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, 123 (5), 555-71

Modulation of the Voltage Sensor of L-type Ca2+ Channels by Intracellular Ca2+

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Modulation of the Voltage Sensor of L-type Ca2+ Channels by Intracellular Ca2+

Dmytro Isaev et al. J Gen Physiol.

Abstract

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.

Figures

F<sc>igure</sc> 1.
Figure 1.
Ca2+ load through the patch pipette blocked Ca2+ current through L-type Ca2+ channels and reduced their OFF gating current. Current traces were elicited by voltage pulses from the holding potential from −90 to 20 mV. Pipette solution had 0.3 mM of free Ca2+ (0.3 Ca, Table I), and bath solution was 0 Na.
F<sc>igure</sc> 2.
Figure 2.
Comparison of Ca2+ current magnitude and availability of the intramembrane charge movement measured in a double-pulse experiment. (A) Currents elicited by the pulse protocol shown at the top were recorded in a cell loaded with Ca2+ through the patch pipette. Pipette solution had 0.1 mM of free Ca2+ (0.1 Ca), and bath solution was 0 Na. The time after establishing the whole-cell recording configuration is indicated near traces. Ca2+ current at the first pulse to +20 mV and gating currents elicited by the second pulse to the reversal potential of ionic currents were decreasing in parallel during the course of experiment. (B) Currents obtained similarly to those shown in A, but in a cell recorded with 1 EGTA pipette solution. While Ca2+ currents at the first pulse to +20 mV slowly run-down, gating currents at the second pulse to the reversal potential did not change. (C) Correlation between the magnitude of Ca2+ current and the gating charge during the second pulse for the cell shown in A. The gating charge was obtained by integrating the ON transient during the second pulse, as described in the text. Linear regression is shown by the thin line, the dashed lines show 99% prediction interval. (D) Correlation between the magnitude of Ca2+ current and the gating charge during the second pulse for the cell shown in B. Lines are as in C.
F<sc>igure</sc> 3.
Figure 3.
Effects of manipulations of intracellular Ca2+ by sodium–calcium exchanger (NCX) on L-type Ca2+ channels. Voltage pulses were from −90 to 20 mV. (A) Activation of NCX reverses the inhibition of Ca2+ current by intracellular Ca2+. Pipette solution was 0.3 Ca. When bathing solution was changed from 0 Na (indicated by open boxes) to 150 Na (indicated by gray boxes), NCX reduced local [Ca2+]. (B) NCX functioning in the reverse mode reversibly blocks Ca2+ currents. Pipette solution: 0 Ca, 20 Na. When bathing solution was changed from 150 Na to 0 Na, NCX increased local [Ca2+].
F<sc>igure</sc> 4.
Figure 4.
Intracellular Ca2+ loaded through NCX reversibly blocked Ca2+ currents and reduced the OFF gating current transients. Voltage pulses were from −90 to 20 mV. Pipette solution was 0 Ca, 20 Na. (A) Protocol of application of bath solutions (boxes) and amplitudes of Ca2+ currents (dots). When bathing solution was changed from 150 Na (gray boxes) to 0 Na (open boxes), NCX functioning in the reversed mode increased local [Ca2+]. When bathing solution was 150 Na, Gd, Ca2+ currents were blocked. (B) Current traces recorded at times indicated by corresponding numbers in A.
F<sc>igure</sc> 5.
Figure 5.
OFF gating currents elicited by repolarizations to −90 mV were reduced by intracellular Ca2+. Bathing solution: Gd. Pipette solutions are indicated in C. (A) Representative tracings for pulses to −40, 0, 40, and 80 mV. (B) Average ON charge transfer functions obtained with different pipette solutions, as illustrated in A. The averaging procedure is described in the text. Different symbol sizes correspond to different [Ca2+], as listed in C. Only the maximal SEM value is shown for clarity. The curves are single Boltzmann fits. V1/2 was: 15 ± 3 mV (1 EGTA), 13 ± 4 mV (0.01 Ca), 7 ± 3 mV (0.1 Ca), and −5 ± 5 mV (0.3 Ca); K was 25 ± 3 mV. Each group had n = 6 cells. (C) Average OFF charge transfer functions. The dashed lines are the fits to the ON charge transfer from B. (D) The difference between ON and OFF charge transfer functions. The solid lines are single Boltzmann fits. V1/2 was: 31 ± 12 mV, and K was 11 ± 3 mV for all fits.
F<sc>igure</sc> 6.
Figure 6.
Combination of high intracellular [Ca2+] and a brief depolarization caused a large negative shift of the voltage distribution of the intramembrane charge movement in L-type Ca2+ channels. (A) Representative tracings of the OFF charge for repolarizations to 40, −40, −120, and −200 mV applied after a 20-ms prepulse to 20 mV. Bathing solution: Gd. Pipette solutions are indicated. (B) Average OFF charge distributions obtained with 1 EGTA (open circles), or 0.3 Ca (filled circles) pipette solutions, as illustrated in A. The curves are single-Boltzmann fits. In 1 EGTA solution: V1/2 = −27 ± 2 mV, K = 27 ± 3 mV, n = 6. With 0.3 Ca solution: V1/2 = −116 ± 6 mV, K = 29 ± 5 mV, n = 4.
F<sc>igure</sc> 7.
Figure 7.
Intracellular Ca2+ accelerates inactivation of gating currents. Bathing solution: Gd. Pipette solutions are indicated. (A) Representative OFF gating current traces elicited by repolarizations to −60 mV are spaced according to the time of conditioning at 20 mV. (B) Average charge transferred during the OFF transients plotted versus logarithm of conditioning duration. Different symbols correspond to different [Ca2+] in the pipette. Charge transfers are normalized to the maximal value in each cell and averaged, n = 4 for 0.3 Ca, n = 7 for other concentrations. The maximal values were achieved at 10 ms with 1 EGTA and 0.01 Ca, at 5 ms with 0.1 Ca, and at 2.5 ms with 0.3 Ca pipette solutions. Only the maximal SEM value is shown for clarity. Curves are single-exponential fits. The time constants were: 670 ± 47 ms in 1 EGTA, 280 ± 35 ms in 0.01 Ca, 77 ± 12 ms in 0.1 Ca, and 8 ± 2 ms in 0.3 Ca.
F<sc>igure</sc> 8.
Figure 8.
The “IQ-AA” mutant, in which Ca2+/calmodulin does not accelerate inactivation of Ca2+ current, was affected in the same manner as the wild-type channel. Both ionic and OFF gating currents were reduced by the Ca2+ load. Experimental protocol was as in Fig. 3 B.
F<sc>igure</sc> 9.
Figure 9.
Ca2+ current promotes inactivated states of the voltage sensor in the Δ1733 deletion mutant channels. (A) Pulse protocols used to monitor availability of ionic currents (current trace 1, test pulse from −150 to 20 mV) and the intramembrane charge movement in inactivated channels (current trace 2, test pulse from −150 to −50 mV). Conditioning pulses were from −90 mV to Vcond. Pipette solution: 1 EGTA. (B) Test currents recorded as shown in A. Conditioning voltages are indicated by the figures above the traces. Traces 3 (test voltage 20 mV) and 4 (test voltage −150 mV) were recorded with 0 Na bath solution. Traces 5 (test voltage −150 mV) were recorded in the same cell after the blockade of ionic currents by Gd bath solution.
F<sc>igure</sc> 10.
Figure 10.
Comparison of the reduction of calcium current and the availability of charge movement from −150 to −50 mV after conditioning at different voltages, measured as in Fig. 9. (A) Current inactivation (circles) and charge 2 (filled bars) in the Δ1733 mutant. Current inactivation was measured as the reduction of the magnitude of test calcium current relative to the magnitude without conditioning (Vcond = −90 mV), charge 2 was measured as the difference between charge transfers at −50 mV obtained with and without (Vcond = −90 mV) conditioning. Before averaging, the increase of the charge movement between −150 and −50 mV, was related to the maximal charge transfer in each cell. The maximal charge transfer was obtained in the Gd bath solution by pulsing from −150 to 60 mV. In the Δ1733 mutant, the charge movement recorded in 0 Na solution (filled bars) was significantly increased by conditioning pulses more positive than −20 mV (P < 0.05, unpaired Student's t test). The charge movement in Gd solution (gray bars) was significantly increased only after 100 mV conditioning. (B) Current inactivation (circles) and charge 2 (bars) in wild-type channels. Current inactivation was similar to that in the Δ1733 mutant, but there was no significant change of charge movement between −150 and −50 mV (0 Na bath solution, open bars).

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