An S6 mutation in BK channels reveals beta1 subunit effects on intrinsic and voltage-dependent gating

Abstract

Large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are exquisitely regulated to suit their diverse roles in a large variety of physiological processes. BK channels are composed of pore-forming alpha subunits and a family of tissue-specific accessory beta subunits. The smooth muscle-specific beta1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of beta1 on channel's gating energetics are not completely understood. One of the difficulties is that it has not yet been possible to measure the effects of beta1 on channel's intrinsic closed-to-open transition (in the absence of voltage sensor activation and Ca(2+) binding) due to the very low open probability in the presence of beta1. In this study, we used a mutation of the alpha subunit (F315Y) that increases channel openings by greater than four orders of magnitude to directly compare channels' intrinsic open probabilities in the presence and absence of the beta1 subunit. Effects of beta1 on steady-state open probabilities of both wild-type alpha and the F315Y mutation were analyzed using the dual allosteric HA model. We found that mouse beta1 has two major effects on channel's gating energetics. beta1 reduces the intrinsic closed-to-open equilibrium that underlies the inhibition of BK channel opening seen in submicromolar Ca(2+). Further, P(O) measurements at limiting slope allow us to infer that beta1 shifts open channel voltage sensor activation to negative membrane potentials, which contributes to enhanced channel opening seen at micromolar Ca(2+) concentrations. Using the F315Y alpha subunit with deletion mutants of beta1, we also demonstrate that the small N- and C-terminal intracellular domains of beta1 play important roles in altering channel's intrinsic opening and voltage sensor activation. In summary, these results demonstrate that beta1 has distinct effects on BK channel intrinsic gating and voltage sensor activation that can be functionally uncoupled by mutations in the intracellular domains.

Figure 1.

The mβ1 subunit promotes BK channel activation in high Ca²⁺ and reduces channel activation in low Ca²⁺. (A) Left, families of BK/α currents evoked by 40-ms depolarizations (20-mV steps over the indicated range) in 7 μM Ca²⁺. Right, normalized G-V relationships (mean ± SEM) of BK/α at indicated Ca²⁺ (n = 12–44). (B) Left, families of BK/α+β1 currents evoked by 40-ms depolarizations (20-mV steps over the indicated range) in 7 μM Ca²⁺. Right, normalized G-V relationships (mean ± SEM) of BK/α+β1 at indicated Ca²⁺ (n = 7–39). (C) V_1/2-Ca²⁺ relationships (mean ± SEM) for BK/α (open symbols) and BK/α+β1 channels (closed symbols). (D) Q-Ca²⁺ relationships (mean ± SEM) for BK/α (open symbols) and BK/α+β1 channels (closed symbols).

Figure 2.

Effects on 0 Ca²⁺ logP_O-V relation by changes in L₀, Vh_O, Vh_C, and z_J. (A) BK channel gating scheme at 0 Ca²⁺ according to the HA model. Channel resides in either open (O) or closed (C) conformation, with zero to four (subscripts) activated (A) voltage sensors. L is the C-O equilibrium constant with all four voltage sensors in the resting (R) state (dashed box). J is the R-A equilibrium constant when channels are closed. D is the allosteric interaction factor between C-O transition and voltage sensor activation. Equilibrium between C-O transitions is allosterically regulated by the states of four independent and identical voltage sensors. (B) Simulated 0 Ca²⁺ logP_O-V relation according to the HA model (L₀ = 1 e⁻⁶, z_L = 0.30 e₀, z_J = 0.58 e₀, Vh_C = +200 mV, Vh_O = +50 mV). Dashed line represents linear fit of the logP_O-V relation at the limiting slope. L₀ (point where dashed line and zero line cross) and z_L (slope of dashed line) are the zero voltage value of L and its partial charge, respectively. (C) Effects on logP_O-V relation of changing L₀ from 1 e⁻⁶ (black) to 1 e⁻⁸ (red). Notice the shift of the limiting slope along the Y axis. (D) Effects on logP_O-V relation of changing Vh_O (−50 mV, red), Vh_C (+100 mV, green), or z_J (0.4 e₀, blue) and leaving other parameters the same as in B (black). Notice reducing Vh_O, but not Vh_C or z_J shifts the steep phase of the logP_O-V relation to more hyperpolarized potentials.

Figure 3.

Evaluating effects of mβ1 on L₀ and Vh_O in the presence of wild type α. (A) Representative single channel currents of BK/α (left) and BK/α+β1 (right) in 0 Ca²⁺. Time scales are 10 ms for +40 mV and 1 ms for −40 and −80 mV, respectively. (B) Burst duration (mean ± SEM) versus voltage for BK/α (n = 5–10) and for BK/α+β1 (n = 3–13). (C) Gap duration (mean ± SEM) versus voltage for BK/α (n = 5–10) and for BK/α+β1 (n = 3–13). (D) LogP_O-V (mean ± SEM, left) and P_O-V relations (mean ± SEM, right panel) of BK/α. P_O between −120 and +100 mV were measured using single channel recordings (n = 2–13). P_O between +110 and +290 mV were measured using macroscopic recordings (n = 12). Linear fit of logP_O–V relation at the “steep phase” (dashed line, left) indicates that the measurement either has reached or is approaching the limiting slope. The solid line represents best fits to the HA model (held z_L = 0.3 e₀, z_J = 0.58 e₀, fitting yielded L₀ = 1 e⁻⁶, Vh_C = +202 mV, and Vh_O = 46 mV). (E) LogP_O-V (mean ± SEM, left) and P_O-V relations (mean ± SEM, right) of BK/α+β1. P_O between −60 and +80 mV were measured using single channel recordings (n = 4–22). P_O between +90 and +310 mV were measured using macroscopic recordings (n = 7). Linear fit of logP_O–V relation at the “steep phase” (dashed line, left panel overlaps with the solid line) indicates that the measurement has not reached the limiting slope. Fits to the HA model were not well constrained, reasonable fits were obtained when L₀ ranged between 1 e⁻¹⁰ and 1 e⁻⁸. The solid line represents one of the fits (held L₀ = 1 e⁻⁹, z_L = 0.3 e₀, z_J = 0–0.58 e₀, fitting yielded Vh_C = +132 mV and Vh_O = −48 mV). (F) Reducing z_J did not improve the fits. Best fits (solid lines) to the HA model (held z_J = 0.37 e₀, z_L = 0.3 e₀, L₀ > 1 e⁻¹³, yielded L₀ = 1e⁻¹³, Vh_C = +192 mV, and Vh_O = −261 mV).

Figure 4.

F315Y mutation greatly increases P_O at 0 Ca²⁺ by increasing L₀. (A) Representative macroscopic (left) and single channel (right) recordings of BK/F315Y at 0 Ca²⁺. (B) Representative single channel currents of BK/α at 0 Ca²⁺ show opening to be much briefer than the F315Y mutant. (C) G-V relations (mean ± SEM) for BK/α (n = 12) and BK/F315Y (n = 13). F315Y mutation left shifts G-V and decreases the apparent voltage dependence. (D) LogP_O-V relations (mean ± SEM) for BK/α (n = 3–12) and BK/F315Y (n = 4–7). (E) Representative logP_O-V relations of BK/F315Y where the limiting slope was fitted to Eq. 4 to estimate z_L and L₀ values (mean ± SEM) are indicated in the figure (n = 6). (F) Best fits to the HA model (held z_J = 0–0.58 e₀, z_L = 0.26 e₀, yielded L₀ = 9 e⁻², z_J = 0.58 e₀, Vh_C = +92 mV, and Vh_O = +35 mV). (G) Best fits to the HA model assuming F315Y does not alter Vh_C and Vh_O (held z_L = 0.26 e₀, Vh_C = +202 mV, and Vh_O = +46 mV, yielded L₀ = 4 e⁻², z_J = 0.36 e₀).

Figure 5.

Evaluating effects of mβ1 on L₀ and Vh_O in the presence of F315Y. (A) An example of single channel recordings of BK/F315Y+β1 at 0 Ca²⁺. Notice that maximum P_O reaches ∼1. (B) Representative macroscopic recordings of BK/F315Y+β1 at 0 Ca²⁺. (C) G-V relation (mean ± SEM) for BK/F315Y (n = 13) and BK/F315Y+β1 (n = 28). (D) Representative single channel recordings for BK/F315Y and BK/F315Y+β1. Notice that β1 dramatically increases the burst durations. (E) Representative logP_O-V relations of BK/F315Y+β1 where the limiting slope were fitted to Eq. 4 to estimate z_L, and L₀ values indicated in the figure represent mean ± SEM (n = 7). (F) Best fits to the HA model (held z_J = 0.58 e₀, z_L = 0.27 e₀ yielded L₀ = 1.8 e⁻³, Vh_C = +72 mV, and Vh_O = −26 mV).

Figure 6.

Effects on V_1/2-Ca²⁺ relations of changing L₀, Vh_O, or J₀. (A) Effects on V_1/2-Ca²⁺ and Q-Ca²⁺ relations by changing L₀. P_O-V relations were simulated based on the HA model and fitted to the Boltzmann function to obtain V_1/2-Ca²⁺ relations. Gating parameters were the same (z_L = 0.30 e₀, z_J = 0.58 e₀, Vh_C = +200 mV, Vh_O = +50 mV, K_C = 13 μM, K_C = 1.3 μM) except for L₀ (L₀ = 1 e⁻⁶, black line; L₀ = 1 e⁻⁸, orange line; L₀ = 1 e⁻⁹, red line). (B) Effects on V_1/2-Ca²⁺ and Q-Ca²⁺ relations by changing L₀ when Vh_C is +400 mV. Gating parameters are same as A except Vh_C is +400 mV. (C) Effects on V_1/2-Ca²⁺ and Q-Ca²⁺ relations by changing Vh_O or J₀. Gating parameters were the same (z_L = 0.30 e₀, L₀ = 1 e⁻⁶, z_J = 0.58 e₀, K_C = 13 μM, K_C = 1.3 μM) except for Vh_C and Vh_O, (Vh_C = +200 mV, Vh_O = +50 mV, black line; Vh_C = +200 mV, Vh_O = −20 mV, solid green line, Vh_C = +130 mV, Vh_O = −20 mV, green dash line). (D) Effects on V_1/2-Ca²⁺ relations by changing J_O and L₀. Black lines (L₀ = 1 e⁻⁶, Vh_C = +200 mV, Vh_O = +50 mV, other parameters as in A); blue line, left (L₀ = 1 e⁻⁹, Vh_C = +130 mV, Vh_O = −20 mV); blue line, right (L₀ = 1 e⁻⁸, Vh_C = +130 mV, Vh_O = −20 mV).

Figure 7.

Intracellular domain deletions of β1 eliminate the leftward shift of the G-V relationship at high Ca²⁺. (A) Cartoon of the β1ΔN₁₁ mutant. (B) Families of BK/α+β1ΔN₁₁ currents evoked by 60-ms depolarizations in 7 μM Ca²⁺. (C) Normalized G-V relationships (mean ± SEM) of BK/α+β1ΔN₁₁ at indicated Ca²⁺ (n = 5–18). (D) V_1/2-Ca²⁺ and Q-Ca²⁺ relationships (mean ± SEM) for BK/α+β1ΔN₁₁ compared with BK/α and BK/α+β1. (E) Cartoon of the β1ΔC₁₁ mutant. (F) Families of BK/α+β1ΔC₁₁ currents evoked by 90-ms depolarizations in 7 μM Ca²⁺. (G) Normalized G-V relationships (mean ± SEM) of BK/α+β1ΔC₁₁ at indicated Ca²⁺ (n = 4–26). (H) V_1/2-Ca²⁺ and Q-Ca²⁺ relationships (mean ± SEM) for BK/α+β1ΔC₁₁ compared with BK/α and BK/α+β1. (I) Cartoon of the β1ΔN₁₀ΔC₁₁ mutant. (J) Families of BK/α+β1ΔN₁₀ΔC₁₁ currents evoked by 150-ms depolarizations in 7 μM Ca²⁺. (K) Normalized G-V relationships (mean ± SEM) of BK/α+β1ΔN₁₀ΔC₁₁ at indicated Ca²⁺ (n = 3–14). (L) V_1/2-Ca²⁺ and Q-Ca²⁺ relationships (mean ± SEM) for BK/α+β1ΔN₁₀ΔC₁₁ compared with BK/α and BK/α+β1.

Figure 8.

Intracellular domain deletions impair β1's ability to reduce L₀ and shift Vh_O. (A) Examples of single channel currents of BK/F315Y+β1ΔN₁₁. (B) Representative logP_O-V relations of BK/α+β1ΔN₁₁ where the limiting slope were fitted to Eq. 4 to estimate z_L. z_L values indicated in the figure represent mean ± SEM (n = 8). (C) Best fits to the HA model (held z_L = 0.24 e₀, z_J = 0–0.58 e₀, yielded L₀ = 5.5e⁻³, z_J = 0.58 e₀, Vh_C = +69 mV, and Vh_O = +15 mV). (D) Examples of single channel currents of BK/F315Y+β1ΔC₁₁. (E) Representative logP_O-V relations of BK/α+β1ΔC₁₁ where the limiting slope was fitted to Eq. 4 to estimate z_L. z_L value indicated in the figure represents mean ± SEM (n = 5). (F) Best fits to the HA model (held z_L = 0.24 e₀, z_J = 0–0.58 e₀, yielded L₀ = 8 e⁻³, z_J = 0.58 e₀, Vh_C = +103 mV and Vh_O = +43 mV). (G) Examples of single channel currents of BK/F315Y+β1ΔN₁₀C₁₁. (H) Representative logP_O-V relations of BK/F315Y+β1ΔN₁₀C₁₁ where the limiting slope was fitted to Eq. 4 to estimate z_L. z_L values indicated in the figure represent mean ± SEM (n = 8). (I) Best fits to the HA model (held z_L = 0.13 e₀, z_J = 0–0.58 e₀, yielded L₀ = 1.3 e⁻², z_J = 0.58 e₀, Vh_C = +98 mV, and Vh_O = +29 mV).