Voltage-Gated Ca2+-Channel α1-Subunit de novo Missense Mutations: Gain or Loss of Function - Implications for Potential Therapies

Abstract

This review summarizes our current knowledge of human disease-relevant genetic variants within the family of voltage gated Ca²⁺ channels. Ca²⁺ channelopathies cover a wide spectrum of diseases including epilepsies, autism spectrum disorders, intellectual disabilities, developmental delay, cerebellar ataxias and degeneration, severe cardiac arrhythmias, sudden cardiac death, eye disease and endocrine disorders such as congential hyperinsulinism and hyperaldosteronism. A special focus will be on the rapidly increasing number of de novo missense mutations identified in the pore-forming α1-subunits with next generation sequencing studies of well-defined patient cohorts. In contrast to likely gene disrupting mutations these can not only cause a channel loss-of-function but can also induce typical functional changes permitting enhanced channel activity and Ca²⁺ signaling. Such gain-of-function mutations could represent therapeutic targets for mutation-specific therapy of Ca²⁺-channelopathies with existing or novel Ca²⁺-channel inhibitors. Moreover, many pathogenic mutations affect positive charges in the voltage sensors with the potential to form gating-pore currents through voltage sensors. If confirmed in functional studies, specific blockers of gating-pore currents could also be of therapeutic interest.

Keywords: calcium-channels; channelopathies; gating pore currents; mutations; neurodevelopmental disorders.

FIGURE 1

Subunit structure of voltage-gated Ca²⁺ channels and their mutation-sensitive regions. (A) The pore-forming α1-subunit determines most of the biophysical properties of voltage-gated Ca²⁺ channels and also carries the binding domains for subtype-selective drugs and toxins. β-and α2δ-subunits associate with α1-subunits of Cav1 and Cav2 channels but not with Cav3 T-type channels. They support channel trafficking to the membrane, fine-tune gating properties but appear to have also channel-independent functions. Here we only discuss pathogenic mutations in α1-subunit genes. (B) Transmembrane folding topology of α1-subunits. Four homologous domains (I-IV) each form a voltage sensing domain (VSD, S1-S4; dotted box). The S6 helices together with the connecting S5-S6 linkers of each domain contribute to the formation of a single central Ca²⁺-selective pore. The S4-S5 linkers in each domain transmit the voltage-dependent conformational changes of the S4-helix movements to the cytoplasmic side of the pore by interactions with adjacent S4-S5 – linkers (Hofer et al., 2020) and S6 helices (Wu et al., 2016; Catterall et al., 2020). Mutations neutralizing the voltage-sensing positive charges in each voltage sensor can open an additional ion conducting pathway (termed ω-pore) through the voltage –sensing domain, which can conduct pathogenic gating pore currents (see Figure 5 and text for details). P1 and P2 are helices contributing to the formation of the external part of the pore. They coordinate the formation of the Ca²⁺-selectivity filter formed by four negatively charged amino acid residues indicated by the red circles. (C) Cryo-electron microscopy structure of the Cav1.1 calcium channel complex purified from rabbit skeletal muscle (PDB 5GJV). Only a top view of the pore-forming α1-subunit is shown to illustrate the position of the four voltage-sensing domains (VSD I – IV, highlighted in different colors). The voltage-sensing positively charged S4 helices of the VSD domains are shown in green (positive charges are not indicated). The central pore-forming region including the intracellular activation gate are formed by the S6 helices indicated in orange. P indicates the ion conducting pathway. The structure is in a presumably inactivated state with S4 voltage-sensors “up” and the activation gate closed (Wu et al., 2016). (D) Most of the missense mutations (in particular GOF mutations) causing Ca²⁺-channelopathies occur in regions important for voltage-dependent channel gating. These functional modules consists of S4 (green) and the cytoplasmic S4-S5 linkers (light green), which are tightly coupled through multiple interactions (not illustrated) to the activation gate formed by the four S6 helices (orange). For clarity the VSD (gray), S4 and S4-S5 linker are only shown for domain I together with all 4 S6 helices (orange). Positions where pathogenic mutations occur in all Cavs (Figures 3, 4) are indicated in blue. In IS4-S5 this represents the position of the FHM1 mutation S218L (CACNA1A) and the CSNB2 mutation S229P (CACNA1F), which both cause GOF (type 2 gating; see text for details). The position of the Timothy Syndrome mutation G402R/S (CACNA1C) is indicated in light blue. Note that pathogenic GOF mutations at the same position also occur in Cav1.3, Cav1.4, and Cav2.3 (Figure 3). The schemes in (C,D) were generated using UCSF Chimera 1.13.1 (Pettersen et al., 2004). The position of mutations in the Cav1.1 α1-subunit is shown based on the sequence alignments in Figures 3, 4 and does not account for potential differences in the folding structure of the different α1-subunits.

FIGURE 2

Typical gain-of-function macroscopic gating changes described for inherited or de novo missense mutations in voltage-gated Ca²⁺ channel α1-subunits. To facilitate discussion the most frequently observed gating changes are classified into types 1-4. For details see text. Mutant current properties are indicated in blue (A–C) or green curves (D), wildtype current in black. (A) Type 1 is characterized by the appearance of a large fraction of a non-inactivating current component (as e.g., during long depolarizations from a negative holding potential to the potential of maximal inward current; right panel). Changes in the voltage-dependence of activation gating (left) may or may not be present (blue curves, left). Note that even if maximal inward current is reduced by a mutation, current amplitude may exceed wildtype current later during depolarization due to the slowly inactivating current component (right, dotted curve). (B) The main feature of type 2 changes is a strong shift of the voltage-dependence of activation to more negative voltages independent of smaller effects on current inactivation during depolarizations. The voltage-dependence of inactivation may or may not be shifted to more negative voltages (left). The voltage range in which steady-state inward current (“window current”) may be observed (i.e., the overlap of voltage-dependence of activation and inactivation curves) is indicated for mutant current (shaded) (C). Some mutations do not affect the voltage-dependence of gating (left) but cause a slowing of inactivation (weaker than in type 1). (D) As described in Figure 5, gating pore currents are enabled by mutations of an S4 gating charge (green lines). The position of the mutation relative to the hydrophobic constriction site (HCS, Figure 5) determines if the pore is open during the hyperpolarized “down”-state of the S4 helix at negative voltages (left) or during the depolarized “up”-state at positive voltages (right). In the “down” state, at potentials near the K⁺-equilibrium potential, inward gating-pore currents would be primarily carried by Na⁺. In the “up” state, at potentials positive to the activation threshold of the channel, potassium outward gating-pore current predominates. However, upon fast repolarization of the action potential to negative potentials Na⁺ inward gating-pore current may predominate until the voltage sensor moves back to the “down” position” (especially relevant in “slowly inactivated” Na⁺-channels,” for details see ref. Sokolov et al., 2008). G/G_max, normalized conductance (steady-state activation); I/I_max, normalized inward current (steady-state inactivation). V₀._5,act, V₀._5,inact, half maximal voltages of activation and inactivation; hp, holding potential; ΔV, depolarization from a negative holding potential to the voltage of maximal inward current (V_max).

FIGURE 3

Position of pathogenic mutations within S6 helices of the ten different Ca²⁺ channel α1-subunits. Sequence alignment and labeling is as described in legend to Figure 4.

FIGURE 4

Position of pathogenic mutations within S4, the S4-S5 linkers and S5 helices of the ten different Ca²⁺ channel α1-subunits. The sequence alignment for the human α1-subunits was generated using Clustal omega and Jalview software (www.jalview.org; Waterhouse et al., 2009). Conserved residues are highlighted in blue. The accession numbers are identical to the alignment in Wu et al. (2016): CACNA1S (S, Cav1.1): Q13698; CACNA1C [C, Cav1.2; contains exon 8 (Splawski et al., 2005)]: Q13936; CACNA1D (D, Cav1.3): Q01668; CACNA1F (F, hCav1.4): O60840; CACNA1A (A, Cav2.1): O00555; CACNA1B (B, Cav2.2): Q00975; CACNA1E (E, hCav2.3): Q15878; CACNA1G (G, hCav3.1): O43497; CACNA1H (H, hCav3.2): O95180; CACNA1I (I, hCav3.3: Q9P0 × 4. All amino acid positions in the text are numbered according to the above Uniprot accession numbers and may differ in the original publications or other databases. Arrows indicate the position of positively charged residues (gating charges) in S4 helices, asterisks indicate the presence of pathogenic gating pore mutations in human Nav1.4 (SCNA4) or Nav1.5 (SCNA5) Na⁺-channels. Numbers on the right indicate the amino acid position of the last residue in each line, blue circles are 10 residues apart. Red lines on the bottom denote secondary structural elements of the rabbit Cav1.1 channel (Wu et al., 2016) with labels placed at the start of a feature. Residues affected by a mutation are highlighted in red. Note that not all highlighted mutations are discussed in the text. For information on mutations in all Ca²⁺ channels refer to the Uniprot-database and to reviews cited in the text.

FIGURE 5

Membrane potential dependent conformations of the voltage-sensor allow pathogenic gating pore current. Simplified scheme illustrating the membrane potential dependent conformations of a single voltage-sensor and the effects of a mutation in gating charge R2. Membrane-spanning S4 helices are shown in green, positively charged residues (mostly arginines spaced at three residue intervals) as blue spheres. Clusters of extracellular and intracellular negative counter-charges (NC) forming ion pair interactions with the gating charges are shown in red. The hydrophobic constriction site is indicated in yellow (HCS). (A) In the resting state, the positively charged S4 helix is pulled inside by the negative resting potential (A, S4 “down”). (B) Upon depolarization, the S4 segment moves outward according to a sliding-helix model (Catterall et al., 2020; B, S4 “up”), and transports positive gating charges through the HCS. Inside and outside the HCS the arginine side chains are stabilized by forming ion pairs with negative charges within the VSD (gray lines). Upon S4 movement the ion pair partners are exchanged and the large arginine side chains seal the VSD and prevent formation of a water filled space through which ions can flow (Catterall et al., 2020). Replacement of a positive gating charge by a smaller or hydrophilic residue can disrupt this seal as exemplified here for a neutralizing mutation in R2 (C). This position of the mutation permits an inward gating-pore current during the hyperpolarized “down”-state of the S4 helix at negative voltages but not during the depolarized “up”-state at positive voltages (D). In contrast, mutations of one of the inner gating charges would permit gating pore current to flow in the activated but not the resting position of S4 (for X-ray structures of mutant voltage-gated Na⁺-channels see Jiang et al., 2018; Jiang D. et al., 2019).