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TASK2 is a member of the two-pore domain [K.sup.+] channel family that plays a role in acid-base homeostasis; TASK2 knockout animals have plasma electrolyte patterns typical of the human clinical condition of renal tubular acidosis. It is expressed preferentially in epithelia, including the proximal tubules of the kidney. In common with the other TASK channels, TASK2 is sensitive to changes in extracellular pH, although the molecular mechanism of such pH sensing is not understood. We have examined the role of charged residues in the extracellular domains in pH sensing using a mutational approach. Mutant channels were expressed in CHO cells and studied by whole-cell and single-channel patch clamp. Neutralization of no single amino acid in isolation gave complete loss of pH sensitivity. However, the combined removal of five charged amino acids in the large extracellular loop linking the first transmembrane and pore domains, the M1-P1 loop, resulted in an essentially pH-insensitive channel, stabilized in the open state. Wild-type channels contain two such loops, but a concatemeric construct, comprised of one wild-type subunit and one containing the five mutations, was fully pH-sensitive, indicating that only one M1-P1 loop is required to yield a fully pH-sensitive channel, demonstrating a regulatory role of this distinctive structure in two-pore domain [K.sup.+] channels. Thus, pH sensing in TASK2 channels is conferred by the combined action of several charged residues in the large extracellular M1-P1 loop. potassium channel | regulation | acid | proton | gating

TASK-2 is a member of the two-pore domain [K.sup.+] (K2P) channel family that is expressed at high levels in several epithelia, including the proximal tubule. In common with the other TASK channels, TASK-2 is sensitive to changes in extracellular pH. We have expressed human TASK-2 in Chinese hamster ovary cells and studied whole cell and single-channel activity by patch clamp. The open probability of [K.sub.2P] channels is generally independent of voltage, yielding linear current-voltage (I-V) curves. Despite these properties, we found that these channels showed distinct inward rectification immediately on the establishment of whole cell clamp, which became progressively less pronounced with time. This rectification was due to intracellular [Na.sup.+] but was unaffected by polyamines or [Mg.sup.2+] (agents that cause rectification in Kir channels). Rectification was concentration- and voltage-dependent and could be reversibly induced by switching between [Na.sup.+]-rich and [Na.sup.+]-free bath solutions. In excised inside-out patches, [Na.sup.+] reduced the amplitude of single-channel currents, indicative of rapid block and unblock of the pore. Mutations in the selectivity filter abolished [Na.sup.+]-induced rectification, suggesting that [Na.sup.+] binds within the selectivity filter in wild-type channels. This sensitivity to intracellular [Na.sup.+] may be an additional potential regulatory mechanism of TASK-2 channels. intracellular sodium; voltage dependent; pore block

TASK2 is a member of the two-pore domain K+ channel family that plays a role in acid-base homeostasis; TASK2 knockout animals have plasma electrolyte patterns typical of the human clinical condition of renal tubular acidosis. It is expressed preferentially in epithelia, including the proximal tubules of the kidney. In common with the other TASK channels, TASK2 is sensitive to changes in extracellular pH, although the molecular mechanism of such pH sensing is not understood. We have examined the role of charged residues in the extracellular domains in pH sensing using a mutational approach. Mutant channels were expressed in CHO cells and studied by whole-cell and single-channel patch clamp. Neutralization of no single amino acid in isolation gave complete loss of pH sensitivity. However, the combined removal of five charged amino acids in the large extracellular loop linking the first transmembrane and pore domains, the M1-P1 loop, resulted in an essentially pH-insensitive channel, stabilized in the open state. Wild-type channels contain two such loops, but a concatemeric construct, comprised of one wild-type subunit and one containing the five mutations, was fully pH-sensitive, indicating that only one M1-P1 loop is required to yield a fully pH-sensitive channel, demonstrating a regulatory role of this distinctive structure in two-pore domain K+ channels. Thus, pH sensing in TASK2 channels is conferred by the combined action of several charged residues in the large extracellular M1-P1 loop. [ABSTRACT FROM AUTHOR]

TASK-2 is a member of the two-pore domain K+ (K2P) channel family that is expressed at high levels in several epithelia, including the proximal tubule. In common with the other TASK channels, TASK-2 is sensitive to changes in extracellular pH. We have expressed human TASK-2 in Chinese hamster ovary cells and studied whole cell and single-channel activity by patch clamp. The open probability of K2p channels is generally independent of voltage, yielding linear currentvoltage (I-V) curves. Despite these properties, we found that these channels showed distinct inward rectification immediately on the establishment of whole cell clamp, which became progressively less pronounced with time. This rectification was due to intracellular Na+ but was unaffected by polyamines or Mg2+ (agents that cause rectification in Kir channels). Rectification was concentration- and voltage-dependent and could be reversibly induced by switching between Na+-rich and Na+-free bath solutions. In excised inside-out patches, Na+ reduced the amplitude of single-channel currents, indicative of rapid block and unblock of the pore. Mutations in the selectivity filter abolished Na+-induced rectification, suggesting that Na+ binds within the selectivity filter in wild-type channels. This sensitivity to intracellular Na+ may be an additional potential regulatory mechanism of TASK-2 channels. [ABSTRACT FROM AUTHOR]