Modulation of CaV2.1 channels by the neuronal calcium-binding protein visinin-like protein-2

Abstract

CaV2.1 channels conduct P/Q-type Ca2+ currents that are modulated by calmodulin (CaM) and the structurally related Ca2+-binding protein 1 (CaBP1). Visinin-like protein-2 (VILIP-2) is a CaM-related Ca2+-binding protein expressed in the neocortex and hippocampus. Coexpression of CaV2.1 and VILIP-2 in tsA-201 cells resulted in Ca2+ channel modulation distinct from CaM and CaBP1. CaV2.1 channels with beta2a subunits undergo Ca2+-dependent facilitation and inactivation attributable to association of endogenous Ca2+/CaM. VILIP-2 coexpression does not alter facilitation measured in paired-pulse experiments but slows the rate of inactivation to that seen without Ca2+/CaM binding and reduces inactivation of Ca2+ currents during trains of repetitive depolarizations. CaV2.1 channels with beta1b subunits have rapid voltage-dependent inactivation, and VILIP-2 has no effect on the rate of inactivation or facilitation of the Ca2+ current. In contrast, when Ba2+ replaces Ca2+ as the charge carrier, VILIP-2 slows inactivation. The effects of VILIP-2 are prevented by deletion of the CaM-binding domain (CBD) in the C terminus of CaV2.1 channels. However, both the CBD and an upstream IQ-like domain must be deleted to prevent VILIP-2 binding. Our results indicate that VILIP-2 binds to the CBD and IQ-like domains of CaV2.1 channels like CaM but slows inactivation, which enhances facilitation of CaV2.1 channels during extended trains of stimuli. Comparison of VILIP-2 effects with those of CaBP1 indicates striking differences in modulation of both facilitation and inactivation. Differential regulation of CaV2.1 channels by CaM, VILIP-2, CaBP1, and other neurospecific Ca2+-binding proteins is a potentially important determinant of Ca2+ entry in neurotransmission.

Figure 1.

Effect of coexpression of VILIP-2 on Ca_V2.1 channels. A, Voltage dependence of activation. Ca_V2.1/β_2a channels was activated by 5 ms pulses to the indicated potentials from a holding potential of -80 mV, and Ca²⁺ tail currents were recorded after repolarization to -40 mV without (gray) or with (black) coexpression of VILIP-2 (±SEM; n ≥ 10). B, I_Ca was evoked by 1 s depolarizing test pulses to +10 mV from a holding potential of -80 mV in tsA-201 cells expressing Ca_V2.1/β_2a channels without (gray) or with (black) VILIP-2. Current records were normalized to the peak inward current and averaged (±SEM; n ≥ 10). C, I_Ba was evoked and analyzed as in B. D, I_Ca was evoked and analyzed as in B for Ca_V2.1ΔCBD/β_2a. E, I_Ca conducted by Ca_V2.1/β_1b channels was evoked and analyzed as in B. F, I_Ba conducted by Ca_V2.1/β_1b channels was evoked and analyzed as in B. G, I_Ba conducted by Ca_V2.1ΔCBD/β_1b channels was evoked and analyzed as in B. H, I, Residual current amplitude at the end of the 1 s pulse (I_Res) was divided by peak current from cells transfected with Ca_V2.1 alone (gray) or with VILIP-2 (black) for the indicated experimental conditions. WT, Wild type. Error bars represent SEM.

Figure 2.

Effect of coexpression of VILIP-2 on paired-pulse facilitation of Ca_V2.1/β_2a channels. Test pulses to potentials ranging from -40 to +80 mV were applied without a prepulse (open symbols) or with a 50 ms prepulse to +10 mV and an 8 ms period at -80 mV before the test pulse (filled symbols). A, Peak inward calcium currents for cells expressing Ca_V2.1/β_2a channels without (gray circles) or with (black squares) VILIP-2. B, Same protocol as that in A, with 10 mm barium as the charge carrier. Insets, Representative calcium or barium currents at 70 mV. Calibration: horizontal, 2 ms; vertical, A, B, left insets, 1 nA; A, B, right insets, 500 pA. Error bars represent SEM.

Figure 3.

Effect of VILIP-2 on facilitation and inactivation of Ca_V2.1 channels during trains of repetitive depolarizations. Test pulses to +10 mV (0 mV for I_Ba) for 5 ms at a frequency of 100 Hz were applied to transfected tsA-201 cells expressing Ca_V2.1 channels only (gray) or Ca_V2.1 channels plus VILIP-2 (black). Peak current amplitudes were normalized to the first pulse in the series and plotted against time (±SEM; n ≥ 10; every 10th SEM is plotted). A, Representative I_Ca measured for the first pulse, the pulse at 355 ms, and the last pulse in a train of stimuli for Ca_V2.1/β_2a alone. B, Representative I_Ca measured for the first pulse, the pulse at 355 ms, and the last pulse in a train of stimuli for Ca_V2.1/β_2a plus VILIP-2. C, I_Ca, Ca_V2.1/β_2a. D, I_Ca, Ca_V2.1/β_1b. E, I_Ba,Ca_V2.1/β_2a. F, I_Ba, Ca_V2.1/β_1b. Error bars represent SEM.

Figure 4.

Effect of VILIP-2 on Ca_V2.1/β_2a channels after trains of repetitive depolarizations. Ca_V2.1/β_2a channel currents were evoked by 1 s test pulses to +10 mV before (gray) or after (black) a 600 ms train of 5 ms depolarizations to +20 mV (+10 mV for I_Ba) at 100 Hz. A 2 min interval was maintained between applications of this protocol to allow the effects of the previous stimuli to return to baseline. Current amplitudes were normalized to the peak current of the first test pulse. Mean normalized currents during test pulse 1 (gray) and test pulse 2 (black) are overlaid. A, I_Ca, Ca_V2.1/β_2a alone. B, I_Ba, Ca_V2.1/β_2a alone. C, I_Ca, Ca_V2.1/β_2a plus VILIP-2. D, I_Ba, Ca_V2.1/β_2a plus VILIP-2. E, Bar graph presenting facilitation as the pulse ratio (P2/P1) at the time of the peak current in each panel (mean ± SEM). Error bars represent SEM.

Figure 5.

Effect of VILIP-2 on Ca_V2.1/β_1b channels after trains of repetitive depolarizations. Ca_V2.1/β_1b channel currents were evoked by 1 s test pulses to +10 mV before (gray) or after (black) a 600 ms train of repetitive depolarization to +20 mV (+10 mV for I_Ba) for 5 ms at 100 Hz. A 2 min interval was maintained between applications of this protocol to allow the effects of the previous stimuli to return to baseline. Current amplitudes were normalized to the peak current of the first test pulse. Mean normalized currents during test pulse 1 (gray) and test pulse 2 (black) are overlaid. A, I_Ca, Ca_V2.1/β_1b alone. B, I_Ba, Ca_V2.1/β_1b alone. C, I_Ca, Ca_V2.1/β_1b plus VILIP-2. D, I_Ba, Ca_V2.1/β_1b plus VILIP-2. E, Bar graph presenting facilitation as the pulse ratio (P2/P1) at the time of the peak current in each panel (mean ± SEM). Error bars represent SEM.

Figure 6.

Association of VILIP-2 with Ca_V2.1 channels via the IQ-like domain and CBD. Transfected cells were cross-linked in situ with DSP, as described in Materials and Methods. A, Lysates from cells transfected with Ca_V2.1 plus VILIP-2 myc or Ca_V2.1ΔCBD plus VILIP-2 myc were subjected to immunoprecipitation by anti-Ca_V2.1 antibodies or control IgG as indicated in the presence of 10 mm EGTA. B, Lysates from cells transfected with Ca_V2.1, Ca_V2.1/1965ST, Ca_V2.1/IM-AA, and Ca_V2.1ΔCBD/IM-AA plus VILIP-2 myc were subjected to immunoprecipitation by anti-Ca_V2.1 antibodies or control IgG in the presence of 10 mm EGTA. Blots were probed with either anti-Ca_V2.1 (top) or anti-myc (bottom). WT, Wild type.

Figure 7.

Effect of CaBP1 on Ca_V2.1/β_1b channels. A, Average, smoothed I_Ca conducted by Ca_V2.1 channels evoked by 1 s depolarizing pulses from a holding potential of -80 mV to +30 mV without (gray) or with (black) coexpression of CaBP1 (±SEM; n = 5-7). B, From a holding potential of -80 mV, test pulses to +10 mV for 5 ms at a frequency of 100 Hz were applied to transfected tsA-201 cells expressing Ca_V2.1 channels alone (gray) or Ca_V2.1 channels plus CaBP1 (black). Peak current amplitudes were normalized to the first pulse in the series and plotted against time of stimulation (±SEM; n = 8-10; every 10th SEM is plotted). C, Averaged, smoothed I_Ba was measured as in A with a test pulse to +20 mV for Ca_V2.1/β_1b channels without (gray) or with (black) coexpression of CaBP1 (±SEM; n = 5-8). D, From a holding potential of -80 mV, test pulses to 0 mV for 5 ms at a frequency of 100 Hz were applied to transfected tsA-201 cells expressing Ca_V2.1/β_1b channels without (gray) or with (black) coexpression of CaBP1. Peak current amplitudes were normalized to the first pulse in the series and plotted against time (±SEM; n = 6-8; every 10th SEM is plotted). Error bars represent SEM.

Figure 8.

Comparison of the structures and modulatory effects of CaM, VILIP-2, and CaBP1. A, Schematic illustration of the structures of CaM, CaBP1, and VILIP-2. EF-hands that are active in binding Ca²⁺ are indicated in gray, and inactive EF-hands are indicated in white. Circles denote the central α helix connecting EF-hands 2 and 3. Bent lines indicate N-terminal myristoylation. The lengths of the line segments approximately correspond to the length of the amino acid sequences. B, Ca²⁺-dependent inactivation of Ca_V2.1/β_2a channels with CaM, CaBP1, or VILIP-2. Overlapped Ca²⁺ and Ba²⁺ currents are plotted. The shaded area indicates the Ca²⁺-dependent increase in inactivation caused by CaM. C, Ca²⁺-dependent facilitation and inactivation of Ca_V2.1/β_2a channels with CaM, CaBP1, or VILIP-2. The CaM and CaBP1 data in B and C were modified from Lee et al. (2000) and (2002), respectively. Error bars represent SEM.