Molecular basis of proton block of L-type Ca²⁺ channels

Hydrogen ions are important regulators of ion flux through voltage- gated Ca²⁺ channels but their site of action has been controversial. To identify molecular determinants of proton block of L-type Ca²⁺ channels, we combined site-directed mutagenesis and unitary current recordings from wild- type (WT) and mutant L-type Ca²⁺ channels expressed in Xenopus oocytes. WT channels in 150 mM K⁺ displayed two conductance states, deprotonated (140 pS) and protonated (45 pS), as found previously in native L-type Ca²⁺ channels. Protein block was altered in a unique fashion by mutation of each of the four P-region glutamates (EI-EIV) that form the locus of high affinity Ca²⁺ interaction. Glu(E) → Gln(Q) substitution in either repeats I or III abolished the high-conductance state, as if the titration site had become permanently protonated. While the EIQ mutant displayed only an ~40 pS conductance, the EIIIQ mutant showed the ~40 pS conductance plus additional pH-sensitive transitions to an even lower conductance level. The EIVQ mutant exhibited the same deprotonated and protonated conductance states as WT, but with an accelerated rate of deprotonation. The EIIQ mutant was unusual in exhibiting three conductance states (~145, 102, 50 pS, respectively). Occupancy of the low conductance state increased with external acidification, albeit much higher proton concentration was required than for WT. In contrast, the equilibrium between medium and high conductance levels was apparently pH-insensitive. We concluded that the protonation site in L-type Ca²⁺ channels lies within the pore and is formed by a combination of conserved P-region glutamates in repeats I, II, and III, acting in concert. EIV lies to the cytoplasmic side of the site but exerts an additional stabilizing influence on protonation, most likely via electrostatic interaction. These findings are likely to hold for all voltage-gated Ca²⁺ channels and provide a simple molecular explanation for the modulatory effect of H⁺ ions on open channel flux and the competition between H⁺ ions and permeant divalent cations. The characteristics of H⁺ interactions advanced our picture of the functional interplay between P-region glutamates, with important implications for the mechanism of Ca²⁺ selectivity and permeation.