2D interfacial exchange-diffusion has the potential to augment spatiotemporal precision of Ca2+ signalling
Abstract
Nano-junctions between the endoplasmic reticulum and the cytoplasmic surfaces of the plasma membrane and other organelles shape the spatiotemporal features of biological Ca2+ signals. Herein we propose that 2D Ca2+ exchange-diffusion on the negatively charged phospholipid surface lining the nano-junctions participates in guiding Ca2+ from its source (channel or carrier) to its target (transport protein or enzyme). Evidence provided by in vitro Ca2+ flux experiments using an artificial phospholipid membrane is presented in support of the above proposed concept, and results from stochastic simulations of Ca2+ trajectories within nano-junctions are discussed to substantiate its possible requirements. Finally, we analyze recent literature on Ca2+ lipid interactions, which suggest that 2D interfacial Ca2+ diffusion may represent an important mechanism of signal transduction in other biological systems characterized by high phospholipid surface-to-aqueous volume ratios.
Introduction
All rapid functions in the body are selectively regulated by the
universal biological messenger: ionic calcium (Ca2+). For this single ionic messenger to harmoniously control such a great range of biological mechanisms, it is crucial that its signals are delivered with pinpoint precision and millisecond timing. The endoplasmic reticulum (ER) is the main organelle that orchestrates this essential spatiotemporal precision of Ca2+ signalling via numerous
different close contact sites or nano-junctions (NJ) with the
plasma membrane (PM), mitochondria, lysosomes and other organelles \cite{23339179}.
NJs have been defined as cytoplasmic sub-compartments where membranes of
different organelles appose each other within the nano-metre scale.
Typically the limiting membranes are separated by 20 nm or less and the
specialized signalling function has been shown to fail at separation
distances greater than 50 nm \cite{15541459,17418403,Pritchard2019}. The main function of the NJs in
cells is to precisely localize the Ca2+ signals to
specific Ca2+ sensors positioned within organellar membrane or
PM domains, while bypassing the bulk cytoplasm. For example,
refilling of the sarcoplasmic reticulum (SR) of vascular smooth muscle
cells, in order to maintain asynchronous
[Ca2+]cyt oscillations, is achieved
by coupling Ca2+ entry via
Ca2+-influx-mode NCX (rNCX) to SERCA at PM-SR NJs,
thus bypassing the bulk cytoplasm \cite{Lee2002}. Employing stochastic particle
simulator modelling software and using known NCX and SERCA turnover
rates and surface densities, it was possible to generate a computational model of this
cellular signalling process, which demonstrated that the rate of
Ca2+ entry via rNCX/SERCA was indeed sufficient for
replacing the Ca2+ released by periodic opening and
closing of IP3Rs during the activation of asynchronous
cytoplasmic Ca2+ waves that stimulate contraction \cite{17418403}.
However, to generate plausible predictions by our computational model,
it was necessary to implement a Ca2+ target size on
SERCA of approximately 20 nm2, which is 2500 times
larger than the area occupied by the dehydrated Ca2+ with a
diameter of 1 Å. Assuming that short range local electrostatic
forces of attraction between the fixed negatively charged binding sites
on the SERCA macromolecule and the positively charged
Ca2+ would increase the effective target size to an area several times larger
than the size of the non-hydrated Ca2+, it would still
be orders of magnitude smaller than 20 nm2. Therefore,
in order to achieve effective functional transfer of
Ca2+ from NCX on the PM to SERCA on the SR, it appears
that an additional, as yet ignored, mechanism is operative to support
the linkage between Ca2+ signalling elements (sources
and sinks) within NJs, besides the proximity of the PM and SR in these
regions. To this end, all available computational modelling data prompt us to conclude that, by itself, a 3D
random walk of the Ca2+ between their sources and sinks on the two closely
apposing membrane surfaces may be insufficient for efficient NJ Ca2+signalling. We thus propose that, in addition, 2D exchange
diffusion of Ca2+ on the targeted surface of the NJ has the potential to increase the efficiency of Ca2+ reaching its target.
Background
It is well established that Ca2+ can move rapidly
through negatively charged solid lattices, such as fluorapatite, by the
process of exchange diffusion \cite{Jay_2011}. An analogous model, featuring negatively charged phospholipids (PLs), has been described some time ago \cite{TOBIAS1962,5723329,5803390}. This
experimental model consists of a millipore filter impregnated with a
mixture of phospholipids of animal origin, separating two aqueous phases
and exhibits properties that are highly relevant to the topic of
Ca2+ movements through narrow aqueous passages lined
by PLs. Its salient feature is that it supports net
transfer of Ca2+ through relatively long PL lined
pores at a much faster rate than would be possible for free diffusion within the limited
adjoining aqueous phase. The mechanism that was presented to explain
Ca2+ transport through this solid ion exchange
membrane involves the association of Ca2+ with a
negatively charged phosphate or carboxyl group of the PL surface on the cis-side of the
membrane, followed by transfer of Ca2+ within a
2-dimensional matrix of similar sites, constituted by the pore-lining PL layers, and a final step of dissociation of
Ca2+ from the negatively charged PL head
groups on the trans-side of the membrane. The rate of this PL-mediated transport of radioactive labeled
Ca2+ through the membrane was decreased by removal of
Ca2+ from the buffered solution on the trans-side of the
membrane. Paradoxically, further removal of remaining
Ca2+ from the trans-solution by the addition of the
soluble, but non-permeant chelator EDTA, increased the rate of
PL-mediated net Ca2+ transport across the PL
lined millipore filter by more than one order of magnitude. The mechanism proposed to explain
the latter observation is that dissociation of Ca2+ from
PL head groups on the trans-side of the membrane is rate-limiting
and addition of a freely diffusible, impermeant Ca2+ binding site on EDTA, to the trans-side of the flux chamber
facilitates the dissociation of PL bound Ca2+. Once
the Ca2+ transported from the cis-side of the
flux chamber has been chelated by EDTA, which is dissolved in a large
volume of buffered solution on the trans-side, it cannot rebind
to the PL membrane, but is replaced by the next Ca2+arriving from the cis-side. In this model system, electro-neutrality is preserved by the
movement of Mg2+ and monovalent cations in the
opposite direction. The mechanism envisioned for the transfer of
Ca2+ from the PL membrane to EDTA involves an intermediary step of partial dissociation from the negative PL site and
simultaneous association with a carboxyl group of EDTA. We propose
herein, that a similar mechanism is involved in the transfer of
Ca2+ bound to PL head-groups of the membranes
lining the NJ to its biological target site on the Ca2+-receptor protein.
Model Description
When we compare the artificial PL-mediated Ca2+ transport in the model described above with biological PL-lined
nano-spaces some striking parallels become obvious. For example, rod outer
segments of the bovine eye feature a 15-nm wide cytoplasmic phase between the intercalated
discs, which stretch for several microns \cite{McLaughlin1981}. The lipid bilayers lining this
narrow space contain 45% phosphatidylethanolamine, 36%
phosphatidylcholine and 16% phosphatidylserine, calculated as percent
of the total PL. At physiological pH, phosphatidylcholine and
phosphatidylethanolamine are zwitterions and phosphatidylserine has one
net negative charge \cite{McLaughlin1981}. In this example, it was calculated that of the
Ca2+ released during stimulation, 90% to 99% was
bound to the PL-head groups of membranes Iining the nano-spaces between
the intercalated discs, which is similar to the high ratio of bound/free
Ca2+ in the above model membrane. Although
historically it has been accepted that such binding slows diffusion, due
to a drastic decrease in freely diffusible Ca2+ in the
aqueous phase, it is also possible that the PL-bound
Ca2+ within the water-PL
membrane interface of the nano-space continues its trajectory by the mechanism of 2D Ca2+ exchange diffusion. We therefore propose that the
mechanism of 2D Ca2+/Mg2+,
K+ exchange diffusion at the aqueous/phospholipid
interfaces of NJs facilitates targeting of Ca2+ receptors located on organellar membranes and the inner PM.
Returning to the example of NCX-mediated SR refilling in vascular smooth
muscle, we propose that Ca2+ enters the NJ via the
reverse mode of NCX located in the junctional domain
of the PM. It will then perform a
3D random walk and when it hits the negatively charged PL membrane boundary of the junctional nano-space proceeds along the lipid-water interface by a series of steps of reversible binding to negatively charged oxygen molecules for some variable time before being released back into the aqueous phase to resume its 3D random walk. After a number of such cycles the Ca2+ is envisioned to hit the ER membrane surface in the proximity of its target protein, in this case SERCA, which will then be reached more effectively by 2D surface exchange diffusion than would be expected if it depended solely on 3D diffusion in the aqueous phase.