Mechanism Underlying pH-Modulation of Ca2+-Dependent Gating in the MthK Channel

MthK is a Ca2+-gated K+ channel whose activity is modulated by cytoplasmic pH. To determine possible mechanisms underlying the channel's pH sensitivity, we recorded current through MthK channels, which were purified from E.coli membranes, reconstituted into liposomes and then incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca2+] (ranging from nominally 0 to 30 mM) at a given pH, in which the solution bathing the cytoplasmic side of the channels was replaced via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca2+] and open probability (Po), with a mean Hill coefficient (nH) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor nH changed significantly as a function of pH over pH ranging from 6.5 to 9.0, suggesting that H+ does not alter either functional coupling or cooperativity in Ca2+-dependent gating. In addition, channel openings were not observed in the nominal absence of Ca2+ at pH up to 9.0. However, increasing pH decreased the EC50 for Ca2+ activation by ∼4.7-fold per 10-fold increase in [H+], displaying a linear relation between log(EC50) and pH over the entire range of pH studied (6.5 to 9.0). Together, these results suggest that H+-binding does not directly modulate either the allosteric coupling between Ca2+-binding and channel opening or the channel's closed-open equibrium. We may account for the pH modulation by assuming that increasing pH yields a relative energetic stabilization of the Ca2+-bound states over unliganded states of the channel.