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Voltage‐gated ion channels, whose first identified function was to generate action potentials, are divided into subfamilies with numerous members. The family of voltage‐gated proton channels (HV) is tiny. To date, all species found to express HV have exclusively one gene that codes for this unique ion channel. Here we report the discovery and characterization of three proton channel genes in the classical model system of neural plasticity, Aplysia californica. The three channels (AcHV1, AcHV2, and AcHV3) are distributed throughout the whole animal. Patch‐clamp analysis confirmed proton selectivity of these channels but they all differed markedly in gating. AcHV1 gating resembled HV in mammalian cells where it is responsible for proton extrusion and charge compensation. AcHV2 activates more negatively and conducts extensive inward proton current, properties likely to acidify the cytosol. AcHV3, which differs from AcHV1 and AcHV2 in lacking the first arginine in the S4 helix, exhibits proton selective leak currents and weak voltage dependence. We report the expansion of the proton channel family, demonstrating for the first time the expression of three functionally distinct proton channels in a single species. [ABSTRACT FROM AUTHOR]

Aspartate key to proton selectivity Voltage-gated proton channels are considered completely selective for protons -- no evidence exists for the permeation of ions other than H.sup.+. A study of the underlying mechanism of the high selectivity of the human voltage-gated proton channel reveals an Asp112 residue as the selectivity filter. When this residue was changed to a neutral amino acid, the mutant channel lost proton selectivity and either became anion-selective or did not conduct any ions. The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel.sup.1, H.sub.V1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes.sup.2,3,4, histamine secretion by basophils.sup.5, sperm capacitation.sup.6, and airway pH.sup.7. The most selective ion channel known, H.sub.V1 shows no detectable permeability to other ions.sup.1. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity.sup.1,8,9,10,11, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions.sup.12. Here we identify aspartate 112 as a crucial component of the selectivity filter of H.sub.V1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of H.sub.V1 requires an acidic group at the selectivity filter., Author(s): Boris Musset [sup.1] , Susan M. E. Smith [sup.2] , Sindhu Rajan [sup.3] , Deri Morgan [sup.1] , Vladimir V. Cherny [sup.1] , Thomas E. DeCoursey [sup.1] Author Affiliations: [...]

Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated [K.sup.+] (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia,J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853-858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PL[A.sub.2]) or the production of superoxide anion ([O.sub.2.sup..-]). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by [Zn.sup.2+] inhibition of NADPH oxidase activity assessed by [H.sub.2][O.sub.2] production, thus validating previous studies showing that [Zn.sup.2+] inhibited [O.sub.2.sup..-] production when assessed by cytochrome creduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and BK inhibitors did not impair antimicrobial activity. In contrast, we present additional evidence that voltage-gated proton channels serve the essential role of charge compensation during the respiratory burst.

Voltage-gated proton channels are expressed highly in rat alveolar epithelial cells. Here we investigated whether these channels contribute to pH regulation. The intracellular pH ([pH.sub.i]) was monitored using BCECF in cultured alveolar epithelial cell monolayers and found to be 7.13 in nominally HC[O.sub.3]-free solutions [at external pH ([pH.sub.o]) 7.4]. Cells were acid-loaded by the N[H.sup.+.sub.4] prepulse technique, and the recovery was observed. Under conditions designed to eliminate the contribution of other transporters that alter pH, addition of 10 [micro]M Zn[Cl.sub.2], a proton channel inhibitor, slowed recovery about twofold. In addition, the [pH.sub.i] minimum was lower, and the time to nadir was increased. Slowing of recovery by Zn[Cl.sub.2] was observed at [pH.sub.o] 7.4 and [pH.sub.o] 8.0 and in normal and high-[K.sup.+] Ringer solutions. The observed rate of [Zn.sup.2+]-sensitive [pH.sub.i] recovery required activation of a small fraction of the available proton conductance. We conclude that proton channels contribute to [pH.sub.i] recovery after an acid load in rat alveolar epithelial cells. Addition of Zn[Cl.sub.2] had no effect on [pH.sub.i] in unchallenged cells, consistent with the expectation that proton channels are not open in resting cells. After inhibition of all known pH regulators, slow [pH.sub.i] recovery persisted, suggesting the existence of a yet-undefined acid extrusion mechanism in these cells. proton conductance; pH regulation; hydrogen ion; acid load; 2',7'bis(2-carboxyethyl)-5(6)-carboxyfluorescein; intracellular pH

Author(s): Thomas E. DeCoursey (corresponding author); Deri Morgan; Vladimir V. Cherny The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O[sub.2 [...]

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion ([O.sub.2.sup.-]) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the [H.sup.+] efflux was thought to be contained within the [gp91.sup.phox] subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function ([COS.sub.phox]). The 7D5 antibody, which detects an extracellular epitope of the [gp91.sup.phox] protein, labeled 96-98% of [COS.sub.phox] cells. NADPH oxidase was functional because [COS.sub.phox] (but not [COS.sub.WT]) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells ([COS.sub.WT]) or [COS.sub.phox] cells studied at [pH.sub.o] 7.0 and [pH.sub.i] 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable [H.sup.+] current in [COS.sub.WT] or [COS.sub.phox] cells. Therefore, [gp91.sup.phox] does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase. KEY WORDS: phagocytes * [gp91.sup.phox * [H.sup.+] channels * superoxide * respiratory burst

Inhibition by polyalent 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(sub 2) reduced the H(super +) current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H(super +) 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(super 2+) were inconsistent with classical voltage-dependent block in which Zn(super 2+) binds within the membrane voltage field. Instead, Zn(super 2+) binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn(super 2+) were strongly pH(sub o) dependent but were insensitive to pH(sub i), suggesting that protons and Zn(super 2+) compete for external sites on H(super +) channels. The apparent potency of Zn(super 2+) in slowing activation was approximately 10X greater at pH(sub o) 7 than at pH(sub o) 6, and approximately 100X greater at pH(sub o), 6 than at pH(sub o) 5. The pH(sub o) dependence suggests that Zn(super 2+), not ZnOH(super +), is the active species. Evidently, the Zn(super 2+) receptor is formed by multiple groups, protonation of any of which inhibits Zn(super 2+) binding. The external receptor bound H(super +) and Zn(super 2+) with pK(sub a) 6.2-6.6 and pK(sub M) 6.5, as described by several models. Zn(super 2+) effects on the proton chord conductance-voltage (g(sub H)-V) relationship indicated higher affinities, pK(sub a) 7 and pK(sub M) 8. CdCl(sub 2) had similar effects as ZnCl(sub 2) and competed with H(super +), but had lower affinity. Zn(super 2+) applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H(super +) currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn(super 2+) receptor may be the same modulatory protonation site(s) at which pH(sub o) regulates H(super +) channel gating.