Beta1 subunits facilitate gating of BK channels by acting through the Ca2+, but not the Mg2+, activating mechanisms

Abstract

The beta1 subunit of BK (large conductance Ca2+ and voltage-activated K+) channels is essential for many key physiological processes, such as controlling the contraction of smooth muscle and the tuning of hair cells in the cochlea. Although it is known that the beta1 subunit greatly increases the open probability of BK channels, little is known about its mechanism of action. We now explore this mechanism by using channels in which the Ca2+- and Mg2+-dependent activating mechanisms have been disrupted by mutating three sites to remove the Ca2+ and Mg2+ sensitivity. We find that the presence of the beta1 subunit partially restores Ca2+ sensitivity to the triply mutated channels, but not the Mg2+ sensitivity. We also find that the beta1 subunit has no effect on the Mg2+ sensitivity of WT BK channels, in contrast to its pronounced effect of increasing the apparent Ca2+ sensitivity. These observations suggest that the beta1 subunit increases open probability by working through the Ca2+-dependent, rather than Mg2+-dependent, activating mechanisms, and that the action of the beta1 subunit is not directly on the Ca2+ binding sites, but on the allosteric machinery coupling the sites to the gate. The differential effects of the beta1 subunit on the Ca2+ and Mg2+ activation of the channel suggest that these processes act separately. Finally, we show that Mgi2+ inhibits, rather than activates, BK channels in the presence of the beta1 subunit for intermediate levels of Cai2+. This Mg2+ inhibition in the presence of the beta1 subunit provides an additional regulatory mechanism of BK channel activity.

Fig. 1.

The β1 subunit partially restores Ca²⁺ sensitivity to TM Ca²⁺-insensitive BK channels. (A and B) Single-channel currents recorded from a TM channel in the absence (A) and presence (B)ofthe β1 subunit at the indicated and +50 mV. (C) Plots of P_o vs. from 10 representative experiments to show the range of restoration of Ca²⁺ sensitivity to the TM channel by the β1 subunit. (D) Plots of P_o vs. V at 0 for TM channels with and without the β1 subunit (data from six patches for each point). The standard errors are often obscured by the symbol.

Fig. 2.

The β1 subunit restores the Ca²⁺ sensitivity to channels in which the four α subunits all have the TM, such that no endogenous α subunits from BK channels are present. (A) Single-channel currents from a TM channel in the presence of the β1 subunit are fully blocked by 1.5 mM TEAo, as indicated in the current amplitude histogram. Data are from the same outside-out patch. (B) Single-channel currents in the presence of the β1 subunit from a TM channel with the TEA binding sites mutated are partially blocked by 1.5 mM TEAo, reducing the current from ≈13 pA to ≈9 pA. Data are from the same outside-out patch. Similar results were obtained in eight additional experiments. If one or more of the subunits did not have the TEA site mutation, then the current would have been ≈7.5 pA or less (39).

Fig. 3.

The β1 subunit still restores Ca²⁺ sensitivity to the TM channel after replacing the only intracellular negative charge on the β1 subunit with a positive charge (E13K) and adding another adjacent positive charge (T14R). (A) Representative single-channel currents recorded from a TM channel in the presence of the doubly mutated β1 subunit. (B) Plots of P_o vs. for TM channels with the doubly mutated β1 subunit.

Fig. 4.

The M513 site is not responsible for the restoration of the Ca²⁺ sensitivity to the TM channel by the β1 subunit. (A) Single-channel currents recorded from a Ca²⁺-insensitive quadra mutant channel made by adding the mutation M513I to the TM channel. (B) The β1 subunit restores Ca²⁺ sensitivity to the quadra mutant channel, +50 mV.

Fig. 5.

The β1 subunit does not restore the Mg²⁺ sensitivity to the TM channel, and it has little effect on the Mg²⁺ sensitivity of WT BK channels. (A) Single-channel currents recorded from WT channels at the indicated at +100 mV. (B and C) Currents from TM channels in the absence (B) and presence (C)ofthe β1 subunit. (D) Plots of P_o vs. for TM and WT channels with and without the β1 subunit at +100 mV. (E) Currents from WT channels in the absence and presence of the β1 subunit with 5.7 μM at –50 mV. (F) Currents from WT channels in the absence and presence of the β1 subunit with 0 and 10 mM at +50 mV. (G) Plots of P_o vs. V from experiments like those in E and F (n ≥ 5 for each point in D and G). The open triangles typically obscure the filled triangles.

Fig. 6.

In the presence of the β1 subunit, inhibits the activity of WT channels for intermediate levels of . (A) Single-channel currents recorded from WT channels in the absence of the β1 subunit at 5.7 μM without and with 10 mM at –30 mV. (B) As in A, except for the presence of the β1 subunit. (C) Plots of P_o vs. V for WT channels with the β1 subunit with 0, 5.7, and 100 μM_, each with and without 10 mM . (D)Asin C, except for the presence of the β1 subunit. (E) Plots of the voltage for half activation, V_0.5, vs. for WT channels with and without the β1 subunit, as indicated. (n ≥ 3 for each point in C–E.)