Voltage-Gated Calcium Channels in Nonexcitable Tissues

Abstract

The identification of a gain-of-function mutation in CACNA1C as the cause of Timothy syndrome, a rare disorder characterized by cardiac arrhythmias and syndactyly, highlighted roles for the L-type voltage-gated Ca²⁺ channel Ca_V1.2 in nonexcitable cells. Previous studies in cells and animal models had suggested that several voltage-gated Ca²⁺ channels (VGCCs) regulated critical signaling events in various cell types that are not expected to support action potentials, but definitive data were lacking. VGCCs occupy a special position among ion channels, uniquely able to translate membrane excitability into the cytoplasmic Ca²⁺ changes that underlie the cellular responses to electrical activity. Yet how these channels function in cells not firing action potentials and what the consequences of their actions are in nonexcitable cells remain critical questions. The development of new animal and cellular models and the emergence of large data sets and unbiased genome screens have added to our understanding of the unanticipated roles for VGCCs in nonexcitable cells. Here, we review current knowledge of VGCC regulation and function in nonexcitable tissues and cells, with the goal of providing a platform for continued investigation.

Keywords: Timothy syndrome; nonexcitable cells; voltage-gated Ca2+ channel.

Figure 1

Voltage-gated Ca²⁺ channel structure. (a) Ribbon diagram of the pore-forming α₁ subunit of a voltage-gated Ca²⁺ channel demonstrating the pseudo-fourfold symmetry and homology of each domain to a K_v channel monomer, as depicted in panel b. (c) Schematic of a domain-swapped architecture, shared by voltage-gated Ca²⁺ channels, and a nondomain-swapped architecture found in other cation channels for comparison. (d) Structure of rabbit Ca_V1.1 (Protein Data Bank: 5GVY) shown from the membrane. The pore-forming α₁ subunit spans the membrane. The individual domains (DI–DIV) are colored in various shades of green, and the intracellular C-terminal domain (CTD) is brown. The extracellular α₂δ subunit is turquoise, the γ subunit is dark purple, and the cytoplasmic β subunit is light purple. (e) View of the α₁ and γ subunits from outside of the cell shows the domain-swapped architecture. For clarity, the α₂δ subunit is not shown. (f) Zoomed image of the highlighted area in panel e, with the pore glutamate residues colored in purple and the Ca²⁺ ions in yellow. Abbreviation: VSD, voltage sensor domain.

Figure 2

Structure and function of Timothy syndrome (TS) mutation. (a) Schematic of the alternative splicing event for exon 8 or 8a in CACNA1C and the exon in the context of the Ca_V1.2 α_1C subunit. (b) Structure of the homologous rabbit Ca_V1.1 (Protein Data Bank: 5GVY) shown from the membrane. Only the α₁ subunit and the β subunit (blue) are shown. The individual α₁ domains (DI–DIV) are colored in various shades of green except for the polypeptide encoded by exon 8 (or exon 8a) in red, with the G406 residue that is mutated to G406R highlighted in blue; the linker between domain I and domain II (I–II linker) is depicted in gray, in which the α interaction domain (AID) is highlighted in black. (c) Voltage-clamp Ca²⁺ current recording from a cardiomyocyte isolated from a mouse with the TS mutation (red) overlaid with a recording from a cardiomyocyte isolated from a wild-type (WT) littermate. The currents are scaled for comparison, and the enhanced Ca²⁺ influx through the TS mutant channel is highlighted in gray (E.Q. Wei and G.S. Pitt, unpublished data).

Figure 3

Tissue expression pattern of Ca_V1.2. (a) Figure and data are adapted from the ARCHS4 web resource (https://amp.pharm.mssm.edu/archs4/gene/CACNA1C) showing CACNA1C expression in various tissues. In red are nonexcitable cell types with expression of Ca_V1.2. Note that expression levels for many of these cell types are like the expression level in the heart, the canonical Ca_V1.2-expressing tissue. In contrast, skeletal muscle myoblasts, which express an alternative L-type voltage-gated Ca²⁺ channel (Ca_V1.1), show no expression (highlighted by gray box). (b) Figure and data for protein detection (adapted from https://www.proteinatlas.org/ENSG00000151067-CACNA1C/tissue) (55) also demonstrate broad tissue expression, with no Ca_V1.2 protein detected in skeletal muscle (highlighted by gray box).

Figure 4

Timothy syndrome (TS) mouse models do not display syndactyly. Images of forelimbs from TS1 and TS2 mice and their respective littermate wild-type (WT) controls.

Figure 5

Ca_V1.2 is expressed during mouse limb development. (a) A Ca_V1.2^lacZ/+ reporter mouse detects Ca_V1.2 expression during mouse limb development. A frozen tissue section (10 μM) from Ca_V1.2^lacZ/+ forelimb at E14.5 was stained with X-gal and counterstained with nuclear fast red. (b) Normal skeletal development of Col2a1-Cre;Cacna1c^flox/flox conditional knockout (cKO) mouse embryos. Double staining with alizarin red and Alcian blue of the whole skeleton (top), hindlimbs and forelimbs (bottom) of Cacna1c^flox/flox (control), and cKO littermate mouse embryos at P0.