Folacci M et al. (2023), Functional Characterization of Four Known Cav2....

XB-ART-59636

Membranes (Basel) 2023 Jan 11;131:. doi: 10.3390/membranes13010096.

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Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders.

Folacci M , Estaran S , Ménard C , Bertaud A , Rousset M , Roussel J , Thibaud JB , Vignes M , Chavanieu A , Charnet P , Cens T .

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Cav2.1 channels are expressed throughout the brain and are the predominant Ca2+ channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four variations (p.A405T in I-II loop and p.R1359W, p.R1667W and p.S1799L in IIIS4, IVS4, and IVS6 helices, respectively) identified in patients exhibiting a wide spectrum of disorders including ataxia symptoms. Functional analysis using two major Cav2.1 splice variants (Cav2.1+e47 and Cav2.1-e47) in Xenopus laevis oocytes, revealed a lack of effect upon A405T substitution and a significant loss-of-function caused by R1359W, whereas R1667W and S1799L caused both channel gain-of-function and loss-of-function, in a splice variant-dependent manner. Structural analysis revealed the loss of interactions with S1, S2, and S3 helices upon R1359W and R1667W substitutions, but a lack of obvious structural changes with S1799L. Computational modeling suggests that biophysical changes induced by Cav2.1 pathogenic mutations might affect action potential frequency in Purkinje cells.

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Species referenced: Xenopus laevis
Genes referenced: cav2
GO keywords: calcium channel activity [+]

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	Figure 1. (Top) Topology of the voltage-gated Cav2.1 subunit with the position of the four pathogenic variants studied: A405T, R1359W, R1667W, and S1799L. The Cavα is formed of four repeats (I to IV) each containing 6 transmembrane helices (S1–S6). The S1–S4 helices constitute the voltage-sensing domain (VSD) able to move in response to membrane potential changes. The four re-entrant P loops between segments S5 and S6 carry the selectivity filter and delineate the extracellular end of the channel pore. The S5 and S6 helices of the four repeats delineate the cytoplasmic end of the channel pore. (Bottom) Amino-acid sequences of the I-II loop, and of the IIIS4, IVS4, and IVS6 helices. Cav2.1 variants referenced in the NCBI ClinVar database are indicated with their accession number. References in italics correspond to variants ‘of uncertain significance’ when subjected to in silico analysis. The ‘R’ above the sequences of the IIIS4 and IVS4 helices indicates the position of a basic residue. The Cav2.1 sequence is numbered according to the NCBI Genbank® sequence AAB64179.
	Figure 2. (A) Representative current traces were recorded from X. laevis oocytes that expressed the indicated Cav2.1+e47 variants with Cavβ4a and Cavα2δ1. Currents were elicited from a holding potential of −100 mV by a two-pulse protocol illustrated on top, and consisting of a 2.5 s-long depolarization from −80 mV to +40 mV, followed by a 400 ms-long depolarization to 0 mV (WT and R1359W) or +10 mV (A405T, R1667W, and S1799L). Scale bars, 200nA. (B) Representative images of HEK cells immunostained with anti-Cav2.1 (red) and anti-ZO-1 (green). Anti-ZO-1 was used to visualize the cell membrane. Scale bars, 10 μm. (C) Quantification of fluorescence intensity realized on individualized cells (n = 10 for each variant). Asterisks and number signs denote significant differences vs. WT (*** p < 0.001) and vs. Cav2.1+e47 splice variants (### p < 0.001), respectively (non-paired Student’s t-test). n.s. = non-significant.
	Figure 3. Voltage dependence of Cav2.1 activation. (Top) Average current-voltage relationships for X. laevis oocytes expressing Cav2.1+47 (left) or Cav2.1−e47 variants (right). (Bottom) Box plots of half-maximal activation potential (Va, left) and slope factor of the Boltzmann curve of channel activation (ka, right) were obtained for all Cav2.1 variants studied. The mean values are given in Table 1. Asterisks and number signs denote significant differences vs. WT ( p < 0.01, * p < 0.001) and vs. Cav2.1+e47 splice variant (# p < 0.05), respectively (non-paired Student’s t-test). n.s. = non-significant.
	Figure 4. Voltage dependence of Cav2.1 inactivation. (Top) Voltage-dependent isochronal inactivation curves for X. laevis oocytes expressing Cav2.1+47 (left) or Cav2.1−e47 (right) variants. (Bottom) Box plots of half-maximal inactivation potential (Vi, left) and slope factor of the Boltzmann curve of channel inactivation (ki, right) were obtained for all Cav2.1 variants studied. The mean values are given in Table 1. Asterisks and number signs denote significant differences vs. WT ( p < 0.01, * p < 0.001) and vs. Cav2.1+e47 (# p < 0.05, ### p < 0.001), respectively (non-paired Student’s t-test). n.s. = non-significant.
	Figure 5. Cav2.1 inactivation kinetics. Box plot of the ratio of remaining current at the end of a 400 ms-long depolarization (R400) to 0 mV (WT, A405T, R1359W, and R1667W Cav2.1 variants) or + 10 mV (S1799L Cav2.1 variants) with respect to the peak current amplitude. Asterisks and number signs denote significant differences vs. WT ( p < 0.01, * p < 0.001), and vs. Cav2.1+e47 (# p < 0.05, ### p < 0.001), respectively (non-paired Student’s t-test). n.s. = non-significant.
	Figure 6. Recovery from inactivation of Cav2.1 variants. (A) Representative current traces were obtained in X. laevis oocytes expressing Cav2.1+47 with Cavβ4a and Cavα2δ1. The experimental protocol, illustrated above traces, consists of a 2.5 s-long depolarization to +10 mV, followed by inter-pulse intervals between 100 ms and 8 s, and a second 100 ms-long depolarization to +10 mV. Scale bars: 200 nA and 2 s. (B) Percentage of current recovery plotted against the inter-pulse duration for Cav2.1+e47 (top) and Cav2.1−e47 variants (bottom). (C) Bar graph showing percent recovery at 8 s for all Cav2.1 variants studied. Asterisks and number signs denote significant differences vs. WT ( p < 0.01, * p < 0.01) and vs. Cav2.1+e47 (## p < 0.01), respectively (non-paired Student’s t-test). n.s. = non-significant.
	Figure 7. Homology model of human Cav2.1 viewed from the side (A) or from the top (B). The IIIS4, IVS4, and IVS6 transmembrane helices carrying the studied mutation are represented in magenta, dark green, and brown, respectively. The four residues R1359 (IIIS4), R1667 (IVS4), S1799L (IVS6), and A405 (I-II loop, indicated with black arrow) are represented in balls. (C,D) Modeling of the voltage sensing domain (VSD) in repeat III of WT Cav2.1 (C), and Cav2.1 R1359W (D), showing putative non-bonded interactions in dash lines: hydrogen bond pairs in green (K1355 in IIIS4), ionic bond pairs in yellow (E1291 in IIIS2, and to a lesser extent, D1317 in IIIS3), hydrophobic interaction in purple (L1251 in IIIS1). Steric clashes (K1358 in IIIS4) are represented by a red dashed line. (E,F) Modeling of the IV-VSD of WT Cav2.1 (E), and Cav2.1 R1667W (F). (G,H) Modeling of the pore domain around the IVS6 helix of WT Cav2.1 (G), and Cav2.1 S1799L (H).
	Figure 8. Computer modeling of human Purkinje cell. (A) Representative firing patterns obtained from simulated Purkinje cell with WT Cav2.1 properties (Va, ka, run 1), with a hyperpolarizing shift of Va (run 2), a depolarization shift of Va (run 3), an increase of ka (run 4), or a combination of both (run 5 and 6). (B) Bar graph showing the mean action potential frequencies (freq) obtained for the different simulations (in Hertz: 57.3 ± 0.8, 47.3 ± 1.0, 86.3 ± 0.7, 55.7 ± 0.9, 43.8 ± 0.8 and 85.5 ± 0.7 for run 1 to run 6, respectively). (C) Bar graph showing the mean action potential areas obtained for the different simulations (in mV.ms: 64.0 ± 0.1, 64.7 ± 0.2, 62.2 ± 0.1, 64.0 ± 0.1, 64.8 ± 0.2, 57.6 ± 0.1 for run 1 to 6, respectively). The number of action potentials analyzed was between 389 and 807. Asterisks denote significant difference vs. run 1 ( p < 0.01, * p < 0.01) (non-paired Student’s t-test).

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	Figure 1. (Top) Topology of the voltage-gated Cav2.1 subunit with the position of the four pathogenic variants studied: A405T, R1359W, R1667W, and S1799L. The Cavα is formed of four repeats (I to IV) each containing 6 transmembrane helices (S1–S6). The S1–S4 helices constitute the voltage-sensing domain (VSD) able to move in response to membrane potential changes. The four re-entrant P loops between segments S5 and S6 carry the selectivity filter and delineate the extracellular end of the channel pore. The S5 and S6 helices of the four repeats delineate the cytoplasmic end of the channel pore. (Bottom) Amino-acid sequences of the I-II loop, and of the IIIS4, IVS4, and IVS6 helices. Cav2.1 variants referenced in the NCBI ClinVar database are indicated with their accession number. References in italics correspond to variants ‘of uncertain significance’ when subjected to in silico analysis. The ‘R’ above the sequences of the IIIS4 and IVS4 helices indicates the position of a basic residue. The Cav2.1 sequence is numbered according to the NCBI Genbank® sequence AAB64179.
	Figure 2. (A) Representative current traces were recorded from X. laevis oocytes that expressed the indicated Cav2.1+e47 variants with Cavβ4a and Cavα2δ1. Currents were elicited from a holding potential of −100 mV by a two-pulse protocol illustrated on top, and consisting of a 2.5 s-long depolarization from −80 mV to +40 mV, followed by a 400 ms-long depolarization to 0 mV (WT and R1359W) or +10 mV (A405T, R1667W, and S1799L). Scale bars, 200nA. (B) Representative images of HEK cells immunostained with anti-Cav2.1 (red) and anti-ZO-1 (green). Anti-ZO-1 was used to visualize the cell membrane. Scale bars, 10 μm. (C) Quantification of fluorescence intensity realized on individualized cells (n = 10 for each variant). Asterisks and number signs denote significant differences vs. WT (*** p < 0.001) and vs. Cav2.1+e47 splice variants (### p < 0.001), respectively (non-paired Student’s t-test). n.s. = non-significant.
	Figure 3. Voltage dependence of Cav2.1 activation. (Top) Average current-voltage relationships for X. laevis oocytes expressing Cav2.1+47 (left) or Cav2.1−e47 variants (right). (Bottom) Box plots of half-maximal activation potential (Va, left) and slope factor of the Boltzmann curve of channel activation (ka, right) were obtained for all Cav2.1 variants studied. The mean values are given in Table 1. Asterisks and number signs denote significant differences vs. WT ( p < 0.01, * p < 0.001) and vs. Cav2.1+e47 splice variant (# p < 0.05), respectively (non-paired Student’s t-test). n.s. = non-significant.
	Figure 4. Voltage dependence of Cav2.1 inactivation. (Top) Voltage-dependent isochronal inactivation curves for X. laevis oocytes expressing Cav2.1+47 (left) or Cav2.1−e47 (right) variants. (Bottom) Box plots of half-maximal inactivation potential (Vi, left) and slope factor of the Boltzmann curve of channel inactivation (ki, right) were obtained for all Cav2.1 variants studied. The mean values are given in Table 1. Asterisks and number signs denote significant differences vs. WT ( p < 0.01, * p < 0.001) and vs. Cav2.1+e47 (# p < 0.05, ### p < 0.001), respectively (non-paired Student’s t-test). n.s. = non-significant.
	Figure 5. Cav2.1 inactivation kinetics. Box plot of the ratio of remaining current at the end of a 400 ms-long depolarization (R400) to 0 mV (WT, A405T, R1359W, and R1667W Cav2.1 variants) or + 10 mV (S1799L Cav2.1 variants) with respect to the peak current amplitude. Asterisks and number signs denote significant differences vs. WT ( p < 0.01, * p < 0.001), and vs. Cav2.1+e47 (# p < 0.05, ### p < 0.001), respectively (non-paired Student’s t-test). n.s. = non-significant.
	Figure 6. Recovery from inactivation of Cav2.1 variants. (A) Representative current traces were obtained in X. laevis oocytes expressing Cav2.1+47 with Cavβ4a and Cavα2δ1. The experimental protocol, illustrated above traces, consists of a 2.5 s-long depolarization to +10 mV, followed by inter-pulse intervals between 100 ms and 8 s, and a second 100 ms-long depolarization to +10 mV. Scale bars: 200 nA and 2 s. (B) Percentage of current recovery plotted against the inter-pulse duration for Cav2.1+e47 (top) and Cav2.1−e47 variants (bottom). (C) Bar graph showing percent recovery at 8 s for all Cav2.1 variants studied. Asterisks and number signs denote significant differences vs. WT ( p < 0.01, * p < 0.01) and vs. Cav2.1+e47 (## p < 0.01), respectively (non-paired Student’s t-test). n.s. = non-significant.
	Figure 7. Homology model of human Cav2.1 viewed from the side (A) or from the top (B). The IIIS4, IVS4, and IVS6 transmembrane helices carrying the studied mutation are represented in magenta, dark green, and brown, respectively. The four residues R1359 (IIIS4), R1667 (IVS4), S1799L (IVS6), and A405 (I-II loop, indicated with black arrow) are represented in balls. (C,D) Modeling of the voltage sensing domain (VSD) in repeat III of WT Cav2.1 (C), and Cav2.1 R1359W (D), showing putative non-bonded interactions in dash lines: hydrogen bond pairs in green (K1355 in IIIS4), ionic bond pairs in yellow (E1291 in IIIS2, and to a lesser extent, D1317 in IIIS3), hydrophobic interaction in purple (L1251 in IIIS1). Steric clashes (K1358 in IIIS4) are represented by a red dashed line. (E,F) Modeling of the IV-VSD of WT Cav2.1 (E), and Cav2.1 R1667W (F). (G,H) Modeling of the pore domain around the IVS6 helix of WT Cav2.1 (G), and Cav2.1 S1799L (H).
	Figure 8. Computer modeling of human Purkinje cell. (A) Representative firing patterns obtained from simulated Purkinje cell with WT Cav2.1 properties (Va, ka, run 1), with a hyperpolarizing shift of Va (run 2), a depolarization shift of Va (run 3), an increase of ka (run 4), or a combination of both (run 5 and 6). (B) Bar graph showing the mean action potential frequencies (freq) obtained for the different simulations (in Hertz: 57.3 ± 0.8, 47.3 ± 1.0, 86.3 ± 0.7, 55.7 ± 0.9, 43.8 ± 0.8 and 85.5 ± 0.7 for run 1 to run 6, respectively). (C) Bar graph showing the mean action potential areas obtained for the different simulations (in mV.ms: 64.0 ± 0.1, 64.7 ± 0.2, 62.2 ± 0.1, 64.0 ± 0.1, 64.8 ± 0.2, 57.6 ± 0.1 for run 1 to 6, respectively). The number of action potentials analyzed was between 389 and 807. Asterisks denote significant difference vs. run 1 ( p < 0.01, * p < 0.01) (non-paired Student’s t-test).