Different sensitivities of human and rat rho(1) GABA receptors to extracellular pH.
We have examined the sensitivity of human and rat homo-oligomeric rho(1) GABA receptors to variations in extracellular pH (pH(o)) using the whole-cell patch clamp technique. The GABA-induced conductance mediated by the rat rho(1) receptor (rho(1)-R) decreased with a decrease in pH(o) between 9.0 to 5.4. Below pH(o) 7.4 the effect of protons on the GABA-induced conductance was apparently competitive, but above pH(o) 7.4 the inhibitory effect of extracellular protons was almost independent on the GABA concentration. Titration of the GABA-induced conductance at 3 microM GABA revealed two protonation sites on rat rho(1)-R with pKa 6.4 and pKa 8.2. At 10 microM GABA the low pKa (6.4) was shifted to a clearly lower value (5.6), but the high pKa was only slightly decreased (from 8.2 to 7.9). Zn(2+) ions were capable of relieving the proton inhibition at low pH(o) indicating that Zn(2+) interacts with the low pKa site. Unlike the rat rho(1)-R, the human rho(1)-R was sensitive only to changes in pH(o) at acidic levels. Proton inhibition of human rho(1)-R was apparently competitive, as observed on rat-rho(1) at acidic pH(o). Titration of the human rho(1)-R gave a single H(+) binding site with a pKa of 6.3, similar to the value for the low pKa on rat rho(1)-R. The pKa value of human rho(1)-R was not dependent on the GABA concentration. A chimeric receptor, consisting of the N-terminal part of the rat rho(1)-R and C-terminal part of the human rho(1)-R, displayed pH(o) sensitivity similar to that observed for rat rho(1)-R. This indicates that the high pKa of rat rho(1)-R is attributable to the 11 amino acid differences between the rat and human rho(1)-R extracellular domains.
Rivera
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Department of Biosciences, Division of Animal Physiology, P.O. Box 17, FIN-00014 University of Helsinki, Finland.
Wegelius
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Reeben
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Kaila
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Michael Pasternack
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004553:Electric Conductivity; D005110:Extracellular Space / Q000737:chemistry / Q000378:metabolism; D018756:GABA Antagonists / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D011522:Protons; D051381:Rats; D018079:Receptors, GABA / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D018756:GABA Antagonists; C106350:GABRR2 protein, human; C491397:Gabrr2 protein, rat; D011522:Protons; D018079:Receptors, GABA; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; D011993:Recombinant Fusion Proteins; D015032:Zinc