The calcium channel gamma subunits comprise an eight-member protein family that share a common topology consisting of four transmembrane domains and intracellular N- and C-termini. Although the first gamma subunit was identified as an auxiliary subunit of a voltage-dependent calcium channel, a review of phylogenetic, bioinformatic, and functional studies indicates that they are a functionally diverse protein family. A cluster containing gamma1 and gamma6 conforms to the original description of the protein family as they seem to act primarily as subunits of calcium channels expressed in muscle. Members of a second cluster (gamma2, gamma3, gamma4, gamma8) function as regulators of AMPA receptor localization and function in the brain and are collectively known as TARPs. The function of members of the third cluster (gamma5, gamma7) remains unclear. Our analysis shows that the members of each cluster contain conserved regulatory motifs that help to differentiate the groups. However, the physiological significance of these motifs in many cases remains to be demonstrated.

The eight members of the calcium channel γ subunit family are integral membrane proteins that regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of γ 2 , γ 3 , γ 4 and γ 8 (the TARPs) modulate AMPA receptor localization and function, the γ 1 and γ 6 subunits conform to the original description of these proteins as regulators of voltage gated calcium channels. We have previously shown that the γ 6 subunit is highly expressed in atrial myocytes and that it is capable of acting as a negative modulator of low voltage activated calcium current. In this study we extend our understanding of γ 6 subunit modulation of low voltage activated calcium current. Using engineered chimeric constructs, we demonstrate that the first transmembrane domain (TM1) of γ 6 is necessary for its inhibitory effect on Cav3.1 current. Mutational analysis is then used to identify a unique GxxxA motif within TM1 that is required for the function of the subunit strongly suggesting the involvement of helix-helix interactions in its effects. Results from co-immunoprecipitation experiments confirm a physical association of γ 6 with the Cav3.1 channel in both HEK cells and atrial myocytes. Single channel analysis reveals that binding of γ 6 reduces channel availability for activation. Taken together, the results of this study provide both a molecular and a mechanistic framework for understanding the unique ability of the γ 6 calcium channel subunit to modulate low voltage activated (Cav3.1) calcium current density. Calcium channel γ subunits comprise a family of eight proteins that share a common topology consisting of four transmembrane domains with intracellular N-and C-terminal ends. The first member of this protein family to be described, γ 1 , was isolated as a subunit of the high-voltage activated (HVA), Cav1.1 calcium channel found in skeletal muscle (Jay et al. 1990). Unlike other calcium channel accessory subunits (β, α 2 δ) which enhance calcium current, γ 1 was shown to accelerate L-type calcium current activation and inactivation in heterologous systems when coexpressed with the Cav1.2 (also an HVA) α 1 subunit (Singer et al. 1991;Eberst et al. 1997). Skeletal muscle isolated from knockout mice lacking the γ 1 gene have increased HVA calcium current density confirming a physiological role of γ 1 as a negative regulator of HVA, L-type calcium current density in developing skeletal myocytes (Freise et al. 2000;Held et al. 2002).Phylogenetic and sequence homology analysis indicates that the recently described γ 6 protein is the closest homologue of γ 1 within the γ subunit family (Burgess et al. 2001;Chu et al. 2001). Both γ 1 and γ 6 have short C-terminal regions that lack the consensus PDZ1-binding motif that is a notable characteristic of the four γ subunits (γ 2 , γ 3 , γ 4 and γ 8 ) known collectively as the TARP proteins that regulate AMPA receptor trafficking and function (Tomita et al. 2003;Osten & Stern-Bach, 2006). The γ 1 and γ 6 subunits also share similarities in their ti...

The calcium channel ␥ 6 subunit modulates low voltage-activated (LVA) calcium current in both human embryonic kidney (HEK) cells and cardiomyocytes, although the mechanism of modulation is unknown. We recently showed that ␥ 6 contains a critical GxxxA motif in the first transmembrane domain (TM1) that is essential for its inhibition of the Cav3.1 (LVA) calcium current. In this study, we tested the hypothesis that an eight-amino acid peptide that contains the GxxxA motif from ␥ 6 TM1 can act as a novel pharmacological inhibitor of the Cav3.1 calcium current by performing whole-cell electrophysiology. Our results demonstrate that the peptide inhibits Cav3.1 current by dynamically binding and dissociating from the Cav3.1 channel in a concentration-dependent but largely voltage-independent manner. By selectively substituting residues within the peptide, we show that both the GxxxA framework and surrounding aliphatic side-chains contribute to the presumably interhelical interactions between ␥ 6 TM1 and the Cav3.1 channel. The fast kinetics of the interaction supports the view that ␥ 6 acts as an endogenous LVA channel antagonist within the plasma membrane, suggesting a mechanism other than regulation of surface expression or membrane trafficking of the poreforming subunit of the channel. We also demonstrate that the peptide has different affinities for Cav3.1 and Cav1.2 calcium currents, which is consistent with the selective effect of ␥ 6 on LVA and high voltage-activated calcium currents in vivo.

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Na þ -activated potassium channels (K Na channels), which are encoded by the Slack and Slick genes contribute to neuronal adaptation during sustained stimulation, and, in auditory brainstem neurons, may regulate the accuracy of timing of action potentials. These channels have been found to be modulated very potently by activation of protein kinase C (PKC) and by receptors linked to activation of this kinase. Activators of PKC increase the amplitude of Slack-B currents and slow their rate of activation, and in contrast, activation of PKC decreases the amplitude of Slick currents. Heteromeric Slack/Slick channels are regulated by PKC to a greater extent than either Slack-B or Slick heteromers (90% decrease in amplitude). Previous experiments using Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) have identified three serine residues in Slack-B that are phosphorylated under basal conditions, but are not within consensus sites for PKC. In order to study the mechanisms of regulation of Slack and Slick channels by phosphorylation, we have begun to identify the specific residues that undergo phosphorylation by protein kinase C. Consensus sequence analysis predicts that there are 13 potential sites of possible PKC phosphorylation in the Slack-B sequence. We have constructed individual site mutants for each of these sites in which the serine/threonines have been mutated to alanines to prevent phosphorylation at these residues. These mutants were expressed in Xenopus oocytes and their response to a PKC-activating phorbol ester (TPA) was characterized by two-electrode whole cell clamp electrophysiology. Of the 13 consensus site mutants, only one generated currents that matched wild-type Slack-B currents in their amplitude and kinetic behavior, but completely failed to respond to application of TPA, suggesting that the phosphorylation state of a single residue regulates Slack-B current amplitude and rate of activation.

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