Can the selectivity of a mechanosensitive channel be modulated by an ion selective antechamber domain?


The goal of this study is to investigate the basis of ion specificity of Eschereschia coli mechanosensitive channel MscS:

  1. Site-directed mutagenesis of antechamber domain pore residues to be either positively charged, negatively charged, or neutral.

  2. Patch clamp electrophysiology to determine either cation, anion, or no preference for mutant and native MscS mechanosensitive channel proteins in Xenopus laevi oocytes.

Background & Significance

Ion exchange between cells and membrane bound cellular compartments is an important cellular process and signal transduction mechanism. There are many well known cellular processes that are controlled by ion fluxes including development, muscular movements, and senses such as touch and hearing. The latter of these processes have been attributed to a specific class of ion channels solving one of the most basal problems in the evolutionary history of the cell, that is osmoregulation. The early cell’s solution to the problem of excess water into itself was to sense the tension and curvature of it’s membrane which is a function of the cell’s interior volume. As the volume increases, tension in the membrane increases and specialized mechanosensitive channels embedded in the membrane are pulled apart acting as a release valve for the contents of the cell to be released relieving the pressure (Kung 2005). This early mechanism is more or less homologous to many other categorically distinct senses due to the fact that many things can affect the forces in cellular membranes. Known membrane deformation actions that are sensed by mechanosensitive channels are mechanical touch (here referring to mechanical processes outside of the membrane), asymmetric membrane lipid composition, and of course osmotic homeostasis (Kung 2005). It has also been suggested that changes in membranes due to temperature changes are detected by mechanosensitive channels (Kung 2005), possibly explaining why lipid soluble compounds such as capsaicin and menthol provide sensations of hot and cold.

Discovery and structural crystallization of the bacterial MscS complex (Bass 2002) (Fig. 1 and 2), and subsequently MscL (Chang 1998), provided a mechanistic rationale for how these proteins accomplish mechanical sensing of the membrane forces. Under deformations and phase transitions membrane forces change position along the normal axis spanning the membrane, we see then that the points of forceful contact with embedded objects changes under different membrane states (Wiggins 2005). The transmembrane helices of MscS, arranged as a turbine, adopt the lowest energy configuration in correspondance with the membrane forces, changing the width of the aperture at the center allowing the passage of particles (Tang 2006, Wiggins 2005, Wiggins 2004). An apt analogy is how the aperture of a camera lens is changed by the concerted twisting of several blocking plates with a specific geometric arrangement.

Many channels act solely on a more enzymatic model where a specific substrate binding makes a conformational energy accessible causing a geometric arrangement to transiently open a gate in which the substrate can then be passed through. While some control of substrate selectivity can be managed in the channel by the size of the opening and the charge of the channel lining these structures are far less specific than substrate binding channels (Maksaev 2013, Maksaev 2012). This disadvantage is offset by an increased possible conductance which may be especially useful for releasing large volumes of substance. One then wonders how a specific stimuli such as temperature change and touch can be parsed if the underlying force is similar?

A full description of the anisotropy of a lipid bilayer is beyond the scope of the problem engaged here but indeed these different stimuli affect membranes differently (Markin 2007). Different force profiles along the normal membrane axis can be distiguished by specific transmembrane arrangements (Tang 2006, Wiggins 2005). But for two mechanosensitive channels with distinguishable affectors we have the same effect: the conductance of bulk cellular contents down a pressure and/or osmotic gradient.

The hypothesis that ion selectivity is achieved in the membrane spanning pore is irrelevant as pores such as MSL10 (Msc-Like 10; discussed further later) from Arabidopsis thaliana are lined with bulky aromatic residues such as phenylalanine (Maksaev 2012, Maksaev 2013). This suggests that size exclusion may be a factor but that charge would be unaffected, which is not the case in MSL10 which shows a strong preference for anions (Maksaev 2012, Maksaev 2013). So the question is how are these channels ion selective?

The answer may lie in the conspicuously large