Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.
cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.
Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Ohio 45267.
D H
DH
Malinowska
E Y
EY
Kupert
A
A
Bahinski
A M
AM
Sherry
J
J
Cuppoletti
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002384:Catalysis; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005753:Gastric Mucosa / Q000378:metabolism*; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins; D014982:Xenopus laevis
D018118:Chloride Channels; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases