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Cavβ surface charged residues contribute to the regulation of neuronal calcium channels
Molecular Brain volume 15, Article number: 3 (2022)
Voltage-gated calcium channels are essential regulators of brain function where they support depolarization-induced calcium entry into neurons. They consist of a pore-forming subunit (Cavα1) that requires co-assembly with ancillary subunits to ensure proper functioning of the channel. Among these ancillary subunits, the Cavβ plays an essential role in regulating surface expression and gating of the channels. This regulation requires the direct binding of Cavβ onto Cavα1 and is mediated by the alpha interacting domain (AID) within the Cavα1 subunit and the α binding pocket (ABP) within the Cavβ subunit. However, additional interactions between Cavα1 and Cavβ have been proposed. In this study, we analyzed the importance of Cavβ3 surface charged residues in the regulation of Cav2.1 channels. Using alanine-scanning mutagenesis combined with electrophysiological recordings we identified several amino acids within the Cavβ3 subunit that contribute to the gating of the channel. These findings add to the notion that additional contacts besides the main AID/ABP interaction may occur to fine-tune the expression and properties of the channel.
Neuronal high-voltage-activated (HVA) calcium channels are multisubunits complexes that support depolarization-induced calcium entry and downstream cellular functions . They are composed of a pore-forming subunit (Cavα1) that consists of four homologous membrane domains, each composed of six transmembrane helices, connected via cytoplasmic linkers (I–II, II–III, and III–IV loops), and cytoplasmic amino- and carboxy termini. They require the co-assembly with ancillary subunits to ensure the proper functioning of the channel. Among these ancillary subunits, the cytoplasmic Cavβ regulates several aspects of HVA channels including their gating properties and expression at the cell surface (for review see ). Cavβ subunits are encoded by four different genes (Cavβ1–4) and belong to the family of membrane-associated guanylate kinase (MAGUK). They consist of a conserved core region formed by Src homology 3 (SH3) and guanylate kinase (GK) domains connected by a HOOK region, flanked by non-conserved amino- and carboxy-termini (Fig. 1a). The molecular assembly of the Cavα1/Cavβ complex relies on a conserved 18 residue sequence within the I–II loop of Cavα1 called α1 interaction domain (AID)  that binds into a hydrophobic groove within the GK domain of Cavβ termed AID-binding pocket (ABP) [4,5,6] (Fig. 1a). This high-affinity interaction is critical for Cavβ-mediated enhancement of Cavα1 surface expression and gating. Mutation of key residues within the ABP that weakens or abolishes AID-ABP interaction severely alters the functional influence of Cavβ . Besides the AID/ABP interaction, additional low-affinity contacts between Cavα1 and Cavβ that do not involve the ABP have been proposed to confer essential Cavβ modulatory properties [8,9,10]. In this study, we aimed to assess the functional importance of Cavβ surface charged residues in the regulation of Cav2.1 channels. To do so, we generated a number of Cavβ3 mutants where surface charged residues, most belonging to the GK domain, were replaced with an alanine (Fig. 1b), and recombinant Cavβ3 were expressed in Xenopus oocytes with Cav2.1 for electrophysiological analyses in the presence of 40 mM barium as charge carrier. Cavβ3 was chosen over Cavβ4 because it induces a more pronounced phenotype on Cav2.1 with faster inactivation kinetics, and also because according to our experience, the association of Cavβ3 with Cav2.1 in expression experiments is more complete than of Cavβ4 which would have made the interpretation of Cavβ4 variants slightly more difficult overall. As expected, the maximal macroscopic conductance (Gmax) in cells expressing Cav2.1 was increased by 3.3-fold (p = 0.0001) in the presence of wild-type (WT) Cavβ3 compared to cells expressing Cav2.1 alone (Fig. 1c, d, Additional file 1: Fig. S1, Additional file 2: Table S1). Similarly, all Cavβ3 variants, except the E347A mutant, produced a significant increase of Cav2.1 conductance indicative of the proper expression of Cavβ3 mutants (Fig. 1d, Additional file 1: Fig. S1, Additional file 2: Table S1). However, Cavβ3-dependent potentiation of Cav2.1 currents was significantly reduced when residues D343, D344, E347, E354 (located in the GK domain), and R358 (located in the N-terminus) were mutated (ranging from 1.4-fold reduction for Cavβ3 R358A to 2.0-fold reduction for Cavβ3 E347A compared to WT Cavβ3) (Fig. 1d, Additional file 1: Fig. S1, Additional file 2: Table S1). While the exact underlying mechanisms have not been further investigated in this study, this alteration is likely to have resulted from either a decreased surface expression of the channel, or from a decreased Cavβ-dependent potentiation of the single channel gating (channel open probability and latency to first channel opening). Consistent with the latest, we observed that while co-expression of WT Cavβ3 produced a 10.7 mV hyperpolarizing shift (p = 0.0001) of the mean-half activation potential of Cav2.1, this effect was significantly reduced when the channel was co-expressed with Cavβ3 D343A, D344A, and E347A (Fig. 1e and f, Additional file 1: Fig. S2, Additional file 2: Table S1). In contrast, mutation of residues E354 and R358 did not alter Cavβ3-mediated hyperpolarization of the voltage-dependence of activation of Cav2.1 suggesting that the effect of Cavβ3 mutants on Cav2.1 conductance may have resulted from distinct gating alteration. In that respect, we note that while Cavβ3 H206A did not alter the maximal macroscopic conductance of Cav2.1-expressing cells (Fig. 1d, Additional file 1: Fig. S1, Additional file 2: Table S1), it reduced the hyperpolarizing shift of the voltage-dependence of activation produced by WT Cavβ3 (Fig. 1f, Additional file 1: Fig. S2, Additional file 2: Table S1). Finally, we assessed the effect of Cavβ3 surface charged residues on the voltage-dependence of inactivation of the channel. Co-expression of WT Cavβ3 produced a 16.7 mV hyperpolarizing shift (p = 0.0001) of the mean-half inactivation potential of Cav2.1 (Fig. 1g and h, Additional file 1: Fig. S3, Additional file 2: Table S1). Although this effect was significantly altered upon mutation of Cavβ3 surface charged residues, the magnitude of this alteration remained modest and all Cavβ3 mutants retained their ability to significantly enhance the voltage-dependence of inactivation of the channel (Fig. 1h, Additional file 1: Fig. S3, Additional file 2: Table S1). Indeed, the weakest enhancement was observed with Cavβ3 E339A and H348A which still produced a 9.1 mV 9.4 mV hyperpolarized shift, respectively, suggesting that Cavβ3 surface charged residues have minimal influence on the voltage-dependence of inactivation of Cav2.1 channels. These data however allow us to conclude that for the Cavβ3 mutations for which there is a reduced Gmax (Fig. 1d), the channels under study remain in the Cav2.1 / Cavβ3 complex form.
While AID-ABP interaction is a prerequisite for Cavβ-dependent modulation of HVA channels, additional interactions are expected to contribute to Cavβ modulatory properties [8, 9]. Here, we reported that Cavβ surface charged residues located outside of the ABP play a significant role in Cavβ3-dependent modulation of Cav2.1 channels. In particular, residues D343, D344, and E347 appear to form a hot-spot at the surface of the GK domain to influence activation of the channel, with limited effect on its inactivation. It is of interest that this cluster of residues is in close proximity to the AID sequence itself (Fig. 1B). These data are consistent with previous studies showing that the effect of Cavβ on the voltage-dependence of Cav2.1 channel activation is largely reconstituted by the core region of Cavβ . The question then arises as to how surface charged residues regulate channel gating. One possibility is via enabling additional low affinity interactions between Cavβ and other parts of Cavα1. For instance, the amino- and carboxy-termini as well as the III–IV loop of Cavα1 have been shown to interact directly with Cavβ [8, 9, 12, 13]. In addition, it was reported that the orientation of Cavβ relative to Cavβ1 is essential for Cavβ-mediated regulation of the channel activation [14, 15]. Therefore, it is a possibility that surface charged residues, by supporting low affinity interactions, may contribute to the proper positioning of Cavβ. Inherent to our study are a number of limitations that will need to be addressed in future studies. First, in addition to Cavβ, Cav2.1 associated with Cavα2δ that on the one hand mediates its own effects on the channel, and on the other hand influences the modulatory input of Cavα2δ. For that reason, Cavα2δ was purposely left out of our experiments to simplify the mechanistic analysis of Cavβ3 variants. However, given the important role of Cavα2δ in the modulation of Cav2.1, the present findings will need to be confirmed in the presence of Cavα2δ where it can be expected that allosteric modulations will add another level of complexity to the regulation described in the present study. Second, in this study we used Cavβ3 because it produces a more pronounced phenotype on Cav2.1 evidenced by faster inactivation kinetics compared for instance to Cavβ4, and also because according to our experience the associated of Cavβ3 with Cav2.1 in expression experiments is more complete than of Cavβ4 which would have made the interpretation of the data more complicated. However, and although Cavβ3 represents a legitimate subunit that does associate with Cav2.1 in native condition, Cavβ4 remains the major isoform found co-associated with Cav2.1 in the brain and therefore it will be important to confirm our findings in the presence of Cavβ4. And third, another potential limitation inherent to our experimental settings is the use of Xenopus oocytes where trace levels of endogenous Cavβ have been reported. While such an endogenous Cavβ is unlikely to have played a major role in the regulation of recombinant Cav2.1 since otherwise we would not have observed any effect of the co-expression of Cavβ3, it would nevertheless be important to reproduce these findings in a mammalian cell line.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
Alpha interaction domain
Membrane-associated guanylate kinase
Src homology 3
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Additional file 1: Fig. S1.
Effect of Cavβ3 mutants on Cav2.1 current density. a Mean current–voltage (I/V) relationship for Cav2.1 channels expressed alone (filled circles) and in the presence of wild-type (WT) Cavβ3 ancillary subunit (open circles). b–k Legend same as in (a) but for Cav2.1 channels expressed with Cavβ3 mutants (open red circles). The smooth lines correspond to the fit of the I/V curve with the modified Boltzmann function (1). The dotted line shows the position of the I/V curve for Cav2.1 expressed with WT Cavβ3 for comparison. Fig. S2. Effect of Cavβ3 mutants on the voltage-dependence of activation of Cav2.1 channels. a Mean normalized voltage-dependence of activation for Cav2.1 channels expressed alone (filled circles) and in the presence of wild-type (WT) Cavβ3 ancillary subunit (open circles). b–k Legend same as in (a) but for Cav2.1 channels expressed with Cavβ3 mutants (open red circles). The smooth lines correspond to the fit of the activation curve with the modified Boltzmann function (2). The dotted line shows the voltage-dependence of activation for Cav2.1 expressed with WT Cavβ3 for comparison. Fig. S3. Effect of Cavβ3 mutants on the voltage-dependence of inactivation of Cav2.1 channels. a Mean normalized voltage-dependence of inactivation for Cav2.1 channels expressed alone (filled circles) and in the presence of wild-type (WT) Cavβ3 ancillary subunit (open circles). b–k Legend same as in (a) but for Cav2.1 channels expressed with Cavβ3 mutants (open red circles). The smooth lines correspond to the fit of the inactivation curve with the two-state Boltzmann function (3). The dotted line shows the voltage-dependence of inactivation for Cav2.1 expressed with WT Cavβ3 for comparison.
Additional file 2: Table S1.
Electrophysiological properties of Cav2.1 channel expressed in Xenopus oocytes in the presence of Cavβ3 mutants. Statistical analysis (one-way ANOVA followed by Dunnett’s post hoc multiple comparisons test) was performed for all Cavβ3 variants against Cavβ3 wild-type (WT): *p < 0.05. β decreased conductance; β depolarized shift of voltage-dependence; β hyperpolarized shift of voltage-dependence.
Additional file 3: Table S2.
Statistical summary. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc multiple comparisons test was used to determine statistical significance between Cavβ3 variants against channel expressed alone (top table) and against channel expressed with wild-type (WT) Cavβ3 (bottom table). Adjusted p values from Dunnett's multiple comparisons test are presented.
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Tran-Van-Minh, A., De Waard, M. & Weiss, N. Cavβ surface charged residues contribute to the regulation of neuronal calcium channels. Mol Brain 15, 3 (2022). https://doi.org/10.1186/s13041-021-00887-3
- Ion channels
- Calcium channels
- Voltage-gated calcium channels
- Cav2.1 channels
- Cavβ subunit
- Alanine-scanning mutagenesis