Cross talk between β subunits, intracellular Ca2+ signaling, and SNAREs in the modulation of CaV 2.1 channel steady-state inactivation

Abstract

Modulation of Ca_V 2.1 channel activity plays a key role in interneuronal communication and synaptic plasticity. SNAREs interact with a specific synprint site at the second intracellular loop (LII-III) of the Ca_V 2.1 pore-forming α_1A subunit to optimize neurotransmitter release from presynaptic terminals by allowing secretory vesicles docking near the Ca²⁺ entry pathway, and by modulating the voltage dependence of channel steady-state inactivation. Ca²⁺ influx through Ca_V 2.1 also promotes channel inactivation. This process seems to involve Ca²⁺ -calmodulin interaction with two adjacent sites in the α_1A carboxyl tail (C-tail) (the IQ-like motif and the Calmodulin-Binding Domain (CBD) site), and contributes to long-term potentiation and spatial learning and memory. Besides, binding of regulatory β subunits to the α interaction domain (AID) at the first intracellular loop (LI-II) of α_1A determines the degree of channel inactivation by both voltage and Ca²⁺ . Here, we explore the cross talk between β subunits, Ca²⁺ , and syntaxin-1A-modulated Ca_V 2.1 inactivation, highlighting the α_1A domains involved in such process. β₃ -containing Ca_V 2.1 channels show syntaxin-1A-modulated but no Ca²⁺ -dependent steady-state inactivation. Conversely, β_2a -containing Ca_V 2.1 channels show Ca²⁺ -dependent but not syntaxin-1A-modulated steady-state inactivation. A LI-II deletion confers Ca²⁺ -dependent inactivation and prevents modulation by syntaxin-1A in β₃ -containing Ca_V 2.1 channels. Mutation of the IQ-like motif, unlike CBD deletion, abolishes Ca²⁺ -dependent inactivation and confers modulation by syntaxin-1A in β_2a -containing Ca_V 2.1 channels. Altogether, these results suggest that LI-II structural modifications determine the regulation of Ca_V 2.1 steady-state inactivation either by Ca²⁺ or by SNAREs but not by both.

Keywords: Ca2+-calmodulin; CaV2.1 domains for SNARE-mediated modulation; CaV2.1 steady-state inactivation; CaVβ subunits; presynaptic voltage-gated CaV2.1 channels; syntaxin-1A.

Figure 1

Location of the known α _1A molecular determinants for the binding of the main intracellular proteins that modulate Ca_V2.1 channel inactivation. (A) Schematic representation of the secondary structure of the Ca_V2.1 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 Ca_V2.1 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).

Figure 2

Steady‐state inactivation of Ca_V2.1 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 Ca_V2.1 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 Ca_V2.1 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 (V_1/2 inactivation) (E) for WT Ca_V2.1 channels containing the β ₃ subunit (WT 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 V_1/2 inact and k_inact values at intermediate Ca²⁺‐buffering condition (1 mmol/L EGTA) were (in mV): WT β ₃ (open circles, n = 18) −23.7 ± 0.83 and −5.16 ± 0.24; WT β ₃ + stx 1A (filled circles, n = 12) −32.74 ± 1.46 and −5.5 ± 0.19, respectively. At high Ca²⁺‐buffering condition (10 mmol/L BAPTA), average V_1/2 inact and k_inact values were (in mV): WT β ₃ (open circles, n = 12) −20.74 ± 1.18 and −4.78 ± 0.16; WT β ₃ + stx 1A (filled circles, n = 8) −25.68 ± 1.13 and −5.25 ± 0.21, respectively. a and b: P < 0.001 and P < 0.05 versus the corresponding control condition (absence of syntaxin‐1A), respectively; c: P < 0.01 when compared to the intermediate Ca²⁺‐buffering condition (1 mmol/L EGTA). No significant difference was found for k_inact values (ANOVA P = 0.17).

Figure 3

Δ451‐457 at α _1A LI‐II promotes a Ca²⁺‐dependent component in the steady‐state inactivation of Ca_V2.1 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 Ca_V2.1 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 V_1/2 inactivation (D) for ΔLI‐II _451–457 Ca_V2.1 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 V_1/2 inact and k_inact 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 + stx 1A (filled circles, n = 9) −30 ± 1.85 and −5.73 ± 0.35, respectively. At high Ca²⁺‐buffering condition (10 mmol/L BAPTA), average V_1/2 inact and k_inact values were (in mV): ΔLI‐II β ₃ (open circles, n = 9) −21.84 ± 0.83 and −4.94 ± 0.47; ΔLI‐II β ₃ + stx 1A (filled circles, n = 8) −27.94 ± 1.15 and −4.97 ± 0.33, respectively. a: P < 0.001 when compared to the intermediate Ca²⁺‐buffering condition (1 mmol/L EGTA); b: P < 0.05 versus the corresponding control condition (absence of syntaxin‐1A). No significant difference was found for k_inact values (ANOVA P = 0.14).

Figure 4

Steady‐state inactivation of Ca_V2.1 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 Ca_V2.1 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 V_1/2 inactivation (D) for ΔLI‐II _451–457 Ca_V2.1 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 V_1/2 inact and k_inact 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, respectively. At high Ca²⁺‐buffering condition (10 mmol/L BAPTA), average V_1/2 inact and k_inact values were (in mV): WT β _2a (open circles, n = 9) −2.78 ± 0.95 and −3.1 ± 0.52; WT β _2a + stx 1A (filled circles, n = 11) −7.57 ± 1.36 and −3.15 ± 0.55, respectively. a: P < 0.001 when compared to the intermediate Ca²⁺‐buffering condition (1 mmol/L EGTA); b: P < 0.05 versus the corresponding control condition (absence of syntaxin‐1A). No significant difference was found for k_inact values (ANOVA P = 0.44).

Figure 5

Steady‐state inactivation of Ca_V2.1 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 Ca_V2.1 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 (V_1/2 inactivation) (D) for mutant ΔLI‐II _451–457 Ca_V2.1 mutant channels containing the β _2a subunit (ΔLI‐II 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 V_1/2 inact and k_inact values at intermediate Ca²⁺‐buffering condition (1 mmol/L EGTA) were (in mV): ΔLI‐II β _2a (open circles, n = 8) −11.47 ± 0.86 and −2.87 ± 0.5; ΔLI‐II β _2a + stx 1A (filled circles, n = 9) −13.57 ± 1.15 and −2.73 ± 0.44, respectively. At high Ca²⁺‐buffering condition (10 mmol/L BAPTA), average V_1/2 inact and k_inact values were (in mV): ΔLI‐II β _2a (open circles, n = 9) −2.58 ± 0.99 and −2.05 ± 0.43; ΔLI‐II β _2a + stx 1A (filled circles, n = 9) −7.45 ± 1.15 and −2.12 ± 0.51, respectively. a: P < 0.001 when compared to the intermediate Ca²⁺‐buffering condition (1 mmol/L EGTA); b: P < 0.01 versus the corresponding control condition (absence of syntaxin‐1A). No significant difference was found for k_inact values (ANOVA P = 0.51).

Figure 6

α _1A IQ‐like motif mutation (IM/EE) remove the Ca²⁺‐dependent component in the steady‐state inactivation of β _2a‐containing Ca_V2.1 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 Ca_V2.1 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 V_1/2 inactivation (D) for IM/EE Ca_V2.1 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 V_1/2 inact and k_inact 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 = 11) −1.25 ± 1.71 and −6.44 ± 0.34, respectively. At high Ca²⁺‐buffering condition (10 mmol/L BAPTA), average V_1/2 inact and k_inact values were (in mV): IM/EE β _2a (open circles, n = 7) 1.4 ± 0.95 and ‐5.8 ± 0.62; IM/EE β _2a + stx 1A (filled circles, n = 9) −4.68 ± 1.71 and ‐6.21 ± 0.42, respectively. a: P < 0.05 versus the corresponding control condition (absence of syntaxin‐1A). No significant difference was found for k_inact values (ANOVA P = 0.82).

Figure 7

Steady‐state inactivation of Ca_V2.1 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 Ca_V2.1 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 V_1/2 inactivation (D) for ΔCBD Ca_V2.1 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 V_1/2 inact and k_inact 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 ± 0.31, respectively. At high Ca²⁺‐buffering condition (10 mmol/L BAPTA), average V_1/2 inact and k_inact values were (in mV): ΔCBD β _2a (open circles, n = 12) −4.43 ± 1.15 and −3.68 ± 0.25; ΔCBD β _2a + stx 1A (filled circles, n = 12) −9.6 ± 1.61 and −4.52 ± 0.5, respectively. a: P < 0.01 when compared to the intermediate Ca²⁺‐buffering condition (1 mmol/L EGTA); b: P < 0.05 versus the corresponding control condition (absence of syntaxin‐1A). k_inact values were significantly higher (P < 0.05) at high Ca²⁺‐buffering condition (10 mmol/L BAPTA) than at intermediate Ca²⁺‐buffering condition (1 mmol/L EGTA). The presence of syntaxin‐1A had no significant effect on k_inact.

Figure 8

Sequence alignment of intracellular loop between domains I and II (LI‐II) of human Ca_V2.1 channel α _1A subunit and rabbit Ca_V1.2 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.

Figure 9

Sequence alignment of intracellular domains (LI‐II, LIII‐IV, and C‐tail) of human Ca_V2.1 channel α _1A subunit and rabbit Ca_V1.1 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 Ca_V1.1 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.