Regulation of recombinant GABA(A) receptor function by calcium ion/phospholipid-dependent protein kinase (PKC) phosphorylation.
Lin, Yu-Fung
1995
Abstract
The $\gamma$-aminobutyric acid type A receptor (GABAR) is a hetero-oligomeric protein possessing an intrinsic chloride ion channel. It has been shown that the $\beta$1 and $\gamma$2L subunits of the GABAR contain phosphorylation sites for PKC. To determine the effect of PKC on GABAR function, whole-cell recordings were obtained from mouse fibroblasts expressing recombinant $\rm\alpha1\beta1\gamma2L$ GABARs and the constitutively active PKC (PKM, 40 nM) was applied via the recording pipette. PKM increased GABAR currents, which was not obtained with co-application of the PKC inhibitory peptide (PKC-I) and PKM. PKM produced an enhancement of the maximal GABA current (100% increase) in a reimpalement study. PKM increased GABAR currents only at high ($>$10 $\mu$M) GABA concentrations, resulting in increases in both EC$\sb{50}$ and maximal GABAR current. To determine whether this enhancement was due to direct phosphorylation of the consensus PKC phasphorylation sites contained in the $\beta$1 (serine 409) and $\gamma$2L (serines 327 and 343) GABAR subunit, site-directed mutagenesis was used to replace specific serines with alanines. Reimpalement studies were performed. No enhancement of the maximal GABA current was produced by PKM in cells expressing $\rm\alpha1\beta1(S409A)\gamma2L$(S327A,S343A) GABARs in which all three PKC phosphorylation sites had been removed. However, enhancement of maximal GABA responses was present at a reduced level in cells expressing GABARs with the $\gamma$2L subunit phosphorylation sites removed ($\rm\alpha1\beta1\gamma2L$(S327A,S343A) GABARs) (47% increase) or with the $\beta$1 subunit phosphorylation site removed $\rm(\alpha1\beta1(S409A)\gamma 2L$ GABARs) (25% increase), indicating that both $\beta$1 and $\gamma$2L subunits are required for a complete, PKC-mediated enhancement with phosphorylation of S409 in $\beta$1 having a greater effect on GABAR currents. To elucidate the molecular mechanism of this enhancement, inside-out patch single-channel recordings were performed on wild-type or mutant recombinant $\rm\alpha1\beta1\gamma2L$ GABARs. PKM treatment increased both the mean open duration and opening frequency of GABA-gated openings on wild-type GABAR channels. No increases were produced, however, with coapplication of PKC-I and PKM. In addition, the PKM effects did not occur in mutant $\rm\alpha1\beta1(S409A)\gamma2L(S327A,S343A)$ GABAR channels with all three putative PKC phosphorylation sites modified. In conclusion, these data suggest that PKC phosphorylation enhances recombinant $\alpha1\beta1\gamma2L$ GABAR function by altering GABAR single-channel gating properties.Other Identifiers
(UMI)AAI9527686
Subjects
Biology, Neuroscience Biology, Animal Physiology
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Thesis
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