A unique mechanism of inactivation gating of the Kv channel family member Kv7.1 and its modulation by PIP2 and calmodulin

Fig. 1. Removal of external Ca2+ elicits a large, voltage-dependent slow inactivation in WT Kv7.1 channels. (A and B) Representative traces of WT Kv7.1 currents recorded from transfected CHO cells with pipette solution containing 5 mM BAPTA in nominally zero free Ca2+ concentration. The concentrations of Ca2+ and Mg2+ of the extracellular solutions are indicated in the panels. From a holding potential of −90 mV, cells were stepped for 3 s from −60 to +60 mV in 10 mV of increments and repolarized for 1.5 s to −60 mV. (C) The percentage of visible voltage-dependent inactivation was quantified from at +60 mV. Results are expressed as means ± SEM. In external solutions containing 1.8 mM Ca2+/1.2 mM Mg2+ (n = 10), 0 mM Ca2+/0 mM Mg2+ (n = 5), 0 mM Ca2+/1.2 mM Mg2+ (n = 13), 0 mM Ca2+/3 mM Mg2+ (n = 4), and 3 mM Ca2+/0 mM Mg2+ (n = 4), the inactivation was, respectively, 8 ± 3, 62 ± 7, 41 ± 4, 35 ± 3, and 0 ± 0% [one-way analysis of variance (ANOVA), F(4, 31) = 31.20, ****P < 0.0001; Dunnett’s multiple comparisons test, adjusted ****P < 0.0001 and **P = 0.0013, respectively]. (D) Normalized conductance-voltage relations of WT Kv7.1 currents in 1.8 mM Ca2+/1.2 mM Mg2+, V50 = −25.8 ± 1.5 mV, and…

Fig. 2. Effects of external Ca2+ removal on inactivation of the Kv7.1 pore helix mutant V307L. (A) Mapping the pore helix residue V307 onto the human Kv7.1 cryo-EM structure (PDB: 6UZZ). SF, selectivity filter. (B) Representative traces of WT Kv7.1 and V307L mutant currents recorded using a similar protocol to that of Fig. 1A, with pipette solution containing 5 mM BAPTA in nominally zero free Ca2+ concentration. The extracellular solution contained either 1.2 mM Mg2+/1.8 mM Ca2+ or 1.2 mM Mg2+/0 mM Ca2+. Insets: Tail currents measured at −60 mV. (C) For WT Kv7.1, there is a significant increase in inactivation in Ca2+-free external solutions from 7 to 33% (n = 9; two-tailed paired t test; ***P < 0.0001, t = 8.060, df = 8); for the V307L mutant, there is also a significant increase in inactivation in Ca2+-free external solutions from 0 to 34% (n = 6; two-tailed paired t test; *P = 0.0188, t = 3.424, df = 5). (D) Representative traces of a CHO cell expressing V307L mutant currents and sequentially perfused with a Ca2+-free external solution containing either 130 mM K+ or 130 mM Rb+. From a holding potential of −90 mV, the membrane was stepped to +60 mV for 2 s and then the tail current was…

Fig. 3. Kinetic model of Kv7.1 inactivation gating and sensitivity to external Ca2+ of mutants F351A and S338F. (A) The model assumes two closed states (C1 and C2); two voltage-dependent open states (O1 and O2); a Ca2+-sensitive, slow, voltage-dependent inactivated state I1; and a Ca2+-insensitive, voltage-dependent fast inactivated state I2. For simplicity, we assumed that no openings occur when the cell is voltage clamped to −90 mV, and consequently, we fixed initial values of all states to zero, except C1. (B) Simulation of the gating behavior of WT Kv7.1 in Ca2 +-free external solutions according to the scheme in (A) generated current kinetics that closely matched the experimental traces. (C) Simulation of the gating behavior of WT Kv7.1 in Ca +-containing external solutions according to the scheme in (A) generated current kinetics that closely matched the experimental traces. (D) Mapping the S6 pore residues S338 and F351 onto the human Kv7.1 cryo-EM structure (PDB: 6UZZ). (E and G) Representative traces of F351A and S338F mutant currents, recorded using a similar protocol to that of Fig. 1A. As indicated, the extracellular solution contained either 1.2 mM Mg2+/1.8 mM Ca2+ or 1.2 mM…

Fig. 4. Mutants in the turret region of Kv7.1 prevent inactivation I1. (A) Mapping residues E295, D317, and W304 in the turret, external pore entrance, and pore helix residue, respectively, onto the human KV7.1 cryo-EM structure (PDB: 6UZZ). (B) Representative traces of WT Kv7.1, E295A, and E295Q mutant currents recorded using a similar protocol to that of Fig. 1A, with pipette solution containing 5 mM BAPTA in nominally zero free Ca2+ concentration. The extracellular solution contained either 1.2 mM Mg2+/1.8 mM Ca2+ or 1.2 mM Mg2+/0 mM Ca2+. (C) Mutant D317N at the external pore entrance recorded as above in an extracellular solution containing 1.2 mM Mg2+/1.8 mM Ca2+ does not yield functional currents. (D) Removal of external Ca2+ produced a significant smaller inactivation I1 in mutants E295A (12%; n = 12), E295Q (10%; n = 5), and E295R (13%; n = 6) than WT (48%; n = 12) [one-way ANOVA and Dunnett’s multiple comparisons test; ****P < 0.0001, t = 3.925 F(7, 71)].

Fig. 5. Internal calcified CaM and PIP2 prevent inactivation I1 of WT Kv7.1 in Ca2+-free external solutions. (A) Representative traces of WT Kv7.1 in 1.8 mM Ca2+/1.2 mM Mg2+ and 0 mM Ca2+/1.2 mM Mg2+ external solutions. (B) Removal of external Ca2+ produced a significant smaller inactivation I1 as indicated (2.9% versus 39.4%, respectively; n = 5, two-tailed unpaired t test; ***P < 0.0001, t = 6.996, df = 8). A significant smaller inactivation I1 was obtained following removal of external Ca2+ upon cotransfection with WT CaM than with CaM1,2,3,4 mutant (1.3% versus 22.2%, respectively; n = 7 to 11, two-tailed unpaired t test; *P = 0.0131, t = 2.789, df = 16, **P = 0.0042, t = 3.684 df = 10). (C) Representative traces of WT Kv7.1 in 0 mM Ca2+/1.2 mM Mg2+ external solutions (left red trace) and WT Kv7.1 cotransfected with the PIP4,5-kinase in 1.8 mM Ca2+/1.2 mM Mg2+ (black trace) and 0 mM Ca2+/1.2 mM Mg2+ external solutions (red trace). (D) Increase in cellular PIP2 by cotransfection with the PIP4,5-kinase significantly reduced the inactivation I1 of WT Kv7.1 in Ca2+-free external solutions (2.9% versus 35.1%, respectively; n = 5 to 10, two-tailed unpaired t test; ***P = 0.0002, t = 5.030,…

Fig. 6. Disrupting the interaction between CaM and the S2-S3 linker triggers inactivation I1. (A) Mapping residues S182 in the intracellular S2-S3 linker, onto the human KV7.1 cryo-EM structure (PDB: 6UZZ); shown in atomic proximity, residues Y100 and N138 of CaM. (B) Representative traces of WT Kv7.1 and the Kv7.1 S2-S3 linker mutant S182A in 1.8 mM Ca2+/1.2 mM Mg2+ external solutions. (C) A significant larger inactivation I1 was observed with the mutant S182A, as compared to WT in 1.8 mM Ca2+/1.2 mM Mg2+ external solutions, when the pipette solution contained either Ca2+-CaM [21.7% versus 0.4%, respectively; n = 5 to 17, one-way ANOVA, F(5, 51) = 15.84; ****P < 0.0001; Tukey’s multiple comparisons test; adjusted *P = 0.024] or BAPTA-CaM [43.1% versus 9.2%, respectively; n = 5 to 17, one-way ANOVA, F(5, 51) = 15.84; ****P < 0.0001; Tukey’s multiple comparisons test, adjusted ****P < 0.0001]. The mutant S182A exhibited a larger inactivation when the pipette solution contained BAPTA-CaM (nominally zero free Ca2+) than Ca2+-CaM (nominally 5 μM free Ca2+) [43.1% versus 21.7%, respectively; n = 17, one-way ANOVA, F(5, 51) = 15.84; ****P < 0.0001; Tukey’s multiple comparisons test; adjusted…