Molecular characterization of the regulatory subunits of voltage-dependent calcium channels from rat heart

Voltage-dependent Ca2+ channels are multimeric proteins consisting of pore-forming α₁ and regulatory α ₂δ, β, and γ subunits. The α₁ subunit is essential for current generation, voltage sensing and drug interaction, whereas the regulatory subunits modulate channel properties including current amplitude, kinetics and voltage-dependence. To date, 10 α₁, 4 α₂δ, 4 β, and 8 γ genes have been identified. Although the a1 subunits have been relatively well characterized, our understanding of the regulatory subunits is still primitive. By inspecting the genes encoding the regulatory subunits systematically, I have identified expression of 4 β, 3 α₂δ and 3 γ subunits from rat heart. Using phylogenetic analysis, I have studied the mammalian γ subunit gene family and proposed the following relationship ((((γ₂, γ ₃), (γ₄, γ₈)), (γ₅, γ ₇)), (γ₁, γ₆)) indicating that these γ subunits evolved from a common ancestral γ subunit. Interestingly, the γ ₆ subunit, which is highly expressed in cardiac muscle, most closely resembles γ₁ and shares with it the lack of a PDZ-binding motif that is characteristic of most other γ subunits. Additionally, the expression of γ₁ mRNA and the long isoform of γ ₆ mRNA is most robust in muscle. These results indicate that γ ₁ and γ₆ may share common physiological functions. Among the three α₂δ subunits, only the α ₂δ-3 subunit mRNA level is upregulated upon IGF-1 stimulation in atrial myocytes, where IGF-1 also enhances LVA Ca2+ current density. Thus, the expression of cardiac LVA current stimulated by IGF-1 is probably regulated by the expression of α₂δ-3. Multiple β subunits and their splice variants are expressed in rat heart. My studies demonstrate that the expression of cardiac β subunits varies during postnatal development. I have shown that β₄ mRNA expression is higher in atria of young animals than that of adult animals, which parallels the postnatal change of LVA current density in atrial myocytes. These results suggest that the β₄ subunit may be involved in atrial LVA current expression. In summary, this work has demonstrated the complexity and expression variation of cardiac Ca2+ channel regulatory subunits, and provides a detailed molecular foundation to study their modulatory effects on Ca2+ channels.