Divalent Cations

Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl 2 reduced the H 1 current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H 1 current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn 2 1 were inconsistent with classical voltage-dependent block in which Zn 2 1 binds within the membrane voltage field. Instead, Zn 2 1 binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn 2 1 were strongly pH o dependent but were insensitive to pH i , suggesting that protons and Zn 2 1 compete for external sites on H 1 channels. The apparent potency of Zn 2 1 in slowing activation was z 10 3 greater at pH o 7 than at pH o 6, and z 100 3 greater at pH o 6 than at pH o 5. The pH o dependence suggests that Zn 2 1 , not ZnOH 1 , is the active species. Evidently, the Zn 2 1 receptor is formed by multiple groups, protonation of any of which inhibits Zn 2 1 binding. The external receptor bound H 1 and Zn 2 1 with pK a 6.2–6.6 and pK M 6.5, as described by several models. Zn 2 1 effects on the proton chord conductance–voltage ( g H –V) relationship indicated higher affinities, pK a 7 and pK M 8. CdCl 2 had similar effects as ZnCl 2 and competed with H 1 , but had lower affinity. Zn 2 1 applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H 1 currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn 2 1 receptor may be the same modulatory protonation site(s) at which pH o regulates H 1 channel gating. key words: metal binding constants • cadmium • pH • hydrogen ion • ion channels I N T R O D U C T I O N Voltage-gated proton channels differ from other voltage-gated ion channels, not only in their extreme selectivity for H 1 , but also in the regulation of their gating by pH o and pH i . The mechanism of permeation is believed to differ radically from traditional ion channels, which comprise water-filled pores through which ions diffuse: proton channels appear to conduct H 1 by a Grotthuss-like mechanism of hopping across a hydrogen-bonded chain spanning the membrane (DeCoursey and Cherny, 1994, 1995, 1997, 1998; Cherny et al., 1995). If ion channels are defined narrowly as waterfilled pores, then proton channels are not ion channels, although they conduct protons passively down their electrochemical gradient, and independently of other ionic species. In spite of these fundamental differences, it is remarkable how closely proton channels resemble other voltage-gated channels. H 1 channels activate upon depolarization with a sigmoidal time course, deactivate exponentially, and exhibit a ColeMoore effect (DeCoursey and Cherny, 1994) practically indistinguishable from the behavior of delayed rectifier K 1 channels (Cole and Moore, 1960). Low pH o shifts the voltage-activation curves of both H 1 channels and other ion channels toward positive potentials and slows activation at a given voltage. Voltage-gated proton channels characteristically are inhibited by extracellular polyvalent cations. The transition metals Zn 2 1 and Cd 2 1 have been used most frequently (Thomas and Meech, 1982; Byerly et al., 1984; Barish and Baud, 1984; Mahaut-Smith, 1989; DeCoursey, 1991; Kapus et al., 1993; Demaurex et al., 1993; DeCoursey and Cherny, 1993; Humez et al., 1995; Gordienko et al., 1996; Nordström et al., 1995), but Cu 2 1 , Ni 2 1 , Co 2 1 , Hg 2 1 , Be 2 1 , Mn 2 1 , Al 3 1 , and La 3 1 have similar effects (Thomas and Meech, 1982; Meech and Thomas, 1987; Byerly and Suen, 1989; Bernheim et al., 1993; DeCoursey and Cherny, 1994; Eder et al., 1995). To the extent that each has been explored, all of these metal cations shift the voltage dependence of activation (channel opening) to more positive potentials and slow the opening rate (Byerly et al., 1984; Barish and Baud, 1984; Meech and Thomas, 1987; Mahaut-Smith, 1989; DeCoursey, 1991; DeCoursey and Cherny, 1993; Demaurex et al., 1993; Kapus et al., 1993; Nordström et Portions of this work were previously published in abstract form (DeCoursey, T.E., and V.V. Cherny. 1999. Biophys . J . 76:A147). Address correspondence to Tom DeCoursey, Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke’s Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. Fax: 312-942-8711; E-mail: [email protected]