Molecular Basis of Proton Block of L-Type Ca 2+ Channels

Hydrogen ions are important regulators of ion flux through voltage-gated Ca 2+ channels but their site of action has been controversial. To idendfy molecular determinants of proton block of L-type Ca 2+ channels, we combined site-directed mutagenesis and unitary current recordings from wild-type (WT) and mutant L-type Ca 2§ channels expressed in Xen0pus 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 Ca 2+ channels. Proton block was altered in a unique fashion by mutation of each of the four P-region glutamates (EI-E/V) that form the locus of high affinity Ca 2+ 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 E / I ~ 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 Ca 2+ channels lies within the pore and is formed by a combination of conserved P-region glutamates in repeats L II, and III, acting in concert. E/Vlies 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 Ca 2+ 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 Ca ~+ selectivity and permeation. KEY W O R D S" ion channels * protonation * P-region * permeation * Xenopus oocytes I N T R O D U C T I O N Extracellular p H falls sharply dur ing episodes of intense neurona l activity (Chesler and Kaila, 1992) or with ischemia in brain or hear t (Katz, 1992; Siesjo et al., 1993). The change in pHo has an significant effect on many kinds of ion channels (Hille, 1992; Traynelis, 1996). However, the mechanism of pH-dependen t control o f channel funct ion is not completely unders tood at the molecular level. Voltage-gated calcium channels are particularly interesting targets of hydrogen ion regulation because of their biological impor tance as a delivery system for a key intraceUular messenger. Increased [H +] strongly inhibits ion permea t ion through open Ca 2+ channels as well as reducing channel opening ( P r o d ' h o m et al., 1987; Krafte and Kass, 1988; K l t ckne r and Isenberg, 1994). The inhibitory effect of extracellular acidification on voltage-gated Ca 2+ channels helps limit Ca 2+ overload and subsequent damage dur ing a metabol ic insult (e.g., Ou-Yang et al., 1994). Ilya Bezprozvanny's present address is Department of Physiology, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, Tx 75235-9040. Address correspondence to Dr. Richard W. Tsien, Department of Molecular and Cellular Physiology, Beckman Center, B105A, Stanford, CA 94305-5426. Fax: 415-725-8021; E-mail: [email protected] The interaction between protons and Ca 2+ channels also shows intriguing biophysical properties. Recordings of unitary currents through L-type Ca 2+ channels by the late Peter Hess and colleagues provided the first direct measurements of the p ro tona t ion /dep ro tona tion rates of a single molecule (Prod 'horn et al., 1987). When p robed with various monovalen t cations as charge carriers, these channels were pro tona ted at a single site with an anomalously high affinity for H + (pK~ > 7.0), resulting in an unusual subconductance state (Prod 'horn et al., 1987; Pie t robon et al., 1989; Prod 'horn et al., 1989). Protons also reduced the unitary fluxes of Ca 2+ and o ther divalent cations, a l though much greater acidification was required for block (Krafte and Kass, 1988; Kuo and Hess, 1993; Klockner and Isenberg, 1994). Despite extensive study, d isagreement remains about the locus of H + block of Ca 2+ channels and the mechanism of inhibition of ion flux. In the prevailing hypothesis, Hess and colleagues proposed that protons titrate an external histidine residue, outside of the permeation pathway, and reduce channel conductance by an allosteric mechanism (Pietrobon et al., 1989). Mutagenesis studies have provided direct suppor t for such an allosteric mechanism in the case of p ro ton block of 363 J. GEH. PHYSIOL. 9 The Rockefeller University Press * 0022-1295/96/11/363/12 $2.00 Volume 108 November 1996 363-374 on Jne 0, 2017 D ow nladed fom Published November 1, 1996