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  • Ca(2+)/Calmodulin kinase II and decreases in intracellular pH are required to activate K(+) channels after substantial swelling in villus epithelial cells.

Ca(2+)/Calmodulin kinase II and decreases in intracellular pH are required to activate K(+) channels after substantial swelling in villus epithelial cells.

The Journal of membrane biology (1999-11-07)
R J MacLeod, J R Hamilton
ZUSAMMENFASSUNG

To assess the activation of the charybdotoxin-insensitive K(+) channel responsible for Regulatory Volume Decrease (RVD) after substantial volume increases, we measured intracellular pH (pH(i)), intracellular calcium ([Ca(2+)](i)) and inhibitors of kinases and phosphoprotein phosphatases in guinea pig jejunal villus enterocytes in response to volume changes. Fluorescence spectroscopy was used to measure pH(i) and [Ca(2+)](i) of cells in suspension, loaded with 2, 7,bis-carboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively, and cell volume was assessed using electronic cell sizing. A modest 7% volume increase or substantial 15 to 20% volume increase caused [Ca(2+)](i) to increase proportionately but the 7% increase caused alkalinization while the larger increases resulted in acidification of approximately 0.14 pH units. Following a 15% volume increase, 1-N-0-bis (5-isoquinoline-sulfonyl)-N-methyl-l-4-phenyl-piperazine (KN-62, 50 microm), an inhibitor of Ca(2+)/calmodulin kinase II, blocked RVD. Gramicidin (0.5 microm bypassed this inhibition suggesting that the K(+) channel had been affected by the KN-62. RVD after a modest 7% volume increase was not influenced by KN-62 unless the cell was acidified. Okadaic acid, an inhibitor of phosphoprotein phosphatases 1 and 2A, accelerated RVD after a 20% volume increase; inhibition of RVD generated by increasing the K(+) gradient was bypassed by okadaic acid. Tyrosine kinase inhibitor, genistein (100 microm) had no effect on RVD after 20% volume increases. We conclude that activation of charybdotoxin-insensitive K(+) channels utilized for RVD after substantial (>7%) 'nonphysiological' volume increases requires phosphorylation mediated by Ca(2+)/calmodulin kinase II and that increases in cytosolic acidification rather than larger increases in [Ca(2+)](i) are a critical determinant of this activation.

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Sigma-Aldrich
KN-62, ≥95%, powder