CaBP1 binds specifically to the CBD of α₁2.1. (a) Diagram of CaBP1 and CaM. The four Ca²⁺-binding EF-hand motifs are shown as boxes, and key structural differences between CaBP1 and CaM are indicated by arrows. (b) Diagram of the rat brain α₁2.1 subunit (rbA) showing the intracellular domains that were tested for interaction with CaBP1 in yeast two-hybrid assays. The amino acid boundaries of the indicated constructs are given in parentheses. (c) β-galactosidase assays of yeast cotransformed with the α₁2.1 constructs shown in (b) and either CaBP1 or control vector (pACT2).

CaBP1 associates with the α₁2.1 subunit in tsA-201 cells and rat brain. (a) Lysates from cells transfected with Ca_v2.1 plus CaBP, CaBP1 alone or Ca_v2.1_ΔCBD plus CaBP1 were subjected to immunoprecipitation (i.p.) with affinity-purified α₁2.1-specific antibodies or control IgG as indicated. Experiments were done with 10 mM EGTA (lanes 1 and 2) or 2 mM Ca²⁺ (lanes 3–6). Blots were probed with α₁2.1- (top) or CaBP1-specific antibodies (bottom). (b) Rat cerebellar proteins immunoprecipitated with α₁2.1-specific antibodies (CNA5) or control IgG were immunoblotted with α₁2.1- (top) or CaBP1-specific antibodies (bottom). Lysate from tsA-201 cells transfected with CaBP1 was used as a control.

CaBP1 colocalizes with α₁2.1 in rat brain sections. Rat brain sections were double-labeled with antibodies specific for CaBP1 and α₁2.1. Labeling for CaBP1 is shown in green (a, d) and for α₁2.1 in red (b, e). In the merged images (c, f), double-labeled structures appear yellow. Representative examples are shown from the molecular layer of the cerebellum (a–c) and the CA1 region of the hippocampus (d–f). Scale bars, 5 μm (a–c) and 50 μm (d–f).

CaBP1 enhances the inactivation of I_Ca in tsA-201 cells transfected with Ca_v2.1 channels. (a) Representative traces of I_Ca from cells transfected with Ca_v2.1 either alone (bottom) or with CaBP1 (top). Currents were evoked by 1-s pulses to the indicated voltages from a holding potential of −80 mV and were scaled for comparison. (b) Time constants for the inactivation of Ca_v2.1 channel currents in the absence of CaBP1. Test currents were evoked by pulses to the indicated voltages as described in (a) and fit with a single exponential function. Data were averaged from 6–18 cells. (c) Time constants for inactivation of I_Ca in cells cotransfected with CaBP1. Test currents were evoked by the same voltages as in (a) and (b), but current traces were fit with a double exponential function. Fast (τ_fast, filled bars) and slow time constants (τ_slow, open bars) were averaged from 7–20 cells.

Fast, Ca²⁺-independent inactivation of Ca_v2.1 channels by CaBP1 differs from the modulation of Ca_v2.1 channels by CaM. (a) Ca_v2.1 channel currents recorded with Ca²⁺ or Ba²⁺ as the permeant ion. Test pulses were applied from a holding voltage of −80 mV to +10 mV (Ca²⁺) or 0 mV (Ba²⁺) for Ca_v2.1 either alone or cotransfected with CaM, or to +20 mV (Ca²⁺) or +10 mV (Ba²⁺) for cells cotransfected with CaBP1, to account for the positive shift in voltage-dependent activation caused by CaBP1. The intracellular solution contained 0.5 mM EGTA. (b) The residual current amplitude at the end of a test pulse (I_res, indicated in a) was normalized to the peak current (I_pk) for cells transfected with Ca_v2.1 either alone or with CaBP1 or CaM. (c) Representative currents evoked by a test pulse to +30 mV (+20 mV for I_Ba) in cells transfected with wild-type or mutant Ca_v2.1 lacking the CBD (Ca_v2.1_ΔCBD) either alone or cotransfected with CaBP1. Intracellular solutions contained 0.5 mM EGTA except where 10 mM BAPTA is indicated and extracellular solutions contained 10 mM Ca²⁺ except where Ba²⁺ is indicated. (d) Current amplitudes at 200 ms (I₂₀₀, indicated in c) were normalized to the peak current (I_pk) and plotted for the different conditions. Recordings were from tsA-201 cells transfected with Ca_v2.1 or Ca_v2.1_ΔCBD either alone (open bars) or with CaBP1 (filled bars). Results represent averages of 5–13 cells. Asterisks indicate statistically significant differences between the paired groups (p ≤ 0.05).

CaBP1 alters the voltage dependence of Ca_v2.1 activation. Tail current–voltage curves from tsA-201 cells transfected with Ca_v2.1 (a–c) or Ca_v2.1_ΔCBD (d) either alone (open circles) or with CaBP1 (filled circles). Test pulses (10 ms) to the indicated voltages were applied from a holding voltage of −80 mV and peak tail currents were measured upon the repolarization of cells to −40 mV, normalized to the largest tail current in the series, and plotted against test voltage. Test pulses were held for 10 ms, as activation of currents was complete but inactivation was minimal during this time. Bath solutions contained 10 mM Ca²⁺ (a, c, d) or Ba²⁺ (b), and intracellular solutions contained 0.5 mM EGTA (a, b, d) or 10 mM BAPTA (c). Each point represents the mean of 7–20 cells.

CaBP1 does not support Ca²⁺-dependent facilitation of Ca_v2.1 channels. (a, b) Voltage dependence of Ca_v2.1 Ca²⁺ currents evoked before (P1, filled circles) and after (P2, open circles) a depolarizing prepulse. Tail currents were measured by repolarizing cells to −40 mV for 5 ms after variable test voltages and normalized to the largest tail current evoked by P1. Inset, representative currents evoked by a test pulse to +10 mV before (filled circles) and after (open circles) the prepulse. Intracellular recording solution contained 0.5 mM EGTA. Results were obtained from cells transfected with Ca_v2.1 either alone (a, n = 7) or with CaBP1 (b, n = 10). (c, d) Ca_v2.1 channel currents elicited by repetitive depolarizations. Test pulses (+20 mV (c) or +10 mV (d) to account for voltage shifts cause by Ba²⁺ substitution) at a frequency of 100 Hz were applied to cells transfected with Ca_v2.1 either alone (open circles) or along with CaBP1 (filled circles). Peak current amplitudes were normalized to the first pulse in the series and plotted against time during the train. Every second data point is shown. Intracellular recording solutions contained 0.5 mM EGTA, and bath solutions contained 10 mM Ca²⁺ (c) or Ba²⁺ (d). In (c), n = 9 for open circles; n = 13 for closed circles. In (d), n = 5 for open circles; n = 11 for closed circles.