γ Subunit of Voltage-activated Calcium Channels*

Voltage-activated Ca channels play a major role in many fundamental physiological processes including neurotransmission, muscle contraction, intracellular signaling, hormone secretion, and development. Therefore, an understanding of the structure and regulation of Ca channels is critical for the comprehension of these physiological phenomena. Five types of high voltage-activated Ca channels (named L-, N-, P-, Q-, and R-type) and one type of low voltage-activated Ca channel (known as T-type) have been identified based on the pharmacological and biophysical characteristics of native currents. Voltage-activated Ca channels (called Ca channel or Cav hereafter) consist of four subunits: 1, , 2 , and . The 1 subunit is the main subunit responsible for ion conduction, voltage sensing, and binding of Ca channel-specific drugs and toxins. Ten 1 subunits have been cloned and classified as 11.1 ( 1S), 11.2 ( 1C), 11.3 ( 1D), 11.4 ( 1F), 12.1 ( 1A), 12.2 ( 1B), 12.3 ( 1E), 13.1 ( 1G), 13.2 ( 1H), and 13.3 ( 1I). The and 2 subunits are auxiliary proteins that modulate the properties of the Ca current (1–3). Four isoforms of the subunit ( 1, 2, 3, and 4) and four isoforms of the 2 subunit ( 2 -1, 2 -2, 2 -3, and 2 -4) have been identified. An additional auxiliary subunit, , initially detected in skeletal muscle Ca channels, has been recently associated with neuronal Ca channels (4–6). The study of Ca channel regulation by auxiliary subunits has been focused on and 2 subunits and demonstrated positive regulation of functional activities of Ca channels by these proteins. The and 2 subunits can increase current amplitude and cause various changes in current kinetics and voltage dependence by increasing the number of channels at the plasma membrane and/or causing allosteric modulations of the 1 subunits (7). The skeletal subunit ( 1) was first purified and characterized through biochemical studies with the other subunits of skeletal muscle Ca channels (8, 9) followed by cloning of its cDNA (10, 11). The subunit has not been as extensively studied as the and 2 subunits. The muscle-preferential expression of 1 might be a reason that 1 has not been studied in as great detail as the other auxiliary subunits. However, recent genetic studies revealed the existence of a subunit isoform in the brain whose lack of expression is responsible for the epileptic and ataxic phenotype of the stargazer mouse (4). The discovery of a novel neuronal ( 2) and its relevance to epilepsy and ataxia has drawn much attention to the study of Ca channel subunits. Subsequently, six additional subunit isoforms have been identified through DNA data base searches based on sequence homology to 1 and 2 (12–16). Furthermore, mutations in Ca channel subunits, including mutations, have recently been implicated in a number of human and/or animal neurological disorders, drawing even more attention to the study of Ca channel modulation by its auxiliary subunits. Characterization and Classification of Subunit Isoforms All eight subunit isoforms ( 1–8) are characterized by four predicted transmembrane domains, intracellular N and C termini, and the first extracellular loop that includes a signature motif (GLWXXC), a conserved N-glycosylation site, and a pair of conserved cysteine residues that may form a disulfide linkage in the first extracellular loop (Fig. 1). Phylogenetic analyses have suggested that all subunits are derived from a single ancestral gene through gene duplication (15, 16). Based on the phylogenetic analyses, sequence homologies, and tissue distribution, the subunits have been categorized into two groups: skeletal ( 1 and 6) and neuronal ( 2–5 and 7–8) (15, 16). The 1 and 6 subunits are classified as skeletal muscle subunits based on their predominant expression in skeletal muscle and relatively short C terminus as well as the high sequence homology (62.5% similarity) between them. In addition, a short form of 6 constituted by two instead of four transmembrane domains was reported (15, 16). The 2–5 and 7–8 subunits are classified as neuronal subunits based on their predominant expression in neuronal tissues, relatively long C terminus, and the sequence homology (29.5–90.7% similarity) among them. The neuronal subunits can be subdivided into two groups. The 2–4 and 8 share higher homology than 5 and 7. PDZ binding motifs at the end of C termini of the neuronal subunits also differentiate the two groups of neuronal subunits. The 2–4 and 8 have the same PDZ binding motif (R(R/K)TTPV) at the end of C termini, which differs from the potential PDZ binding motif (S(T/ S)SPC) of the 5 and 7 (16). The genes of 5 and 7 subunits share a distinct intron/exon configuration, a five-exon structure rather than the four-exon structure of the other genes (16, 17). In addition, 8 differs from the other neuronal subunits because of its unusual translation initiation codon ( 3 AXXCTGG 4) and as it contains the longest C terminus (16). The Nand C-terminal regions of 8 also have extensive C/G nucleotide-rich regions (15). There has been confusion about the sequences of 5 and 7. The amino acid sequence of the mouse 5 reported by Klugbauer et al. (14) was quite different from that of human 5 reported by Burgess et al. (13, 15). Chu et al. (16) performed a comprehensive analysis of the difference and suggested a new version of the 5 sequence (16). Based on the new study it seems that the mouse 5 reported by Klugbauer et al. (14) does not actually belong to the family and that the C-terminal sequence of human 5 by Burgess et al. (13, 15) should be modified (16). The C-terminal sequence of 7 was also modified as 5 by two recent studies (16, 17). Therefore, the new sequences of 5 and 7 genes are different from those of the original reports. The new sequences of 5 and 7 have much longer C termini than those of the originals, resulting in C-terminal length similar to that of the other neuronal subunits. In addition, it has been suggested that may belong to the claudin family, a group of cell adhesion proteins in tight junctions (15, 18). Claudins are similar to neuronal subunits in that they have four predicted transmembrane domains, a C-terminal PDZ binding domain, and the first extracellular loop that includes a signature motif (GLWXXC) and a pair of conserved cysteine residues. On the other hand, the phylogenetic relationship of the subunits to claudin families is much lower compared with that among subunits (15). However, the possibility exists that indeed the family may be a subfamily of the claudin superfamily.