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Titolo:
PKA site mutations of ROMK2 channels shift the pH dependence to more alkaline values
Autore:
Leipziger, J; MacGregor, GG; Cooper, GJ; Xu, J; Hebert, SC; Giebisch, G;
Indirizzi:
Yale Univ, Dept Cellular & Mol Physiol, New Haven, CT 06520 USA Yale UnivNew Haven CT USA 06520 r & Mol Physiol, New Haven, CT 06520 USA Vanderbilt Univ, Div Nephrol, Nashville, TN 37322 USA Vanderbilt Univ Nashville TN USA 37322 v Nephrol, Nashville, TN 37322 USA
Titolo Testata:
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
fascicolo: 5, volume: 279, anno: 2000,
pagine: F919 - F926
SICI:
0363-6127(200011)279:5<F919:PSMORC>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
CORTICAL COLLECTING DUCT; CONDUCTANCE K+ CHANNEL; MEMBRANE PATCHES; PROTEIN-KINASE; PHOSPHORYLATION; EXPRESSION; CLONING; CELLS;
Keywords:
phosphorylation; potassium ion channel; inward-rectifier; potassium ion secretion; intracellular pH;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
22
Recensione:
Indirizzi per estratti:
Indirizzo: Leipziger, J Univ Freiburg, Inst Physiol, Hermann Herder Str 7, D-79104 Freiburg, Germany Univ Freiburg Hermann Herder Str 7 Freiburg Germany D-79104
Citazione:
J. Leipziger et al., "PKA site mutations of ROMK2 channels shift the pH dependence to more alkaline values", AM J P-REN, 279(5), 2000, pp. F919-F926

Abstract

Close similarity between the rat native low-conductance K+ channel in the apical membrane of renal cortical collecting duct principal cells and the cloned rat ROMK channel strongly suggest that the two are identical. Prominent features of ROMK regulation are a steep pH dependence and activation by protein kinase A (PKA)-dependent phosphorylation. In this study, we investigated the pH dependence of cloned renal K+ channel (ROMK2), wild-type (R2-WT), and PKA site mutant channels (R2-S25A, R2-S200A, and R2-S294A). Ba2+-sensitive outward whole cell currents (holding voltage -50 mV) were measured in two-electrode voltage-clamp experiments in Xenopus laevis oocytes expressing either R2-WT or mutant channels. Intracellular pH (pH(i)) was measuredwith pH-sensitive microelectrodes in a different group of oocytes from thesame batch on the same day. Resting pH(i) of R2-WT and PKA site mutants was the same: 7.32 +/- 0.02 (n = 22). The oocytes were acidified by adding 3 mM Na butyrate with external pH (pH(o)) adjusted to 7.4, 6.9, 6.4, or 5.4. At pHo 7.4, butyrate led to a rapid (tau: 163 +/- 14 s, where t means time constant, n = 4) and stable acidification of the oocytes (Delta pH(i) 0.13 /- 0.02 pH units, where Delta means change, n = 12). Intracellular acidification reversibly inhibited ROMK2-dependent whole cell current. The effective acidic dissociation constant (pK(a)) value of R2-WT was 6.92 +/- 0.03 (n= 8). Similarly, the effective pK(a) value of the N-terminal PKA site mutant R2-S25A was 6.99 +/- 0.02 (n = 6). The effective pK(a) values of the twoCOOH-terminal PKA site mutant channels, however, were significantly shifted to alkaline values; i. e., 7.15 +/- 0.06 (n = 5) for R2-S200A and 7.16 +/- 0.03 (n = 8) for R2-S294A. The apparent Delta pH shift between the R2-WT and the R2-S294A mutant was 0.24 pH units. In excised inside-out patches, alkaline pH 8.5 activated R2-S294A channel current by 32 +/- 6.7%, whereas in R2-WT channel patches alkalinzation only marginally increased current by 6.5 +/- 1% (n = 5). These results suggest that channel phosphorylation may substantially influence the pH sensitivity of ROMK2 channel. Our data are consistent with the hypothesis that in the native channel PKA activation involves a shift of the pK(a) value of ROMK channels to more acidic values, thus relieving a H+-mediated inhibition of ROMK channels.

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Documento generato il 26/11/20 alle ore 11:45:21