Synergistic interplay of Gβγ and phosphatidylinositol 4,5-bisphosphate dictates Kv7.4 channel activity

Figures

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  Fig. 1. PIP2 depletion reduces Kv7.4 currents. A Examples of whole cell K+ currents from HEK293 Kv7.4 cells evoked by step depolarisation from −60 mV to +20 mV in the absence (a) and presence of 20 μM wortmannin (b). Currents were recorded every 15 s and wortmannin applied after 60 s. Initial current trace is shown in black in both subpanels. Subsequent traces after 5-, 10- and 20-min intervals are shown in blue, green and red, correspondingly. Subpanel c shows the mean amplitude of K+ current at +20 mV in the absence (black) and presence of wortmannin (red). Each point is the mean ± s.e.m. of four cells. B Representative traces of Kv7.4 currents evoked by steps from −60 mV to a range of potential (−90 to +40 mV) in control (a) and after depletion of PIP2 by the cells preincubation with wortmannin + short (≤30 s) application of trypsin (b). The mean data are shown in subpanel c with control (black, n = 22), wortmannin alone (green, n = 34), wortmannin plus trypsin (red, n = 36) and linopirdine (purple, n = 14). C Example of cell-attached patch recording from HEK293 Kv7.4 cell showing the effect of 20 μM wortmannin. Long-term trace is shown in subpanel a. Representative expanded 1.75-s…

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  Fig. 2. PIP2 depletion prevents activation of whole-cell Kv7.4 currents by Gβγ. A Representative whole-cell currents evoked by depolarisation from −60 to +20 mV in Kv7.4 HEK293 cells under control conditions (a) and after incubation in wortmannin (20 μM, b). Right-hand panels show cells perfused internally with Gβγ. B Mean data for the effect of intracellular perfused active Gβγ on whole-cell currents recorded at +20 mV in the absence and presence of 20 μM wortmannin (n = 5)

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  Fig. 3. Gβγ enhancement of Kv7.4 channels in excised patches is prevented by PIP2 depletion. A Representative inside-out patch recording showing stimulatory action of Gβγ. Expanded 1.75 s segments of channel openings in the absence (b) and presence of 2 ng/ml Gβγ (c) are taken from long-term recording (a). Closed state and multiple open states are denoted by C and O1–O2. B Mean concentration effect for Gβγ subunits (n = 4–7) fitted with a best-fit sigmoidal to the available data. C Representative inside-out patch recording from cell preincubated with wortmannin before (b) and after (c) Gβγ application. Subpanels b and c are expanded 2.5-s segments of channel openings taken from long-term recording (a). Closed state and multiple open states are denoted by C and O1–O2. D, Representative inside-out patch recording from cell preincubated with wortmannin before (b) and after (c) PIP2 application. Subpanels b and c are expanded 1.35-s segments of channel openings taken from long-term recording (a). Closed state and open states are denoted by C and O1

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  Fig. 4. Gβγ inhibition abolishes Kv7.4 currents without change in PIP2 levels. A In-cell Western analysis showing influence of Gβγ inhibitors on PIP2 level in HEK293 Kv7.4 cells (n = 11–22). B Mean I-V relationships of HEK293 Kv7.4 currents evoked from holding potential −60 mV before and after application of M201 (50 μM). C Mean data for the effect for mechanistically different inhibitors of Gβγ on whole-cell K+ currents recorded at +20 mV (n = 5–7). D Representative inside-out patch recording showing inhibitory action of 50 μM M201 on single channel activity. Subpanels b and c are representative 1.5-s segments of channel openings before and after drug application taken from long-term recording (a). Closed and open states are denoted by C and O1. E Representative inside-out patch recording showing inhibitory action of 10 μM Grk2i on single channel activity. Subpanels b and c are expanded 2.8-s segments of channel openings before and after drug application taken from long-term recording (a). Closed state and multiple open states are denoted by C and O1–O3. F Mean apparent open probability for Kv7.4 channels in control conditions (green column, n = 14) and after application of three…

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  Fig. 5. Gβγ inhibition prevents activation of Kv7.4 channels by exogenous PIP2. A Representative inside-out patch recording showing stimulatory action of PIP2. Subpanels b and c are 1.75-s segments of channel openings in the absence (b) and presence of PIP2 (100 μM, c) taken from long-term recording (a). Closed state and multiple open states are denoted by C and O1–O3. B Mean concentration-effect for PIP2 (n = 4–11). C Representative inside-out patch recording showing that in the continued presence of 50 μM M201 (continuation of recording from patch shown in Fig. 4D), 100 μM PIP2 applied to the patch failed to activate the channels. Subpanel b is a 1.5-s segment of channel openings taken from long-term recording (a). Closed state and multiple open states are denoted by C and O1. D Representative inside-out patch recording showing that in the continued presence of 100 μM gallein, 100 μM PIP2 applied to the patch failed to activate the channels. Subpanel b is a 2.5-s segment of channel openings taken from long-term recording (a). E Representative inside-out patch recording showing that in the continued presence of 100 μM Grk2i (continuation of recording from patch shown in Fig. 4E), 100 μM…

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  Fig. 6. PIP2 and Gβγ are synergistic regulators of Kv7.4 channels. A Representative inside-out patch recording showing lack of effect of 1 ng/ml Gβγ on channel activity. Subpanel b is an expanded 1.65-s segment of channel openings taken from long-term recording (a). Closed and open states are denoted by C and O1. B Application of 3 μM PIP2 in the presence of 1 ng/ml Gβγ (patch from (A)) significantly increased channel activity. Subpanel b is an expanded 1.65-s segment of channel openings taken from long-term recording (a). Closed state and multiple open states are denoted by C and O1–O3. C Mean apparent open probability for Kv7.4 in control conditions (n = 16), after application of low concentrations of Gβγ (n = 8) and PIP2 alone (n = 8) and in combination (n = 6). D Mean apparent open probability for Kv7.4 activated by PIP2 alone (n = 4–11, dark blue columns) and in the presence of 1 ng/ml Gβγ (cyan columns, n = 4–10)

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  Fig. 7. Effect of Kv7 channel openers in PIP2- and Gβγ-depleted cells. Mean data showing action of Retigabine, S-1 and NS15370 in HEK293 Kv7.4 cells under various conditions (control, preincubated with wortmannin alone, preincubated with wortmannin + short (≤30 s) application of trypsin, preincubated with gallein). Currents after treatment with pan-Kv7 channel blocker linopirdine are shown for reference. *, **, *** and **** denote p < 0.05 – p < 0.0001, respectively, compared to controls