Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl- channel.
Rabbit and human ClC-2G Cl- channels are voltage sensitive and activated by protein kinase A and low extracellular pH. The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G Cl- channel and to determine which amino acid residues play a role in this acid activation. Channel open probability (Po) at +/-80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H+ concentration (that is, extracellular pH, pHtrans), with an apparent acidic dissociation constant of pH 4.95 +/- 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increased Po threefold at pHtrans 7.4, at which the channel normally exhibits low Po. With extracellular pH reduction (protonation) or amidation, increased Po was due to a significant increase in open time constants and a significant decrease in closed time constants of the channel gating, and this effect was insensitive to applied voltage. With the use of site-directed mutagenesis, the extracellular region EELE (amino acids 416-419) was identified as the pH sensor and amino acid Glu-419 was found to play the key or predominant role in activation of the ClC-2G Cl- channel by extracellular acid.
Stroffekova
K
K
Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576, USA.
Kupert
E Y
EY
Malinowska
D H
DH
Cuppoletti
J
J
D000818:Animals; D000090007:CLC-2 Chloride Channels; D002234:Carbodiimides / Q000494:pharmacology; D018118:Chloride Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D018075:RNA, Complementary; D011817:Rabbits; D014982:Xenopus laevis
D000090007:CLC-2 Chloride Channels; D002234:Carbodiimides; D018118:Chloride Channels; D018075:RNA, Complementary; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; D004533:Egtazic Acid; D017868:Cyclic AMP-Dependent Protein Kinases