Protons Block BK Channels by Competitive Inhibition with K+ and Contribute to the Limits of Unitary Currents at High Voltages

Abstract

Proton block of unitary currents through BK channels was investigated with single-channel recording. Increasing intracellular proton concentration decreased unitary current amplitudes with an apparent pKa of 5.1 without discrete blocking events, indicating fast proton block. Unitary currents recorded at pHi 8.0 and 9.0 had the same amplitudes, indicating that 10−8 M H+ had little blocking effect. Increasing H+ by recording at pHi 7.0, 6.0, and 5.0 then reduced the unitary currents by 13%, 25%, and 53%, respectively, at +200 mV. Increasing K+i relieved the proton block in a manner consistent with competitive inhibition of K+i action by H+i. Proton block was voltage dependent, increasing with depolarization, indicating that block was coupled to the electric field of the membrane. Proton block was not described by the Woodhull equation for noncompetitive voltage-dependent block, but was described by an equation for cooperative competitive inhibition that included voltage-dependent block from the Woodhull equation. Proton block was still present after replacing the eight negative charges in the ring of charge at the entrance to the intracellular vestibule by uncharged amino acids. Thus, the ring of charge is not the site of proton block or of competitive inhibition of K+i action by H+i. With 150 mM symmetrical KCl, unitary current amplitudes increased with depolarization, reaching 66 pA at +350 mV (pHi 7.0). The increase in amplitude with voltage became sublinear for voltages >100 mV. The sublinearity was unaffected by removing from the intracellular solutions Ca2+ and Ba2+ ions, the Ca2+ buffers EGTA and HEDTA, the pH buffer TES, or by replacing Cl− with MeSO3−. Proton block accounted for ∼40% of the sublinearity at +200 mV and pH 7.0, indicating that factors in addition to proton block contribute to the sublinearity of the unitary currents through BK channels.