Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel

2010 
MthK is a Ca2+-gated K+ channel whose activity is inhibited by cytoplasmic H+. To determine possible mechanisms underlying the channel’s proton sensitivity and the relation between H+ inhibition and Ca2+-dependent gating, we recorded current through MthK channels 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 [H+], in which the solutions bathing the cytoplasmic side of the channels were changed 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 [H+] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca2+ was not H+ sensitive over pH ranging from 7.3 to 9.0. However, increasing [H+] raised the EC50 for Ca2+ activation by ∼4.7-fold per tenfold increase in [H+], displaying a linear relation between log(EC50) and log([H+]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H+ binding does not directly modulate either the channel’s closed–open equilibrium or the allosteric coupling between Ca2+ binding and channel opening. We can account for the Ca2+ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca2+ allosterically activates MthK, whereas H+ opposes activation by destabilizing the binding of Ca2+.
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