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 channel1, hHV1. This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes2–4, histamine secretion by basophils5, sperm capacitation6, and airway pH7. The most selective ion channel known, HV1 shows no detectable permeability to other ions1. Opposing classes of selectivity mechanisms postulate that (a) a titratable amino acid residue in the permeation pathway imparts proton selectivity1, 8–11, or (b) water molecules “frozen” in a narrow pore conduct protons while excluding other ions12. Here we identify Aspartate112 as a crucial component of the selectivity filter of hHV1. When a neutral amino acid replaced Asp112, 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 Asp185 did not impair proton selectivity, suggesting that Asp112 plays a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp112, the mutant channel was still anion permeable. Evidently, the proton specificity of hHV1 requires an acidic group at the selectivity filter.

Tandem pore domain acid-sensitive K ؉ channel 3 (TASK-3) is a new member of the tandem pore domain potassium channel family. A cDNA encoding a 365-amino acid polypeptide with four putative transmembrane segments and two pore regions was isolated from guinea pig brain. An orthologous sequence was cloned from a human genomic library. Although TASK-3 is 62% identical to TASK-1, the cytosolic C-terminal sequence is only weakly conserved. Analysis of the gene structure identified an intron within the conserved GYG motif of the first pore region. Reverse transcriptase-polymerase chain reaction analysis showed strong expression in brain but very weak mRNA levels in other tissues. Cellattached patch-clamp recordings of TASK-3 expressed in HEK293 cells showed that the single channel currentvoltage relation was inwardly rectifying, and open probability increased markedly with depolarization. Removal of external divalent cations increased the mean single channel current measured at ؊100 mV from ؊2.3 to ؊5.8 pA. Expression of TASK-3 in Xenopus oocytes revealed an outwardly rectifying K ؉ current that was strongly decreased in the presence of lower extracellular pH. Substitution of the histidine residue His-98 by asparagine or tyrosine abolished pH sensitivity. This histidine, which is located at the outer part of the pore adjacent to the selectivity filter, may be an essential component of the extracellular pH sensor.

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