Differential Regulation of PI(4,5)P2 Sensitivity of Kv7.2 and Kv7.3 Channels by Calmodulin

Figures

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  Figure 1. Characterization of calmodulin-dependent Kv7.2 potentiation. (A) Effect of CaM on Kv7.2 current density as a function of the amount of channel cDNA transfected. Mean current density (pA/pF) in cells transfected with channels only (black symbols) or together with 3 μg CaM cDNA per 35 mm dish (red symbols). (B) Effect of increasing CaM on Kv7.2 density. Half maximal CaM effect was obtained approximately when transfecting cells at a 1:2 (w/w) Kv7.2/CaM cDNA ratio. Inset: Representative current traces recorded from HEK293T cells transfected with 0.15 μg Kv7.2 cDNA and, where indicated, with 3 μg CaM cDNA, to illustrate current density quantification. Maximum current was measured at −30 mV as the difference in the amplitude after a pre-pulse to −110 mV to close all channels and after a prepulse to +30 mV to reach maximum Popen (arrow).

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  Figure 2. Effect of calmodulin on Kv7.2 current density. (A) Representative current traces measured in response to the indicated voltage protocol in cells expressing Kv7.2, Kv7.2 + CaM or Kv7.2 + CaM1234. (B) Comparison of the effect on current density of endogenous CaM (gray) WT CaM (red), CaM1234 (blue). (C) Voltage-dependence of activation measured under, resting (black) and elevated (red, CaMWT and blue CaM1234) CaM levels. The V1/2 (mV) under resting and elevated CaMWT and CaM1234 conditions were (number of experiments in brackets): −23.2 ± 0.8 (10), −29.4 ± 1.1 (8), and −39.4 ± 1.2 (8).

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  Figure 3. Effect of reduced calmodulin levels on Kv7.2 current density. (A) Representative current traces measured in response to the indicated voltage protocol in cells expressing Kv7.2 or Kv7.2 + neurogranin (sponge, green). (B) Comparison of the effect on current density of endogenous CaM (gray) and reduced CaM levels (+Sponge, Green). (C) The V1/2 (mV) under reduced and resting CaM conditions were (number of experiments in brackets): −27.8 ± 0.9 (8) and −15.6 ± 2.1 (8).

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  Figure 4. Effect of acute reduction of PI(4,5)P2 levels on calmodulin-dependent regulation of Kv7.2 current density. (A) Scheme of the voltage protocol used to illustrate current inhibition quantification. (B) Averaged current reduction after activation of DrVSP at +100 or +120 mV under resting conditions (gray symbols) and with CaM1234 (blue symbols). (C) Averaged current reduction in the presence of the CaM sponge (green symbols). (D) Comparison of current density with CaM1234 (blue) and the CaM-sponge (green) in cells co-expressing PIP5K (purple). (E) Averaged current reduction after activation of DrVSP in the presence PIP5K in control and cells co-expressing CaM1234 or the CaM-sponge (magenta lines).

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  Figure 5. The F24A mutation results in larger current density and increased resistance to acute PI(4,5)P2 depletion independently of CaM elevation. (A) Averaged current density of Kv7.2 channels (gray column), F24A mutation under resting conditions (orange) and co-expressing CaM1234 (blue). (B) Top, cartoon of a channel subunit. Bottom, sensitivity of the F24A mutant to DrVSP activation under resting conditions (orange), and with co-expression of CaM1234 (blue). The reference effect on wt Kv7.2 is plotted in gray color.

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  Figure 6. Effect of alterations of calmodulin levels on Kv7.2/3 current density. (A) Comparison of the effect on current density of CaM1234 (blue) and the CaM-sponge (green). (B) Activation voltage-dependency measured under reduced (green), resting (gray) and elevated (blue, CaM1234) CaM levels. The V1/2 (mV) under low, resting and elevated CaM conditions were (number of experiments in brackets): −26.5 ± 2.0 (7), −30.5 ± 1.2 (9), and −34.1 ± 1.7 (6).

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  Figure 7. Effect of alterations of PI(4,5)P2 levels on calmodulin-dependent regulation of Kv7.2/3 current density. (A) Comparison of the effect on current density of CaM1234 (blue) and the CaM-sponge (green) in cells co-expressing PIP5K (purple). (B) Averaged current reduction after activation of DrVSP at different voltages in resting conditions (gray symbols) and with CaM1234 (blue symbols). (C) Averaged current reduction after activation of DrVSP in resting conditions (gray symbols) and with a CaM sponge (green symbols).

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  Figure 8. Effect of calmodulin on Kv7.3T current density. (A) Comparison of the effect on current density CaM1234 (blue) and the CaM-sponge (green) on Kv7.3T. (B) Activation voltage-dependency measured under reduced (green), resting (gray) and elevated (blue, CaM1234) CaM levels. The V1/2 (mV) under low, resting and elevated CaM conditions were (number of experiments in brackets): −41 ± 1.1 (10), −34 ± 1.4 (11), and −49 ± 1.1 (10).

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  Figure 9. Effect alterations of PI(4,5)P2 levels on calmodulin-dependent regulation of Kv7.3T current density. (A) Comparison of the effect on current density of CaM1234 (blue) and the CaM-sponge (green) in cells co-expressing PIP5K (purple). (B) Averaged current reduction after activation of DrVSP at different voltages in resting conditions (gray symbols) and with CaM1234 (blue symbols). (C) Averaged current reduction after activation of DrVSP in resting conditions (gray symbols) and with a CaM sponge (green symbols).

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  Figure 10. Model of apocalmodulin regulation of Kv7 PI(4,5)P2 efficacy. Illustration of the proposed equilibrium between three functional Kv7 channels pools. The PI(4,5)P2 efficacy depends upon the abundance of apoCaM and subunit composition. Pool 1 and pool 3 represent the currents observed after expression of a CaM sponge or overexpression of CaM1234, respectively.