and cinacalcet are structurally related
phenylalkylamines, containing two aromatic rings connected by a flexible
chain with a positively charged amino group. Studies have revealed
calcimimetics and calcilytics target a similar interaction sites in a
crevice formed by transmembrane domains (TMs) 3, 5, 6, and 7. The
binding pocket for both calcimimetics and calcyliytics of the CaSR has
been suggested to involve Trp-818, Phe-821, Glu-837, and Ile-841 located
in transmembranes (TM) 6 and TM7, with differences observed between each
family of compounds (petrel and ruat evernote).~All four compounds are
exhibit ionic interactions with a negatively charged amino acid
(Glu-837) located in TM7
(https://academic.oup.com/edrv/article/32/1/3/2354777). The binding
sites for all four compounds are largely overlapping but not identical
and located within a bundle formed by the seven transmembrane domains.
The shared binding pocket for all four CaSR allosteric modulators points
to, though does not prove, the existence of a structurally similar
binding pocket on a separate GPCR mediating this effect. It is not yet
known how if AEA interacts with the same allosteric modulator site on
the CaSR, though the presence of a positively charged amino group may
indicate similarities in the mechanism of binding.

\subsubsection{GPR55, GPR18}

{\label{656734}}

GPR55, GPR119, and GPR18 are three cannabinoid-like orphan GPCRS (Mackie
and Stella 2006; Pertwee, 2010). Little is yet known of the
physiological functions of these receptors. All three receptors are
expressed in brain tissue. AEA is identified as a partial agonist of
GPR18, a GPCR expressed in the central nervous system, including the
cortex (Vassilatis et al., 2003). GPR18 was only recently deorphanized
and its role in physiological processes consequentially unexplored.
GPR18 serves roles in microglial migration in the central nervous
system. GPR18 is suggested to have a sympathoinhibitory role in neurons
of the rostral ventrolateral medulla
(https://www.ncbi.nlm.nih.gov/pubmed/24431468). However, further
physiological roles for this receptor are as yet unexplored. GPR119 is
also expressed in the cortex (Bonini 2002 Methods of identifying
compounds that bind to SNORF25 receptors.). Research on GPR 119 has
focused on it's role in the modulation of insulin release by pancreatic
\textbackslash{}selectlanguagegreek\selectlanguage{greek}β-\selectlanguage{english}\textbackslash{}selectlanguageenglishcells
and of GLP-1 secretion by gut enteroendocrine cells
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268073/). It's action in
the cortex is not yet known. GPR55 was identified as a cannabinoid
receptor. Activation stimulates RhoA, cdc42, rac1.Expressed in the brain
(M. Sawzdargo, et al.Identification and cloning of three novel human G
protein-coupled receptor genes GPR52, PsiGPR53 and GPR55: GPR55 is
extensively expressed in human brain). These three GPCRs serve as
putative targets for AEA to stimulate the pathway identified in this
research. Exploring the role of these receptors in the pathway will
require the development of knockout mice and/or highly specific receptor
inhibitors. Without these tools, testing for VGSC inhibition via GPR18,
GPR55, or GPR119 will be difficult to conclude.

GPR18 is Galpha i/o coupled and Gq

\par\null

Studies of~ G\textbackslash{}selectlanguagegreek\selectlanguage{greek}\emph{βγ}
\selectlanguage{english}\textbackslash{}selectlanguageenglish~ binding to VGKCs and VGCCs
indicate that there is no consensus sequence for protein-protein
interactions~\cite{Lin_2011}, rather a structurally flexible protein
interaction hot spot that accommodates different structures on the beta
gamma particle\cite{DeLano_2002}. This is unsurprising given the
diversity in protein sequence and structure that are known to be
accommodated by this single binding site.

\par\null

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4237895/

GPR18 deorphanized when it was found to be activated by AEA, NAGly,
abn-CBD

expressed in the CNS including cortex (Vassilatis et al., 2003).

(https://onlinelibrary.wiley.com/doi/full/10.1002/mnfr.201500449)

GLP-1 se\textbackslash{}selectlanguageenglishrqb

\subsubsection{}

{\label{648195}}

EC50 for AEA on GPR55 is 18 nM
(https://link.springer.com/chapter/10.1007\%2F978-3-319-20825-1\_1) but
also activated by AM 251

EC50 AEA on GPR18 3.83 uM
(https://onlinelibrary.wiley.com/doi/full/10.1002/mnfr.201500449)
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806671/)

an style='fon3rb

\par\null

\subsubsection{GPRC6A}

{\label{755423}}

\par\null

\subsection{Future Experiments}

{\label{391218}}

As illustrated by our lack of complete understanding about the identity
and composition of the inward current following mGlu1 and mGlu5 receptor
activation, there is still much to be answered regarding Group I mGluR
activation in dopamine neurons. The data presented in this dissertation
add new insights for understanding how these receptors modulate dopamine
neuron activity. But, these data also present new questions that hadn't
been appreciated before. The most relevant and approachable questions
will now be addressed, along with ideas for experiments

\subsection{Summary and Conclusions}

{\label{218703}}

In this work, I identify five ligands that inhibit VGSC current via a
G-protein pathway. All of the ligands are known to act on the CaSR with
either negative or positive allosteric effects. The action of synthetic
modulators of the CaSR, cinacalcet, calhex, NPS 2143, and Calindol, were
characterized in Chapter 1. All produce complete inhibition of VGSC
current with time in a dose-dependent fashion. The inhibition by
Cinacalcet, NPS 2143, and calindol was alleviated in the presence of
G-protein signaling inhibitor GDPbetaS, indicating a G-protein dependent
mechanism. This finding became more intriguing as I determined that
cinacalcet, Calindol, NPS 2143, and calhex exert their effects
independently of the CaSR, their known target GPCR.~ I investigated the
identity of the target GPCR for these ligands by first looking at
candidates within the Class C GPCR family, second, eliminating a class
of G-protein with PTx, and third, determining the involvement of
downstream effector proteins with PKA and PKC inhibitors. Using this
methodology, data showed that cinacalcet does not act through mGluR1,
mGlurR5, or GABAB receptors. Cinacalcet inhibition of VGSCs also does
not require Gi/o G-proteins, eliminating a class of G-protein and
narrowing the range of GPCR candidates. Lastly, cinacalcet inhibition of
VGSCs does not require the activation of PKA or PKC. These findings
leave a number of possibilities for the composition of this pathway: Gq,
G12, and Gs G-proteins, staurosporine-resisitance kinases,
phospholipases, Gby signaling, intracellular calcium-signaling, and a
multitude of other factors, some of which I will explore in this
discussion. The kinetics of cinacalcet-induced inhibition were
quantified in-depth. It was shown that cinacalcet produces inhibition of
VGSC current by shifting the steady-state inhibition of the channels
toward more polarized potentials. This inhibition could therefore be
completely reversed by long hyperpolarizing steps. This finding first
suggests that cinacalcet pushes VGSCs into a deeply inactivated state.
In Chapter 2, I show that this type of inhibition is also enacted by
endogenous cannabinoid anandamide. This is important as AEA is natively
present in the cortex and this establishes that the pathway may play a
role in synaptic communication. AEA, as the other CaSR modulators,
produced complete inhibition of VGSC current over time. This inhibition
was relieved with GDPbetaS and independent of the CaSR. Surprisingly,
inhibition was also independent of AEA target CB1. Like cinacalcet, AEA
shifted inactivation of VGSCs and this inactivation-produced inhibition
was completely reversed with strong hyperpolarizing pulses. While it
cannot be conclusively stated that AEA, Cincalcet, Calindol, Calhex, and
NPS 2143 act via the same pathway and same GPCR, it is highly likely
given their commonality as CaSR allosteric modulators and the highly
similar way in which they affect VGSC inactivation. While NPS 2143,
cinacalcet, calhex, and Calindol aided in the characterization of this
pathway, AEA's participation in this pathway is the finding that
emphasizes the importance of this pathway to synaptic physiology. Here,
we establish a pathway from ligand to G-protein to changes in VGSC
output. This pathway may represent a novel homeostatic mechanism to
reduce neuronal excitability.

\subsection{Extra Information without a
home}

{\label{621237}}

\par\null

Gallein is a cell-permeable, small molecule inhibitor of betagamma
signaling. It binds to betagamma particle with high affinity Kd
\textasciitilde{}400 nM. Gallein acts by blocking the interactions
between Gbetagamma and its effectors. Gallein does not promote
dissociation of Galpha from Gbetagamma.

Since then, beta gamma has been shown to act on Adenylyl cyclase (AC)
I'' (Tang and Gilman, 1991; Sunahara and Taussig, 2002), G
protein-coupled receptor kinase 2 (GRK2) (Pitcher et al., 1992),
phospholipase C (PLC)
\textbackslash{}selectlanguagegreek\selectlanguage{greek}β\selectlanguage{english}\textbackslash{}selectlanguageenglish2
and
\textbackslash{}selectlanguagegreek\selectlanguage{greek}β\selectlanguage{english}\textbackslash{}selectlanguageenglish3
isoforms (Camps et al., 1992; Park et al., 1993; Smrcka and Sternweis,
1993), inwardly rectifying potassium channels (GIRK) (Logothetis et al.,
1987; Nakajima et al., 1996), phosphoinositide 3-kinase
\textbackslash{}selectlanguagegreek\selectlanguage{greek}γ
\selectlanguage{english}\textbackslash{}selectlanguageenglish(PI3K\textbackslash{}selectlanguagegreek\selectlanguage{greek}γ\selectlanguage{english}\textbackslash{}selectlanguageenglish)
(Stephens et al., 1994, 1997), and N-type calcium channels (Ikeda,
1996)''

Proteomic methods and yeast two-hybrid screening have revealed multiple
novel G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishbinding proteins. These include PDZ
domain containing proteins (Li et al., 2006); guanine exchange factors
(GEFs) for small G proteins such as P-Rex1 (Mayeenuddin et al., 2006),
FLJ00018, also known as pleckstrin homology domain containing family G
member 2 (a
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ-\selectlanguage{english}\textbackslash{}selectlanguageenglishactivated
Rac and Cdc42 guanine nucleotide exchange factor) (Ueda et al., 2008),
and p114-RhoGEF (Niu et al., 2003); protein kinase D (PKD) (Jamora et
al., 1999); receptor for activated C kinase 1 (RACK1) (Dell et al.,
2002); soluble NSF attachment protein (SNAP) receptor (SNARE) complex
(Yoon et al., 2007); and a Radil-Rap1A complex (Ahmed et al.,
2010).~\textbackslash{}selectlanguagegreek\selectlanguage{greek}\textbf{A striking observation
for all of these Gβγ binders is that no readily apparent consensus
sequence or structure mediates binding of these proteins to Gβγ.}

\selectlanguage{english}\textbackslash{}selectlanguage

english~Gby has been shown to modulation 1.2 and 1.2 NaV. InaP
persistent is increased when NaV1.2 coexpressed with Gby (Ma et al
1997).

This increase was prevented by a putative
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ-\selectlanguage{english}\textbackslash{}selectlanguageenglishbinding
peptide from the C terminus of the Nav1.2 channel, suggesting that
G-protein \textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits interact with that
C-terminal site during modulation (Ma et al., 1997). Consistent with
this idea, G-protein \textbackslash{}selectlanguagegreek\selectlanguage{greek}β
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits immunoprecipitate with
sodium channel \textbackslash{}selectlanguagegreek\selectlanguage{greek}α
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits from cortical neuron
preparations (Marin et al., 2001).

We show that both Nav1.1 and Nav1.2 conduct~\emph{I} NaP~(Nav1.1
\textgreater{} Nav1.2) that can be modulated by
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ,
\selectlanguage{english}\textbackslash{}selectlanguageenglishbut Nav1.4 and Nav1.5 do not.

Analysis of channel chimeras reveals that the transmembrane bodies of
Nav1.4 and Nav1.5 channels are responsible for their lack of G-protein
modulation rather than their C-terminal domains (Scheuer, MAntegazza,
Powell, Yu.

Modulation by free G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishin neurons might occur locally in
membrane microdomains (Galbiati et al., 2001) strategically positioned
to affect action potential generation in the axon hillock, action
potential back-propagation at dendritic branches, or other localized
electrical events. Such tightly delimited modulation might be critically
important for integrative properties of the neuron but would

The cocrystal structures reveal that effector/binding proteins share a
critical interaction interface on the top of the torus of
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}β
\selectlanguage{english}\textbackslash{}selectlanguageenglishcreated by the
\textbackslash{}selectlanguagegreek\selectlanguage{greek}β
\selectlanguage{english}\textbackslash{}selectlanguageenglishpropeller fold that binds to switch
II helix of the G\textbackslash{}selectlanguagegreek\selectlanguage{greek}α
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits (Fig. 1).

As discussed, small-molecule G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishinhibitors (M119/gallein) have been
used extensively to investigate G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishfunctions in cell biological and
animal models of diseases. In the course of these studies, many
questions concerning off-target effects have been addressed. For
example, in the presence of M119/gallein, the following were observed:

1. Unimpaired isoproterenol- and
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}α\selectlanguage{english}\textbackslash{}selectlanguageenglishs-dependent
cAMP production (Casey et al., 2010).

2. Unimpaired {[}d-Ala2,N-MePhe4,Gly-ol{]}-enkephalin (a
\textbackslash{}selectlanguagegreek\selectlanguage{greek}μ-\selectlanguage{english}\textbackslash{}selectlanguageenglishopioid
receptor-specific agonist),
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}α\selectlanguage{english}\textbackslash{}selectlanguageenglishi-dependent
decrease in cAMP and unimpaired
\textbackslash{}selectlanguagegreek\selectlanguage{greek}μ-\selectlanguage{english}\textbackslash{}selectlanguageenglishopioid
receptor,
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ-\selectlanguage{english}\textbackslash{}selectlanguageenglishdependent
analgesia, as discussed under Biased Agonist Signaling (Mathews et al.,
2008).

3. No effect of compounds on \textbackslash{}selectlanguagegreek\selectlanguage{greek}δ
\selectlanguage{english}\textbackslash{}selectlanguageenglishand
\textbackslash{}selectlanguagegreek\selectlanguage{greek}κ
\selectlanguage{english}\textbackslash{}selectlanguageenglishopioid receptor signaling (Mathews
et al., 2008).

4. Unimpaired fMLP- and
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ-\selectlanguage{english}\textbackslash{}selectlanguageenglishdependent
extracellular signal-regulated kinase activation in HL60 neutrophil-like
cells (Bonacci et al., 2006).

5. Unimpaired M3 muscarinic acetylcholine receptor,
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}α\selectlanguage{english}\textbackslash{}selectlanguageenglishq-
dependent Ca2+ regulation (Bonacci et al., 2006).

6. Unimpaired stromal cell-derived factor-1- and
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}α\selectlanguage{english}\textbackslash{}selectlanguageenglishi-dependent
inhibition of cAMP levels (Kirui et al., 2010).

All these data indicate that GPCRs function normally in the presence of
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishinhibitors and that there is
selectivity for different G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishtargets.

GABAB activates GiRKS baclofen Betterl + Tiao 2006 Pharmacol Ther

Kir3 type channel

Not blocked by galleon (casey, blaxall Circ res 2010) shows selectivity
of Gbetagamma

\textbf{Lehmann} ~\emph{et al} ~(2008) Small molecule disruption of G
protein \textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunit signaling inhibits
neutrophil chemotaxis and inflammation. Mol.Pharmacol.~73 ~410
PMID:~18006643

\textbf{Ukhanov} ~\emph{et al} ~(2011) Phosphoinositide
3-kinase-dependent antagonism in mammalian olfactory receptor neurons.
J.Neurosci.~31 ~273 PMID:~21209212

;\textless{}authorr\_

\par\null

Regulation of VGSCs by second messenger systems may have wide-ranging
effects on cellular activity

5HT regulation is not enough to affect somatic action potentials of
pyramidal neurons in PFC slice but may recue action potential amplitude
back-propogating into apical dendrite (Carr 2002) This reduced dendritic
excitability to negatively modulate acivity-dependent dendritic synaptic
plasticity

Na channels regulate not only somatic spike generation but also
dendritic integration of synaptic input (Schwindt and Crill, 1995;
Stuart and Sakmann, 1995; Lipowsky et al., 1996;Gonzalez-Burgos and
Barrionuevo, 2001),

ty as CaSR allor*b

The experiments in this dissertation demonstrate\ldots{}. . However, it
is less clear \ldots{}..Future experiments blocking the activity of the
endogenous Isl1, Lhx3 or LMO4 enhancers and then examining the
expression of each of these genes and motor neuron specification will
allow us to better assess the role of each enhancer in motor neuron
specification. Likewise, blocking these enhancers could prove to be an
effective mechanism for knocking out, or knocking down Isl1, Lhx3 or
LMO4 expression relatively late in motor neuron development. This late
alteration in gene expression could be a useful model for learning more
about the roles of 105 each of these genes in motor neuron development
and motor neuron subtype specification.

Regulation of VGSCs by second messenger systems may have wide-ranging
effects on cellular activity

5HT regulation is not enough to affect somatic action potentials of
pyramidal neurons in PFC slice but may recue action potential amplitude
back-propogating into apical dendrite (Carr 2002) This reduced dendritic
excitability to negatively modulate acivity-dependent dendritic synaptic
plasticity

Na channels regulate not only somatic spike generation but also
dendritic integration of synaptic input (Schwindt and Crill, 1995;
Stuart and Sakmann, 1995; Lipowsky et al., 1996;Gonzalez-Burgos and
Barrionuevo, 2001),

Since then, beta gamma has been shown to act on Adenylyl cyclase (AC)
I'' (Tang and Gilman, 1991; Sunahara and Taussig, 2002), G
protein-coupled receptor kinase 2 (GRK2) (Pitcher et al., 1992),
phospholipase C (PLC)
\textbackslash{}selectlanguagegreek\selectlanguage{greek}β\selectlanguage{english}\textbackslash{}selectlanguageenglish2
and
\textbackslash{}selectlanguagegreek\selectlanguage{greek}β\selectlanguage{english}\textbackslash{}selectlanguageenglish3
isoforms (Camps et al., 1992; Park et al., 1993; Smrcka and Sternweis,
1993), inwardly rectifying potassium channels (GIRK) (Logothetis et al.,
1987; Nakajima et al., 1996), phosphoinositide 3-kinase
\textbackslash{}selectlanguagegreek\selectlanguage{greek}γ
\selectlanguage{english}\textbackslash{}selectlanguageenglish(PI3K\textbackslash{}selectlanguagegreek\selectlanguage{greek}γ\selectlanguage{english}\textbackslash{}selectlanguageenglish)
(Stephens et al., 1994, 1997), and N-type calcium channels (Ikeda,
1996)''

tr9X

Modulation by free G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishin neurons might occur locally in
membrane microdomains (Galbiati et al., 2001) strategically positioned
to affect action potential generation in the axon hillock, action
potential back-propagation at dendritic branches, or other localized
electrical events. Such tightly delimited modulation might be critically
important for integrative properties of the neuron but would

Proteomic methods and yeast two-hybrid screening have revealed multiple
novel G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishbinding proteins. These include PDZ
domain containing proteins (Li et al., 2006); guanine exchange factors
(GEFs) for small G proteins such as P-Rex1 (Mayeenuddin et al., 2006),
FLJ00018, also known as pleckstrin homology domain containing family G
member 2 (a
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ-\selectlanguage{english}\textbackslash{}selectlanguageenglishactivated
Rac and Cdc42 guanine nucleotide exchange factor) (Ueda et al., 2008),
and p114-RhoGEF (Niu et al., 2003); protein kinase D (PKD) (Jamora et
al., 1999); receptor for activated C kinase 1 (RACK1) (Dell et al.,
2002); soluble NSF attachment protein (SNAP) receptor (SNARE) complex
(Yoon et al., 2007); and a Radil-Rap1A complex (Ahmed et al., 2010). A
striking observation for all of these
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishbinders is that no readily apparent
consensus sequence or structure mediates binding of these proteins to
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ.

\selectlanguage{english}\textbackslash{}selectlanguage

english

Gby has been shown to modulation 1.2 and 1.2 NaV. InaP persistent is
increased when NaV1.2 coexpressed with Gby (Ma et al 1997).

This increase was prevented by a putative
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ-\selectlanguage{english}\textbackslash{}selectlanguageenglishbinding
peptide from the C terminus of the Nav1.2 channel, suggesting that
G-protein \textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits interact with that
C-terminal site during modulation (Ma et al., 1997). Consistent with
this idea, G-protein \textbackslash{}selectlanguagegreek\selectlanguage{greek}β
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits immunoprecipitate with
sodium channel \textbackslash{}selectlanguagegreek\selectlanguage{greek}α
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits from cortical neuron
preparations (Marin et al., 2001).

We show that both Nav1.1 and Nav1.2 conduct~\emph{I} NaP~(Nav1.1
\textgreater{} Nav1.2) that can be modulated by
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ,
\selectlanguage{english}\textbackslash{}selectlanguageenglishbut Nav1.4 and Nav1.5 do not.

Analysis of channel chimeras reveals that the transmembrane bodies of
Nav1.4 and Nav1.5 channels are responsible for their lack of G-protein
modulation rather than their C-terminal domains (Scheuer, MAntegazza,
Powell, Yu.

Modulation by free G\textbackslash{}selectlanguagegreek\selectlanguage{greek}βγ
\selectlanguage{english}\textbackslash{}selectlanguageenglishin neurons might occur locally in
membrane microdomains (Galbiati et al., 2001) strategically positioned
to affect action potential generation in the axon hillock, action
potential back-propagation at dendritic branches, or other localized
electrical events. Such tightly delimited modulation might be critically
important for integrative properties of the neuron but would

(392 receptors in mice, 367 in
humans)https://www.ncbi.nlm.nih.gov/pmc/articles/PMC153653/

The cocrystal structures reveal that effector/binding proteins share a
critical interaction interface on the top of the torus of
G\textbackslash{}selectlanguagegreek\selectlanguage{greek}β
\selectlanguage{english}\textbackslash{}selectlanguageenglishcreated by the
\textbackslash{}selectlanguagegreek\selectlanguage{greek}β
\selectlanguage{english}\textbackslash{}selectlanguageenglishpropeller fold that binds to switch
II helix of the G\textbackslash{}selectlanguagegreek\selectlanguage{greek}α
\selectlanguage{english}\textbackslash{}selectlanguageenglishsubunits (Fig. 1).

\par\null

We found cinacalcet-induced inhibition occurs independently of not only
the CaSR, but also class C GPRCS mGluR1, mGluR5, and the GABAB receptor.
Eliminating mGluR1 as the target of cinacalcet allows us to establish
this as a separate pathway for G-protein-dependent modulation of VGSC
current. Furthermore, mAchR M1, D1 receptor, 5-HT2a/c receptor, and
mGluR1 activation were all shown to depend on either PKA or PKC activity
for VGSC inhibition (Cantrell, Ma, Scheuer and Catterall 1996, Cantrell,
Smith, Goldin, Scheuer and Catterall 1997, Carr, Cooper, Ulrich,
Spruston and Surmeier 2002, Carlier, Sourdet, Boudkkazi,
D\textbackslash{}selectlanguagengerman\selectlanguage{ngerman}église, Ankri,
Fronzaroli-Molinieres and Debanne 2006) whereas VGSC inhibition by
cinacalcet occurred in the absence of PKA and PKC activity.. In
addition, stimulation of mAchR receptors and dopamine D1/D5 receptors
did not reduce VGSC in the same neurons that responded to cinacalcet
(Fig. 9a,c,d). This further distinguishes this pathway from all other
G-protein processes for VGSC modulation for which data currently exists
(Cantrell and Catterall 2001).

VGSC current inhibition that relies on slow inactivation has been shown
to reduce a neuron's ability to sustain trains of spikes (Carr, Day,
Cantrell, Held, Scheuer, Catterall and Surmeier 2003). We predict that
the strong, slow inhibition of VGSC by CaSR modulators should have
similar effects.

\par\null

Indeed actions in the brain are of such concern that new CaSR
modulators, that are not lipophilic and thus less likely to cross the
blood brain barrier, are being synthesized and tested clinically
(Martin, Bell, Pickthorn, Huang, Vick, Hodsman and Peacock 2014). Our
findings may describe mechanisms of off-target effects of cinacalcet.

\par\null

Reduction in peak VGSC current could serve as a neuroprotective
safeguard, preventing excitotoxicity during periods of intense synaptic
transmission. Consequently, the G-protein pathway investigated here may
represent an avenue for new therapies characterized by neuronal
hyperexcitability such as epilepsy, migraine, chronic pain, and periodic
paralysis.

\par\null

https://www.sciencedirect.com/science/article/pii/S0006899305013107?via\%3Dihub

Kim et al. show that AEA blocks TTX-S and TTX-R VGSCs in rat dorsal root
ganglion neurons of the periphery. Conc-dep manner with Kd 5.4 uM on
TTX-S neurons. Produced hyperpolarization of steady-state inactivation.
AEA affected inactivated Na channels rather than resting- indication for
use-dependence. Inhibition not reversed by CB1 antagonist AM 251, CB2
antagonist AM 630, or Capsazepine (VR1 antagonist). They conclude this
then means direct action but do not explore other G-protein dependent
pathways.

\par\null

family C GPCRs also includes a amino acid- and divalent cation-sensing
receptor called GPRC6A

\textbackslash{}selectlanguage

english \nocite{*}

\selectlanguage{english}
\FloatBarrier
\bibliographystyle{plainnat}
\bibliography{bibliography/converted_to_latex.bib%
}

\end{document}

