Voltage-gated Na and Ca2+ channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca2+ and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.

The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor's perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex. Applying this principle to the long-standing controversy of calmodulin binding to ion channels, we find a surprising Ca2+-induced switch in calmodulin stoichiometry with Ca2+ channels—one calmodulin binds at basal cytosolic Ca2+ levels while two calmodulins interact following Ca2+ elevation. This feature is curiously absent for the related Na channels, also potently regulated by calmodulin. Overall, our assay adds to a burgeoning toolkit to pursue quantitative biochemistry of dynamic signalling complexes in living cells.

The High Voltage-Activated (HVA) subgroup of voltage-gated calcium channels contain an a1 subunit, which forms the selective pore and determines the main functional properties of the channel. The a1 subunit is associated with auxiliary subunits including b and a2d, which modulate trafficking and functional properties of the channels. There are four known genes encoding a2d subunits, which are believed to have similar structure. They consist of two peptides: the highly glycosylated a2 which is entirely extracellular is disulfide-bonded to a d subunit that links the protein into the plasma membrane. The a2 and d peptides are encoded by a single gene as an uninterrupted a2d pre-protein, which is further processed post-translationally. We have recently shown that a 2 d subunits are glycosyl phophatidyl inositol (GPI)-achored proteins (Davies et al., 2010, PNAS 107:1654-1659, and this is essential for their function, and explains their localization in lipid raft fractions (Davies et al, 2006, J. Neurosci. 26: 8748-8757). We further studied the mechnism and consequences of the proteolytic cleavage of the a 2 d subunits (a 2 d-1, -2 and -3)and identified the cleavage sites in a 2 d-2 and a 2 d-3. Western blots from brain tissue revealed only the mature form of the protein strongly associated with lipid rafts. However, transfecting mammalian cells with cDNA for a2d-1,-2,and -3 resulted in incomplete cleavage (~40-60% processing) most probably due to limitations of the cleavage-mediating protease(s) in heterologous expression systems. The mature form is localized in lipid rafts, suggesting that maturation of the protein might occur in localized membrane domains. Moreover, electrophysiological recordings demonstrated that proteolytic cleavage is key to the function of these subunits. We are currently examining the nature of the protease(s) involved in this proteolytic processing.