Large-conductance Ca2+- and voltage-gated K+ channels form and break interactions with membrane lipids during each gating cycle

Abstract

Membrane depolarization and intracellular Ca²⁺ promote activation of the large-conductance Ca²⁺- and voltage-gated (Slo1) big potassium (BK) channel. We examined the physical interactions that stabilize the closed and open conformations of the ion conduction gate of the human Slo1 channel using electrophysiological and computational approaches. The results show that the closed conformation is stabilized by intersubunit ion-ion interactions involving negative residues (E321 and E324) and positive residues (³²⁹RKK³³¹) at the cytoplasmic ends of the transmembrane S6 segments ("RKK ring"). When the channel gate is open, the RKK ring is broken and the positive residues instead make electrostatic interactions with nearby membrane lipid oxygen atoms. E321 and E324 are stabilized by water. When the ³²⁹RKK³³¹ residues are mutated to hydrophobic amino acids, these residues form even stronger hydrophobic interactions with the lipid tails to promote the open conformation, shifting the voltage dependence of activation to the negative direction by up to 400 mV and stabilizing the selectivity filter region. Thus, the RKK segment forms electrostatic interactions with oxygen atoms from two sources, other amino acid residues (E321/E324), and membrane lipids, depending on the gate status. Each time the channel opens and closes, the aforementioned interactions are formed and broken. This lipid-dependent Slo1 gating may explain how amphipathic signaling molecules and pharmacologically active agents influence the channel activity, and a similar mechanism may be operative in other ion channels.

Keywords: BK; KCa1.1; Slo1; electrophysiology; molecular dynamics.

Fig. 1.

Snapshots of simulated hSlo1 without divalent cations and with Ca²⁺ and Mg²⁺. (A) Side view of hSlo1 without divalent cations (Left) and with Ca²⁺ and Mg²⁺ (Right). Ions outside of the selectivity filter and water are not shown. The channel without divalent cations has only one K⁺ in the filter; the outer K⁺ ions left the pore earlier in the simulation. (B) RKK residues viewed from the intracellular side. The cytoplasmic domain is not shown. ³²⁹RKK³³¹, E321, and E324 are shown using spheres. The residues interacting with those with opposite charges are shown in bold. These results are 80 ns after the start of simulation. (C) Probability of interactions of ³²⁹RKK³³¹ with E321 and E324 in hSlo1 without divalent cations (Left) and with Ca²⁺ and Mg²⁺ (Right) analyzed every 20 ps. The cutoff distance was 3.8 Å. Similar results were obtained in all four simulation runs in each condition.

Fig. 2.

Double mutations of E321 and E324 in the RKK ring. (A) K⁺ currents from WT, E321Q:E324Q (EE:QQ), E321A:E324A (EE:AA), E321D:E324D (EE:DD), and E321R:E324R (EE:RR). (B) Voltage dependence of steady-state activation. See SI Appendix, Table S1 for V_0.5 and Q_app. (C) Voltage dependence of current kinetics. The blue dashed curves represent WT. The results shown are means ± SD. Results were obtained with 0 Ca²⁺ with 11 mM EGTA.

Fig. 3.

Triple mutations of the RKK segment. (A) K⁺ currents from R329K:K330K:K331K (RKK:KKK), R329R:K330R:K331R (RKK:RRR), R329Q:K330Q:K331Q (RKK:QQQ), R329N:K330N:K331N (RKK:NNN), R329A:K330A:K331A (RKK:AAA), R329V:K330V:K331V (RKK:VVV), R329E:K330E:K331E (RKK:EEE), and R329D:K330D:K331D (RKK:DDD). (B) Voltage dependence of current activation from the double mutants indicated. See SI Appendix, Table S1 for V_0.5 and Q_app. (C) Voltage dependence of current kinetics. The blue dashed curves represent WT. The results shown are means ± SD. Results were obtained with 0 Ca²⁺ with 11 mM EGTA.

Fig. 4.

RKK neutralization mutants are open at extreme negative voltages. (A) Openings at –240 mV from R329:I:K330I:K331I (RKK:III, Top Left) and R329L:K330L:K331L (RKK:LLL, Top Right). The dashed horizontal lines indicate the 0-pA level. Each patch contained one channel. All-points amplitude histograms are also shown (Bottom). (B) Voltage dependence of P_o for RKK:III and RKK:LLL (n = 6–13). The curves are Boltzmann-type fits (SI Appendix, Table S1). The data for RKK:III and RKK:LLL are shown with ±5 mV offsets for clarity. (C) Mean burst open and interburst closed durations for RKK:III and RKK:LLL at −240 mV. The thick horizontal bars indicate the mean values. (D) Relationship between V_0.5 and Wimley–White hydrophobicity (∆G_wif) of the amino acid in the RKK segment in the mutants where each of the three ³²⁹RKK³³¹ residues is replaced with the amino acid indicated. The electrophysiological results are means ± SD. The Leu and Ile results were from single-channel measurements. Results were obtained with 0 Ca²⁺ with 11 mM EGTA.

Fig. 5.

Gating cycle-dependent interactions of the RKK segment with membrane lipids. (A) Snapshots of the simulation results from hSlo1 WT without divalent cations (Left), WT with Ca²⁺ and Mg²⁺ (Center), and RKK:LLL without divalent cations (Right). ³²⁹RKK³³¹, E321, E324, the interacting lipid molecules, and pore K⁺ ions are depicted using spheres. The ion conduction pathway runs in the center in the upper half of each image. (B) The RKK interaction partners under the three conditions indicated. Lipid carbon atoms are shown in pink. See SI Appendix, Fig. S10 for the remaining subunits. (C) Probability of the interactions between lipid O atoms and the residues indicated under the divalent cation-free condition (blue) and the Ca²⁺- and Mg²⁺-bound condition (red). The results are from an illustrative 100-ns run analyzed every 0.2 ns. (D) The selectivity filter residues viewed from the extracellular side. ²⁸⁹GYG²⁹¹ are shown using spheres. Ions, water, and lipid molecules are not shown. Similar results were obtained in all four simulation runs in each condition.