Partially active channels produced by pka site mutation of the
cloned renal k+ channel romk2 (kir1.2).
Macgregor, Gordon G., Jason Z. Xu, Carmel M. McNicholas, Gerhard
Giebisch and Steven C. Hebert.
Department of Cellular and Molecular Physiology1, Yale University,
New Haven, CT and Division of Nephrology2, Vanderbilt University,
Nashville, TN.
APStracts 5:0107F, 1998.
The activity of the cloned renal K+ channel (ROMK2) is dependent on a
balance between phosphorylation and dephosphorylation. There are only
three protein kinase A (PKA) sites on ROMK2, the phosphorylated
residues being S25, S200 and S294 (35). We previously mutated these
sites from serine to alanine to study the contribution of each site
to overall channel function. Here we have studied each of these
single PKA-site mutants using the single-channel configuration of the
patch clamp technique. Both C-terminal mutations at sites S200A and
S294A showed a decreased open channel probability (Po) while the N
-terminal mutation at site S25A showed no change in Po compared to
wild-type ROMK2. The decrease in Po for the S200A and S294A mutants
was caused by the additional presence of a long closed state. In
contrast, the occurrence of the S25A channel was about 66% less,
suggesting fewer active channels at the membrane. The S200A and S294A
channels had different kinetics compared to wild-type ROMK2 channels,
showing an increased occurrence of sublevels. Similar kinetics were
observed when wild-type ROMK2 was excised and exposed to
dephosphorylating conditions indicating that these effects are
specifically a property of the partially phosphorylated channel and
not due to an unrelated effect of the mutation.
Received 10 March 1998; accepted in final form 8 June 1998.
APS Manuscript Number F61-8.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 16 June 1998