Role of Charged Residues in the S1–S4 Voltage Sensor of BK Channels

Abstract

The activation of large conductance Ca2+-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K+ (KV) channels. Yet BK and KV channels share many conserved charged residues in transmembrane segments S1–S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (Po) and IK kinetics (τ(IK)) over an extended voltage range in 0–50 μM [Ca2+]i. mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of PO. The voltage dependence of PO and τ(IK) at extreme negative potentials was also reduced, implying that the closed–open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and KV channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to KV channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1–S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3–7 kcal mol−1, indicating a strong contribution of non–voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.

Operation of the voltage sensor of a human voltage- and Ca 2+ -activated K + channel

“…4). When direct comparison of parameters can be made, the fitting of scheme IV is in congruence with the findings of a previous model of BK Ca activation (44). Specifically, the Pore Domain voltage dependence (L 0 equivalent to V half = 818 mV at 25°C, z L = 0.49 e 0 ; scheme IV: V half = 820 mV, q = 0.49 e 0 ) and the VSD-Pore interaction (factor D equivalent to −80 meV at 25°C; scheme IV H 4P : −100 meV).…”

“…1C), hydropathy analysis (12), and NMR spectroscopy (53) suggest that the extracellular linker between S3 and S4 in BK Ca is only three residues long. Thus, the voltage-evoked movements of S3, which bears the voltage-sensing D186 (44), could contribute to the fluorescence deflections reported from position 202.…”

“…Having characterized the voltage-dependent rearrangements of S4 (51, 52), we sought to resolve those of S2, which contains residues that make a significant contribution to BK Ca gating charge displacement (44). Fig.…”

“…This marked loss of valence (>78%) is compatible with the prominent role of D153 in the voltage dependence of BK Ca channel activation. The residual voltage dependence likely arose from another gating charge in S2, R167 (44).…”

“…Recently, extensive electrophysiological analysis of the role of charged VSD residues in BK Ca voltage-dependent activation (44) revealed that only one residue in the BK Ca S4 senses voltage (R213) and that a greater portion of the total gating charge movement is contributed by residues outside this segment: one in S3 (D186) and two in S2 (D153, R167) (Fig. 1B).…”