Cross talk betweenβsubunits, intracellular Ca²⁺signaling, and SNAREs in the modulation of CaV2.1 channel steady‐state inactivation

Figures

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  Figure 1. Location of the known α 1A molecular determinants for the binding of the main intracellular proteins that modulate CaV2.1 channel inactivation. (A) Schematic representation of the secondary structure of the CaV2.1 α 1A channel subunit, showing the location of the known α 1A molecular determinants for the binding of three cytosolic proteins involved in the regulation of CaV2.1 channel inactivation: (1) regulatory β subunits (which binds to the α‐interaction domain or AID, located in the cytoplasmic loop connecting domains I and II (LI‐II) of α 1A); (2) SNARE proteins (that interacts with the synprint site, within the intracellular loop connecting domains II and III (LII‐III) of α 1A); and (3) the Ca2+‐calmodulin (Ca2+‐CaM) complex (which binds to the IQ‐like motif and the Calmodulin‐Binding Domain (CBD) site, at the carboxyl tail of α 1A). (B) Location of the α 1A LI‐II deletion around the A454 residue (ΔLI‐II 451–457) (also depicted at panel A).

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  Figure 2. Steady‐state inactivation of CaV2.1 channels containing the regulatory β 3 subunit is independent of intracellular Ca2+ signaling and is favored by syntaxin‐1A (A) Voltage protocol for the study of CaV2.1 channels steady‐state inactivation (see Methods for further details). Representative normalized Ca2+ current traces recorded at intermediate (1 mmol/L EGTA) (B) or high (10 mmol/L BAPTA) (C) intracellular Ca2+‐buffering conditions from a HEK 293 cell expressing CaV2.1 channels composed of wild‐type α 1A, β 3, and α 2 δ subunits (WT β 3) either in the absence (left) or presence (right) of syntaxin‐1A (stx 1A). Currents were elicited by 50 ms depolarizing steps to +20 mV applied after 30‐sec depolarizing prepulses to the indicated voltages. Amplitudes of currents elicited by test pulses to +20 mV after the different prepulses were normalized to the maximum current amplitude obtained after a 30‐sec prepulse to −80 mV in order to generate the corresponding mean steady‐state inactivation curves (D), which were fitted to a single Boltzmann function (see Methods, eq. (1)) to estimate the half‐inactivation potentials (V1/2 inactivation) (E) for WT CaV2.1 channels containing the β 3…

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  Figure 3. Δ451‐457 at α 1A LI‐II promotes a Ca2+‐dependent component in the steady‐state inactivation of CaV2.1 channels containing the auxiliary β 3 subunit, and it prevents syntaxin‐1A‐mediated modulation. Typical normalized Ca2+ current traces recorded at intermediate (1 mmol/L EGTA) (A) or high (10 mmol/L BAPTA) (B) intracellular Ca2+‐buffering conditions from a HEK 293 cell expressing CaV2.1 channels composed of mutant ΔLI‐II 451–457 α 1A, β 3, and α 2 δ subunits (ΔLI‐II β 3) either in the absence (left) or presence (right) of syntaxin‐1A (stx 1A). Currents were elicited by 50‐ms depolarizing steps to +20 mV applied after 30‐sec depolarizing prepulses to the shown voltages. Corresponding mean normalized steady‐state inactivation curves (C), and derived V1/2 inactivation (D) for ΔLI‐II 451–457 CaV2.1 mutant channels containing the β 3 subunit (ΔLI‐II β 3) in the absence (open circles) or presence (filled circles) of syntaxin‐1A (stx 1A), at the above indicated intracellular Ca2+‐buffering conditions. Average V1/2 inact and kinact values at intermediate Ca2+‐buffering condition (1 mmol/L EGTA) were (in mV): ΔLI‐II β 3 (open circles, n = 14) −29.58 ± 1.35 and −4.73 ± 0.16; ΔLI‐II β 3 +…

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  Figure 4. Steady‐state inactivation of CaV2.1 channels containing the regulatory β 2a subunit presents a Ca2+‐dependent component and no regulation by syntaxin‐1A. Illustrative normalized Ca2+ current traces recorded at intermediate (1 mmol/L EGTA) (A) or high (10 mmol/L BAPTA) (B) intracellular Ca2+‐buffering conditions from a HEK 293 cell expressing CaV2.1 channels composed of WT α 1A, β 2a, and α 2 δ subunits (WT β 2a) either in the absence (left) or presence (right) of syntaxin‐1A (stx 1A). Currents were elicited by 50‐ms depolarizing steps to +20 mV applied after 30‐sec depolarizing prepulses to the indicated voltages. Corresponding mean normalized steady‐state inactivation curves (C), and estimated V1/2 inactivation (D) for ΔLI‐II 451–457 CaV2.1 mutant channels containing the β 2a subunit (WT β 2a) in the absence (open circles) or presence (filled circles) of syntaxin‐1A (stx 1A), at the above indicated intracellular Ca2+‐buffering conditions. Average V1/2 inact and kinact values at intermediate Ca2+‐buffering condition (1 mmol/L EGTA) were (in mV): WT β 2a (open circles, n = 12) −12.08 ± 1.36 and −2.43 ± 0.41; WT β 2a + stx 1A (filled circles, n = 11) −14.53 ± 1.02 and −2.33 ± 0.28,…

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  Figure 5. Steady‐state inactivation of CaV2.1 channels formed by mutant α 1A ΔLI‐II 451–457 and β 2a subunits remains Ca2+‐dependent and syntaxin‐1A‐insensitive. Representative normalized Ca2+ current traces recorded at intermediate (1 mmol/L EGTA) (A) or high (10 mmol/L BAPTA) (B) intracellular Ca2+‐buffering conditions from a HEK 293 cell expressing CaV2.1 channels composed of mutant ΔLI‐II 451–457 α 1A, β 2a, and α 2 δ subunits (ΔLI‐II β 2a) either in the absence (left) or presence (right) of syntaxin‐1A (stx 1A). Currents were elicited by 50‐ms depolarizing steps to +20 mV applied after 30‐sec depolarizing prepulses to the indicated voltages. Amplitudes of currents elicited by test pulses to +20 mV after the different prepulses were normalized to the maximum current amplitude obtained after a 30‐sec prepulse to −80 mV in order to generate the corresponding mean steady‐state inactivation curves (C), which were fitted to a single Boltzmann function (see Methods, eq. (1)) to estimate the half‐inactivation potentials (V1/2 inactivation) (D) for mutant ΔLI‐II 451–457 CaV2.1 mutant channels containing the β 2a subunit (ΔLI‐II β 2a) in the absence (open circles) or presence (filled circles)…

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  Figure 6. α 1A IQ‐like motif mutation (IM/EE) remove the Ca2+‐dependent component in the steady‐state inactivation of β 2a‐containing CaV2.1 channels, and it allows modulation by syntaxin‐1A. Representative normalized Ca2+ current traces recorded at intermediate (1 mmol/L EGTA) (A) or high (10 mmol/L BAPTA) (B) intracellular Ca2+‐buffering conditions from a HEK 293 cell expressing CaV2.1 channels composed of mutant IM/EE α 1A, β 2a, and α 2 δ subunits (IM/EE β 2a) either in the absence (left) or presence (right) of syntaxin‐1A (stx 1A). Currents were elicited by 10‐ms depolarizing steps to +20 mV applied after 30‐sec depolarizing prepulses to the shown voltages. Corresponding mean normalized steady‐state inactivation curves (C), and derived V1/2 inactivation (D) for IM/EE CaV2.1 mutant channels containing the β 2a subunit (IM/EE β 2a) in the absence (open circles) or presence (filled circles) of syntaxin‐1A (stx 1A), at the above indicated intracellular Ca2+‐buffering conditions. Average V1/2 inact and kinact values at intermediate Ca2+‐buffering condition (1 mmol/L EGTA) were (in mV): IM/EE β 2a (open circles, n = 10) 4.19 ± 0.8 and ‐6.17 ± 0.5; IM/EE β 2a + stx 1A (filled circles, n =…

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  Figure 7. Steady‐state inactivation of CaV2.1 channels formed by mutant α 1A ΔCBD and β 2a subunits still shows a Ca2+‐dependent component and no regulation by syntaxin‐1A. Typical normalized Ca2+ current traces recorded at intermediate (1 mmol/L EGTA) (A) or high (10 mmol/L BAPTA) (B) intracellular Ca2+‐buffering conditions from a HEK 293 cell expressing CaV2.1 channels composed of mutant ΔCBD α 1A, β 2a, and α 2 δ subunits (ΔCBD β 2a) either in the absence (left) or presence (right) of syntaxin‐1A (stx 1A). Currents were elicited by 50‐ms depolarizing steps to +20 mV applied after 30‐sec depolarizing prepulses to the shown voltages. Corresponding mean normalized steady‐state inactivation curves (C), and derived V1/2 inactivation (D) for ΔCBD CaV2.1 mutant channels containing the β 2a subunit (ΔCBD β 2a) in the absence (open circles) or presence (filled circles) of syntaxin‐1A (stx 1A), at the above indicated intracellular Ca2+‐buffering conditions. Average V1/2 inact and kinact values at intermediate Ca2+‐buffering condition (1 mmol/L EGTA) were (in mV): ΔCBD β 2a (open circles, n = 8) −12.48 ± 1.07 and −2.23 ± 0.25; ΔCBD β 2a + stx 1A (filled circles, n = 6) −14.75 ± 1.53 and −2.59 ±…

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  Figure 8. Sequence alignment of intracellular loop between domains I and II (LI‐II) of human CaV2.1 channel α 1A subunit and rabbit CaV1.2 channel α 1C subunit. Alignments were performed with Clustal Omega (www.ebi.ac.uk/Tools/msa/clustalo/). AID sequences are shown in purple, and rabbit (rb) α 1C LI‐II site for Ca2+‐calmodulin binding is highlighted in green. Position of the human (h) α 1A LI‐II deletion around A454 (in red) (ΔLI‐II 451–457) is shown in orange. “*” identical residues; “:” conservative substitutions (same amino acid group); “.” semi‐conservative substitution (similar shapes). LI‐II residues appear in bold.

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  Figure 9. Sequence alignment of intracellular domains (LI‐II, LIII‐IV, and C‐tail) of human CaV2.1 channel α 1A subunit and rabbit CaV1.1 channel α 1S subunit. Alignments were performed with Clustal Omega (www.ebi.ac.uk/Tools/msa/clustalo/). AID sequences at LI‐II are shown in purple, and the human (h) α 1A LI‐II deletion around A454 (in red) (ΔLI‐II 451–457) is highlighted in orange. Amino acids involved in the physical interaction between LIII‐IV and C‐terminal domain (CTD) of rabbit (rb) α 1S, according to cryo‐EM structural data (Wu et al. 2016), and the homologous sequences in hα 1A are shown in brown. Sequences of the IQ (rbα 1S) and IQ‐like (hα 1A) motifs are shown in green. hα 1A CBD sequence is depicted in blue. Cytoplasmic segments that were invisible in the cryo‐EM structure of CaV1.1 rbα 1S subunit (Wu et al. 2016) are shown in gray. “*” identical residues; “:” conservative substitutions (same amino acid group); “.” semi‐ conservative substitution (similar shapes). LI‐II, LIII‐IV, and CTD residues appear in bold.