Swain SM et al. (2015), Ca(2+)/calmodulin regulates Kvβ1.1-mediated ina...

XB-ART-53370

Sci Rep 2015 Jan 12;5:15509. doi: 10.1038/srep15509.

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Ca(2+)/calmodulin regulates Kvβ1.1-mediated inactivation of voltage-gated K(+) channels.

Swain SM , Sahoo N , Dennhardt S , Schönherr R , Heinemann SH .

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A-type K(+) channels open on membrane depolarization and undergo subsequent rapid inactivation such that they are ideally suited for fine-tuning the electrical signaling in neurons and muscle cells. Channel inactivation mostly follows the so-called ball-and-chain mechanism, in which the N-terminal structures of either the K(+) channel's α or β subunits occlude the channel pore entry facing the cytosol. Inactivation of Kv1.1 and Kv1.4 channels induced by Kvβ1.1 subunits is profoundly decelerated in response to a rise in the intracellular Ca(2+) concentration, thus making the affected channel complexes negative feedback regulators to limit neuronal overexcitation. With electrophysiological and biochemical experiments we show that the Ca(2+) dependence is gained by binding of calmodulin to the "chain" segment of Kvβ1.1 thereby compromising the mobility of the inactivation particle. Furthermore, inactivation regulation via Ca(2+)/calmodulin does not interfere with the β subunit's enzymatic activity as an NADPH-dependent oxidoreductase, thus rendering the Kvβ1.1 subunit a multifunctional receptor that integrates cytosolic signals to be transduced to altered electrical cellular activity.

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	Figure 1. Whole-cell recordings of Kv1.1 currents from HEK 293T cells.(a) Part of the N-terminal sequence of rat Kvβ1.1 protein with score pattern resulting from a search for potential calmodulin binding sites according to Mruk et al. (2014)25; the tallest bar refers to a score value of 9. Within this motif, either the marked arginine residues (RRR) or both phenylalanines (FF) were mutated to asparagine and serine, respectively. (b) Kv1.1 channels were coexpressed with Kvβ1.1 wild type (wt) or mutants RRR and FF in HEK 293T cells; currents were measured upon depolarization to 50 mV. The pipette solution contained 100 μM EGTA. Current traces for the indicated Kvβ1.1 subunits before (black) and after (red) extracellular application of 1 μM ionomycin. The grey trace in the left panel (Ctrl) indicates Kv1.1 currents without Kvβ subunits. (c) Inactivation time constants, based on single-exponential fits from data as shown in panel b. (d) Fractional change in peak current at 50 mV upon ionomycin application. Data in c and d are mean ± s.e.m. with n indicated in parentheses. Two-sided paired t-test between control and ionomycin application in c, Wilcoxon signed rank test in d: *P < 0.001, P < 0.01, *P < 0.05.
	Figure 2. Whole-cell recordings of Kv1.4 current from HEK 293T cells.(a) Kv1.4 channels were expressed in HEK 293T cells alone (left) or in combination with Kvβ1.1-C7S (center) or mutant Kvβ1.1-C7S-RRR (right); currents were measured upon depolarization to 50 mV before (black) and after (red) extracellular application of 1 μM ionomycin. The pipette solution contained 100 μM EGTA. (b) Inactivation time constants, based on single-exponential fits from data as shown in panel a. (c) Fractional change in peak current at 50 mV upon ionomycin application. Data in b and c are mean ± s.e.m. with n indicated in parentheses. Two-sided paired t-test between control and ionomycin application in b, Wilcoxon signed rank test in c: *P < 0.01; P < 0.05.
	Figure 3. Calmodulin (CaM) is required for Ca2+-dependent inactivation mediated by Kvβ1.1.(a–d) Inside-out patch recordings from Xenopus oocytes coexpressing Kv1.1 with Kvβ1.1 wild type (wt) (a,b) and mutants RRR (c) and FF (d). Current traces were obtained at 50 mV. The bath solution facing the intracellular side contained no Ca2+ and no CaM (Ctrl, black), 1 μM Ca2+ (green), 1 μM CaM (blue), or 1 μM Ca2+ plus 1 μM CaM (red). (e) Relative change in fast inactivation time constant for Kvβ1.1 wild type and the mutants for the indicated application of intracellular Ca2+ and CaM. Data are mean ± s.e.m. with n indicated in parentheses. Two-sided paired t-test between control and ionomycin application: **P < 0.01. The intracellular solution contained 1 mM glutathione (reduced). (f) SDS PAGE of the GST-pull-down assay to test for binding of recombinant CaM to sepharose-bound GST alone (GST) or GST-fused Kvβ1.1 variants (wt, RRR, FF). Precipitation of CaM was only observed with GST-fused wild-type Kvβ1.1 in the presence of Ca2+ ions (arrow). GST fusions of the Kvβ1.1 mutants RRR or FF did not co-precipitate CaM. The lane labeled “CaM” shows recombinant CaM as a control.
	Figure 4. Enzymatic activity of Kvβ1.1-C7S variants.(a) Superposition of current traces at 50 mV of Kv1.1 coexpressed in HEK 293T cells with Kvβ1.1-C7S (wt-C7S) or its mutants RRR and FF right after establishment of the whole-cell configuration (black) and about 150 s thereafter (blue). The pipette solution contained 1 mM of the substrate 4CY. (b) As in a, but with mutant Kvβ1.1-C7S-E349K. (c) Relative change in peak current from experiments as in a and b for Kv1.1 (no β) and coexpression with the indicated variants of Kvβ1.1-C7S. (d) Superposition of current traces at 50 mV for Kv1.1 plus Kvβ1.1-C7S-E349K before (black) and after extracellular application of 1 μM ionomycin (red). (e) Fold change of peak current upon ionomycin application for Kv1.1 (no β) and coexpression with the indicated variants of Kvβ1.1-C7S. The pipette solution contained 100 μM EGTA. Data in c and e are mean ± s.e.m. with n indicated in parentheses. Deviation from unity was tested with a Wilcoxon signed rank test: *P < 0.01, P < 0.05.

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	Figure 1. Whole-cell recordings of Kv1.1 currents from HEK 293T cells.(a) Part of the N-terminal sequence of rat Kvβ1.1 protein with score pattern resulting from a search for potential calmodulin binding sites according to Mruk et al. (2014)25; the tallest bar refers to a score value of 9. Within this motif, either the marked arginine residues (RRR) or both phenylalanines (FF) were mutated to asparagine and serine, respectively. (b) Kv1.1 channels were coexpressed with Kvβ1.1 wild type (wt) or mutants RRR and FF in HEK 293T cells; currents were measured upon depolarization to 50 mV. The pipette solution contained 100 μM EGTA. Current traces for the indicated Kvβ1.1 subunits before (black) and after (red) extracellular application of 1 μM ionomycin. The grey trace in the left panel (Ctrl) indicates Kv1.1 currents without Kvβ subunits. (c) Inactivation time constants, based on single-exponential fits from data as shown in panel b. (d) Fractional change in peak current at 50 mV upon ionomycin application. Data in c and d are mean ± s.e.m. with n indicated in parentheses. Two-sided paired t-test between control and ionomycin application in c, Wilcoxon signed rank test in d: *P < 0.001, P < 0.01, *P < 0.05.
	Figure 2. Whole-cell recordings of Kv1.4 current from HEK 293T cells.(a) Kv1.4 channels were expressed in HEK 293T cells alone (left) or in combination with Kvβ1.1-C7S (center) or mutant Kvβ1.1-C7S-RRR (right); currents were measured upon depolarization to 50 mV before (black) and after (red) extracellular application of 1 μM ionomycin. The pipette solution contained 100 μM EGTA. (b) Inactivation time constants, based on single-exponential fits from data as shown in panel a. (c) Fractional change in peak current at 50 mV upon ionomycin application. Data in b and c are mean ± s.e.m. with n indicated in parentheses. Two-sided paired t-test between control and ionomycin application in b, Wilcoxon signed rank test in c: *P < 0.01; P < 0.05.
	Figure 3. Calmodulin (CaM) is required for Ca2+-dependent inactivation mediated by Kvβ1.1.(a–d) Inside-out patch recordings from Xenopus oocytes coexpressing Kv1.1 with Kvβ1.1 wild type (wt) (a,b) and mutants RRR (c) and FF (d). Current traces were obtained at 50 mV. The bath solution facing the intracellular side contained no Ca2+ and no CaM (Ctrl, black), 1 μM Ca2+ (green), 1 μM CaM (blue), or 1 μM Ca2+ plus 1 μM CaM (red). (e) Relative change in fast inactivation time constant for Kvβ1.1 wild type and the mutants for the indicated application of intracellular Ca2+ and CaM. Data are mean ± s.e.m. with n indicated in parentheses. Two-sided paired t-test between control and ionomycin application: **P < 0.01. The intracellular solution contained 1 mM glutathione (reduced). (f) SDS PAGE of the GST-pull-down assay to test for binding of recombinant CaM to sepharose-bound GST alone (GST) or GST-fused Kvβ1.1 variants (wt, RRR, FF). Precipitation of CaM was only observed with GST-fused wild-type Kvβ1.1 in the presence of Ca2+ ions (arrow). GST fusions of the Kvβ1.1 mutants RRR or FF did not co-precipitate CaM. The lane labeled “CaM” shows recombinant CaM as a control.
	Figure 4. Enzymatic activity of Kvβ1.1-C7S variants.(a) Superposition of current traces at 50 mV of Kv1.1 coexpressed in HEK 293T cells with Kvβ1.1-C7S (wt-C7S) or its mutants RRR and FF right after establishment of the whole-cell configuration (black) and about 150 s thereafter (blue). The pipette solution contained 1 mM of the substrate 4CY. (b) As in a, but with mutant Kvβ1.1-C7S-E349K. (c) Relative change in peak current from experiments as in a and b for Kv1.1 (no β) and coexpression with the indicated variants of Kvβ1.1-C7S. (d) Superposition of current traces at 50 mV for Kv1.1 plus Kvβ1.1-C7S-E349K before (black) and after extracellular application of 1 μM ionomycin (red). (e) Fold change of peak current upon ionomycin application for Kv1.1 (no β) and coexpression with the indicated variants of Kvβ1.1-C7S. The pipette solution contained 100 μM EGTA. Data in c and e are mean ± s.e.m. with n indicated in parentheses. Deviation from unity was tested with a Wilcoxon signed rank test: *P < 0.01, P < 0.05.