Molecular characterization of the neuronal P/Q-type and skeletal muscle L-type voltage gated calcium channels

Voltage gated calcium channels allow depolarization induced calcium entry into cells and are mediators of a number of important physiological processes including muscle contraction, neurotransmitter release, secretion and regulation of gene expression. All known high voltage gated calcium channels have a similar composition of the four domain pore forming α₁ and auxiliary α ₂δ, β and γ subunits. This study examined subunit composition and intersubunit interactions of the voltage gated calcium channels. Genetic and molecular approaches were used to characterize the two domain form of the pore forming subunit of P/Q type channels. The two domain protein is a product of CACNA1A gene, which also encodes the full length protein, as revealed by absence of the truncated protein in CACNA1A null mice. Cell expression studies with a recombinant two domain form of α₁ demonstrated that the protein can associate with β. Investigation of the functional properties revealed that the protein trafficked to the plasma membrane. However, it did not conduct calcium currents, but generated gating currents and inhibited activity of P/Q type channels. These studies suggest a physiological role for the truncated two domain subunit in both calcium independent and dependent exocytotic events. Subunit interactions within skeletal muscle calcium channels, in particular interactions of γ subunits, have been characterized using the γ₁ null mice, which have no discernible phenotype. Adenoviral expression was used to introduce γ₁ and γ₂ into skeletal muscle of γ₁ null mice. Biochemical and immunofluorescence analysis revealed that γ₁ incorporated into the channel, but γ₂ did not. Investigations with chimeric subunits of γ ₁ and γ₂ revealed that the N terminal half of γ ₁ allows subunit interaction and restoration of calcium conductance in γ₁ null myotubes. α₂δ and γ ₁ subunits were not associated in the muscular dysgenesis mice that lack α₁1.1. α₁1.1 and γ₁ associated directly in a cell expression system. Interestingly, α ₁1.1 and γ₂ also associated in a cell expression system, suggesting that loss of γ₁ in muscle is being compensated for by another γ subunit. Since subunit interactions are conserved across voltage gated calcium channels, these studies have broad implications for subunit heterogeneity and intersubunit interactions.