The guanylate kinase domain of the beta-subunit of voltage-gated calcium channels suffices to modulate gating

Abstract

Inactivation of voltage-gated calcium channels is crucial for the spatiotemporal coordination of calcium signals and prevention of toxic calcium buildup. Only one member of the highly conserved family of calcium channel beta-subunits--Ca(V)beta--inhibits inactivation. This unique property has been attributed to short variable regions of the protein; however, here we report that this inhibition actually is conferred by a conserved guanylate kinase (GK) domain and, moreover, that this domain alone recapitulates Ca(V)beta-mediated modulation of channel activation. We expressed and refolded the GK domain of Ca(V)beta(2a), the unique variant that inhibits inactivation, and of Ca(V)beta(1b), an isoform that facilitates it. The refolded domains of both Ca(V)beta variants were found to inhibit inactivation of Ca(V)2.3 channels expressed in Xenopus laevis oocytes. These findings suggest that the GK domain endows calcium channels with a brake restraining voltage-dependent inactivation, and thus facilitation of inactivation by full-length Ca(V)beta requires additional structural determinants to antagonize the GK effect. We found that Ca(V)beta can switch the inactivation phenotype conferred to Ca(V)2.3 from slow to fast after posttranslational modifications during channel biogenesis. Our findings provide a framework within which to understand the modulation of inactivation and a new functional map of Ca(V)beta in which the GK domain regulates channel gating and the other conserved domain (Src homology 3) may couple calcium channels to other signaling pathways.

Fig. 1.

Domain structure, purification, and binding assay of Ca_Vβ constructs. (A) Schematic representation of Ca_Vβ_2a, Ca_Vβ_1b, and derived protein constructs used in this study. Ca_Vβ consists of two highly conserved regions, D1 and D2 (boxes), which are connected and flanked by variable regions (continuous lines). The gray box is the SH3 module; the black box is the GK module. The two cysteine residues at the N terminus of Ca_Vβ_2a that undergo palmitoylation are indicated by arrows. (B) A ribbon diagram of the crystal structure of Ca_Vβ in complex with AID (PDB accession code 1T3L). (C) Size-exclusion chromatography elution profile on the Superdex 200 10/30 column (GE Healthcare) of refolded Ca_Vβ_2a-GK and Ca_Vβ_1b-GK. Here 1 indicates the void volume, 2 indicates the elution volume of albumin (67 kDa), and 3 indicates the elution volume of ovalbumin (43 kDa). The inset shows Coomassie-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) gels of the indicated proteins. Numbers indicate the molecular mass of standards, in kDa. (D) SDS/PAGE gel of the binding reaction with the indicated proteins. Yellow fluorescence protein (YFP)-Ca_Vβ_2a-GK is seen to bind specifically to GST–loop I-II (last lane). The binding assay was repeated three times.

Fig. 2.

Refolded Ca_Vβ_2a-GK and Ca_Vβ_2a-SH3-GK shift the current–voltage relationship of Ca_V1.2-mediated currents. (A) Representative gating and ionic currents traces from oocytes expressing Ca_V1.2 cRNA alone and after injection of either Ca_Vβ_2a-SH3-GK or Ca_Vβ_2a-GK. Currents were evoked by 50-ms pulses to −30, 0, and +30 mV from a holding potential of −80 mV. (B) Normalized current–voltage plot from oocytes expressing the subunit combinations Ca_V1.2 cRNA (n = 11), Ca_V1.2 + Ca_Vβ_2a-SH3-GK (n = 14), and Ca_V1.2 + Ca_Vβ_2a-GK (n = 16). For comparison, normalized current–voltage curves for Ca_V1.2 + Ca_Vβ_2a, either injected as a protein (dashed line) or co-injected as cRNA (continuous line) are shown (see Table S1 for details).

Fig. 3.

Ca_Vβ_2a-GK covalently linked to Ca_V1.2 WT, but not to Ca_V1.2 W470S, increases peak current amplitudes and shifts the current–voltage relationship. (A) Representative gating and ionic current traces from oocytes expressing Ca_V1.2 WT covalently linked to either Ca_Vβ_2a (Ca_V1.2-Ca_Vβ_2a) or Ca_Vβ_2a-GK (Ca_V1.2-Ca_Vβ_2a-GK), and Ca_V1.2 W470S covalently linked to Ca_Vβ_2a-GK (Ca_V1.2 W470S-Ca_Vβ_2a-GK). Currents were evoked by 50-ms pulses to −30, 0, and +30 mV from a holding potential of −80 mV. (B) Ionic current from oocytes expressing the different constructs were normalized by charge movement (I/Q) and plotted versus voltage. For Ca_V1.2-Ca_Vβ_2a, the peak I/Q was 2.44 ± 0.44 nA/pC (n = 17); for Ca_V1.2-Ca_Vβ_2a-GK, it was 2.71 ± 0.52 nA/pC (n = 17); and for Ca_V1.2 W470S-Ca_Vβ_2a-GK, it was 0.35 ± 0.03 nA/pC (n = 17). For comparison, the average I/Q from 15 oocytes expressing Ca_V1.2 alone are shown as dashed lines (0.30 ± 0.06 nA/pC). (C) Normalized tail currents from oocytes expressing the different constructs. The continuous lines correspond to the fit of the sum of two Boltzmann distributions, and the dashed line corresponds to the fit obtained from Ca_V1.2-expressing oocytes (see Fig. S4 and Table S1 for details). The fit to Ca_V1.2 W470S-Ca_Vβ_2a-GK was excluded from the plot for clarity.

Fig. 4.

Ca_Vβ-GK slows inactivation of Ca_V2.3-mediated currents. (A) Representative current traces from oocytes expressing Ca_V2.3 cRNA alone or after injection of the specified protein during a 10-s pulse to 0 mV from a holding potential of −90 mV. (B) Average decay times to half-peak current amplitude (t½) for the different subunit combinations: Ca_V2.3 cRNA, t½ = 0.33 ± 0.03 s (n = 26); Ca_V2.3 + Ca_Vβ_2a-GK cRNA, t½ = 0.41 ± 0.05 s (n = 13); Ca_V2.3 + Ca_Vβ_2a-GK, t½ = 1.85 ± 0.44 s (n = 13); Ca_V2.3 + Ca_Vβ_2a-SH3-GK, t½ = 3.57 ± 0.42 s (n = 21); Ca_V2.3 + Ca_Vβ_2a cRNA, t½ = 4.11 ± 0.65 s (n = 12); Ca_V2.3 + Ca_Vβ_2a, t½ = 4.76 ± 0.70 s (n = 16); Ca_V2.3 + Ca_Vβ_2a-SH3, t½ = 0.35 ± 0.04 s (n = 13). The t½ values for Ca_V2.3 + Ca_Vβ_2a-GK, Ca_V2.3 + Ca_Vβ_2a-SH3-GK, and Ca_V2.3 + Ca_Vβ_2a were significantly different from those measured in oocytes expressing Ca_V2.3 alone (t test; P < .01). (C) Time course of inhibition of inactivation by Ca_Vβ_2a-GK. Each bar corresponds to the average t½ measured at different time intervals after protein injection. The first bar includes recordings from 12–50 min (n = 4), and the second bar includes recordings from 51–100 min (n = 6) and every 100 min thereafter (n = 7, 2, and 4, respectively). The dashed line corresponds to t½ for Ca_V2.3 alone.

Fig. 5.

Ca_Vβ-GK shifts midpoint voltage for the steady-state inactivation of Ca_V2.3-mediated currents. (A) Representative traces of Ca_V2.3-mediated currents in the presence of the specified protein during a steady-state inactivation pulse protocol. This consisted of a 10-s conditioning period to voltages of increasing amplitude, from −120 mV to +30 mV in 15-mV increments, followed by a 0.4-s test pulse to 0 mV. Pulses were delivered once every 50 s from a holding potential of −90 mV. (B) Average steady-state inactivation curves from oocytes expressing Ca_V2.3 alone (n = 22) or after injection of full-length Ca_Vβ_2a (n = 13), Ca_Vβ_2a-SH3-GK (n = 23), or Ca_Vβ_2a-GK (n = 15). The continuous lines correspond to Boltzmann distributions plus a noninactivating current component that best described each set of data. For comparison, the Boltzmann distributions that best described the Ca_V2.3 + Ca_Vβ_2a cRNA data (dashed line) also are shown. The V½ values for Ca_V2.3 + Ca_Vβ_2a-GK and Ca_V2.3 + Ca_Vβ_2a-SH3-GK are significantly different than the V½ measured in oocytes expressing Ca_V2.3 alone (t test; P < .01) (see Table S2 for details).

Fig. 6.

Ca_Vβ_1b-GK slows inactivation of Ca_V2.3-mediated currents and shifts the steady-state inactivation toward more depolarized potentials. (A) Representative current traces from a Ca_V2.3-expressing oocytes injected with Ca_Vβ_1b-GK during a 10-s pulse to 0 mV from a holding potential of −90 mV. (B) Current traces evoked with the steady-state inactivation pulse protocol from the same oocytes shown in (A). (C) Average steady-state inactivation curve from oocytes expressing Ca_V2.3 and injected with Ca_Vβ_1b-GK protein (n = 14). For comparison, the Boltzmann distributions that best describe Ca_V2.3 and Ca_V2.3 + Ca_Vβ_2a data from Fig. 5 also are shown (dashed lines). The V½ value for Ca_V2.3 + Ca_Vβ_1b-GK was significantly different from that measured in oocytes expressing Ca_V2.3 alone (t test; P < .01) (see Table S2 for details).

Fig. 7.

The Ca_V2.3-inactivation phenotype induced by full-length Ca_Vβ_1b and Ca_Vβ_2aC3,4S depends on the time of injection. (A) Representative current traces from oocytes expressing Ca_V2.3 channels with Ca_Vβ_1b or Ca_Vβ_2aC3,4S injected either 2–7 h before recording (late injection) or co-injected with Ca_V2.3-encoding cRNA (co-injection). Currents were evoked by a 10-s pulse to 0 mV from a holding potential of −90 mV. (B) Average t½ for both of the subunit combinations shown in (A). Using Ca_Vβ_1b, the t½ values for co-injection (0.29 ± 0.02 s; n = 11) and late injection (2.19 ± 0.25 s; n = 14) differed significantly. This was true for experiments with Ca_Vβ_2aC3,4S as well (co-injection: 0.82 ± 0.08 s, n = 13; late injection: 2.86 ± 0.48 s, n = 16; t test; P < .01). (C) Steady-state inactivation curves from oocytes either co-injected or late-injected with Ca_Vβ_1b. The continuous lines correspond to the Boltzmann distributions that best describe each set of data. For comparison, the Boltzmann distributions that best describe the Ca_V2.3 data from Fig. 5 are shown (dashed lines). (D) As in C, but for Ca_Vβ_2aC3,4S. With both proteins, the V½ values from the co-injection experiments were significantly more negative than those from the late-injection experiments (t test; P < .01). For Ca_Vβ_2a C3,4S, the V½ values from both the late-injection and co-injection experiments were significantly different from those values for Ca_V2.3 alone (t test; P < .01). The V½ value for Ca_Vβ_1b differed from that of Ca_V2.3 alone only in the co-injection experiments. With both proteins, t½ values in late-injection and co-injection experiments differed significantly from each other and from those values for Ca_V2.3 alone (t test; P < .01) (see Table S2 for details).