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, 286 (16), 13945-53

CaBP1 Regulates Voltage-Dependent Inactivation and Activation of Ca(V)1.2 (L-type) Calcium Channels

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CaBP1 Regulates Voltage-Dependent Inactivation and Activation of Ca(V)1.2 (L-type) Calcium Channels

Shimrit Oz et al. J Biol Chem.

Abstract

CaBP1 is a Ca(2+)-binding protein that regulates the gating of voltage-gated (Ca(V)) Ca(2+) channels. In the Ca(V)1.2 channel α(1)-subunit (α(1C)), CaBP1 interacts with cytosolic N- and C-terminal domains and blunts Ca(2+)-dependent inactivation. To clarify the role of the α(1C) N-terminal domain in CaBP1 regulation, we compared the effects of CaBP1 on two alternatively spliced variants of α(1C) containing a long or short N-terminal domain. In both isoforms, CaBP1 inhibited Ca(2+)-dependent inactivation but also caused a depolarizing shift in voltage-dependent activation and enhanced voltage-dependent inactivation (VDI). In binding assays, CaBP1 interacted with the distal third of the N-terminal domain in a Ca(2+)-independent manner. This segment is distinct from the previously identified calmodulin-binding site in the N terminus. However, deletion of a segment in the proximal N-terminal domain of both α(1C) isoforms, which spared the CaBP1-binding site, inhibited the effect of CaBP1 on VDI. This result suggests a modular organization of the α(1C) N-terminal domain, with separate determinants for CaBP1 binding and transduction of the effect on VDI. Our findings expand the diversity and mechanisms of Ca(V) channel regulation by CaBP1 and define a novel modulatory function for the initial segment of the N terminus of α(1C).

Figures

FIGURE 1.
FIGURE 1.
CaBP1 regulates both CDI and VDI. A, representative normalized currents elicited by 400-ms pulses from −80 to +20 mV in Xenopus oocytes expressing LNTα1C (left panel) or SNTα1C (right panel) without (control, black) or with CaBP1 (gray). Currents were recorded in 40 mm Ca2+ solution (ICa) or Ba2+ solution (IBa). α2δ-1 and CaV β2b-subunits were coexpressed in all experiments done in Xenopus oocytes. B, summary of changes in r400 at different voltages in the presence (○) or absence (●) of CaBP1. Data are presented as means ± S.E. (n = 14–39). C, summary of the effects of CaBP1 on r400 in the LNTα1C and SNTα1C isoforms in +20 mV. Bars represent means ± S.E.; the numbers within the bars indicate n. In B and C, the statistical significance of differences between the control and CaBP1 was determined by t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 2.
FIGURE 2.
Kinetic analysis of CaBP1-induced changes in VDI in LNTα1C. A, records of IBa from a representative cell expressing LNTα1C evoked by 10-s depolarizing voltage steps from −80 mV to different voltages. Net IBa, obtained by subtraction of Cd2+-insensitive currents, is shown in gray. The decay time course was fitted to a two-exponential function (see supplemental Table S1 for fitted parameters), and the fitted curve (black line) is shown superimposed on the original trace. B, CaBP1 accelerates the kinetics of decay of IBa of LNTα1C. Currents were elicited by 10-s pulses from −80 mV to various voltages. Traces, averaged from 10–13 oocytes, show the decay phase of IBa, starting from the peak and to the end of the voltage step. For clarity, S.E. is shown only for every 20th acquisition point. Student's t test was used to determine statistical significance at the t = 2000 ms time point. C, summary of the two-exponential fit analysis of IBa decay in LNTα1C. Averaged values of Aslow and Afast (the contribution of each kinetic component), C (the non-inactivating current), and τslow and τfast (the time constants) at different voltages are shown with (○) or without (●) CaBP1. Statistical significance between values was calculated at each voltage using a non-paired t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 3.
FIGURE 3.
Mapping of the CaBP1-binding site in the N terminus of α1C. A, upper, linear representation of the cytosolic N termini of LNTα1C (aa 1–154) and SNTα1C (aa 1–124). Most of the deletion constructs used in this study are indicated below the sequences. The locations of parts of the N terminus with known function (NTI module and CaM-binding site/NSCaTE) and of the newly identified CaBP1-binding segment are indicated by boxes. Lower, schematic representation of the GST-fused segments of LNTα1C used in this study. B, in vitro synthesized, 35S-labeled CaBP1 binds to the distal part of the N terminus in a Ca2+-independent manner. The two gels are from one experiment. The upper panels show the autoradiograms of 35S-radiolabeled CaBP1 bound to the GST fusion proteins indicated on the bottom. The GST fusion proteins eluted from the beads at the end of experiment are shown in the lower panels, after staining the gel with Coomassie Blue. Interactions between CaBP1 and the GST fusion proteins were measured with the addition of 1 mm CaCl2 or EGTA as indicated. input represents the signal from one-sixtieth of the total labeled protein used in the reaction.
FIGURE 4.
FIGURE 4.
CaBP1 depolarizes voltage dependence of activation. A, IBa was evoked by depolarizing pulses from a holding voltage of −80 mV to various voltages in oocytes expressing LNTα1C, SNTα1C, and the Δ46-LNTα1C and Δ139-LNTα1C constructs. Normalized current-voltage (I-V) curves averaged from 9–62 oocytes are shown. I-V curves were fitted to the Boltzmann equation in each cell. The values of Va and ka, averaged from the individual fit in all cells, were used to draw the solid lines of the I-V curves. B, conductance-voltage (G-V) curves. Each point shows the average G/Gmax at each voltage calculated for the same cells as in A. The solid lines have been drawn using the Boltzmann equation for G/Gmax utilizing the values of Ka and Va obtained in the fits of the I-V curves. The Boltzmann fit parameters and number of cells analyzed are shown in Table 2.
FIGURE 5.
FIGURE 5.
N terminus of α1C is involved in the regulation of VDI by CaBP1. A, comparison of the time course of decay of IBa in various channel constructs tested. Currents were recorded at +10 (black) and +20 (dark gray) mV in the indicated constructs expressed in oocytes without CaBP1. In addition, currents recorded at +20 mV in the presence of CaBP1 are presented (light gray). Currents were normalized and averaged (n = 14). S.E. values are shown for each 20th point (n = 4–14 oocytes). B, the CaBP1-induced shift in activation does not account for its effect on VDI. Normalized IBa recorded at +10 mV without CaBP1 (black) is compared with IBa recorded at +20 mV in the presence of CaBP1 (gray) in representative cells. C, summary of the experiments shown in A. Values of r2000 measured at +10 mV (black bars) or +20 mV (dark gray bars) and +20 mV when coexpressing CaBP1 (light gray bars) are summarized. The statistical significance of the differences between the control at +10 or +20 mV and CaBP1 at +20 mV (t test) is shown above each pair of bars. The net effect of CaBP1, i.e. the difference in r2000 in CaBP1 (20 mV) versus the control (LNTα1C at 10 mV) is shown by white bars. Negative values indicate acceleration of VDI by CaBP1. Compared with LNTα1C, the effect of CaBP1 on Δ46-LNTα1C and Δ139-LNTα1C was significantly different, as determined by one-way analysis of variance followed by the Bonferroni t test. These statistical differences are indicated under the corresponding white bars. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

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