ABBREVIATIONS: nAchR, nicotinic acetylcholine receptor; EGTA, ethylene glycol bis(8-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; GABA, ‘p’- aminobutync acid; GABAR, -aminobutync acid type A receptor; HEPES, 4-(2-hydroxyethyl)-1-piperarmneethanesulfonic acid; PKA, cydic AMP- dependent protein kinase; PKC, protein kinase C; NMDA, N-methyl-D-aspartate; AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionate. 1202 0026-895X/93/061202-09$03.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 44:1202-1210 Enhancement of Recombinant ‘y-Aminobutyric Acid Type A Receptor Currents by Chronic Activation of cAMP-Dependent Protein Kinase TIMOTHY P. ANGELOTTI, MICHAEL D. UHLER, and ROBERT L. MACDONALD Departments of Pharmacology (T.P.A.), Biological Chemistry (M.D.U.), Neurology (R.L.M.), and Physiology (R.L.M.) and The Mental Health Research Institute (M.D.U.), University of Michigan Medical School, Ann Arbor, Michigan 48109 Received August 2, 1 993; Accepted September 7, 1993 SUMMARY Ul , f31 , and ‘y2S ‘y-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNAs were transiently expressed in derivative cell lines of mouse L929 fibrobiasts, which possessed different levels of the catalytic subunit of cAMP-dependent protein kinase (PKA). These cell lines included L929 (intermediate levels of kinase), Ca12 (elevated levels of kinase), and RAB1O (low levels of kinase) cells. Pharmacological analysis of GABA-evoked whole-cell currents revealed that, compared with expression in L929 and RABI 0 cells, expression of al /31 y2S GABARs in Cal 2 cells produced a selective enhancement of single whole-cell current amplitudes. No other pharmacological properties (Hill slope, EC, or diazepam sensitivity) of the expressed al /31 ‘y2S GABARs were modified. The GABAR current enhancement in Cal 2 cells was blocked by substitution of a flu subunit mutated at the PKA consensus phosphorylation site, Ser#{176} [$1 (S409A)], for the wild-type 31 subunit. Interestingly, enhancement was specific for GABARs containing all three subunits, because it was not seen after expression of al flu or al $1(S409A) GABAR subunit combinations. Single-channel conductance and gating properties were not different for al 131y2S or al $1(S409A)’y2S GABARs expressed in each cell line, suggesting that PKA did not enhance whole-cell currents by altering these properties of GABARs. These results suggested that unlike acute application of PKA, which has been shown to produce a decrease in GABAR current, chronic elevation of PKA activity can result in enhance- ment of GABAR currents. More importantly, this effect occurred only with GABARs composed of al f31 ‘y2S subunits and not cd $1 subunits and was mediated by a single amino acid residue (Ser#{176}) of the 31 subunit. Rapid modulation of ligand-gated ion channel function by phosphorylation has been shown to occur through several dif- ferent mechanisms. For example, tyrosine phosphorylation of the nAchR increased the rapid phase of receptor desensitization (1). Enhancement of native glycine (2) and non-NMDA recep- tor currents by PKA (3, 4) and of recombinant kainate/AMPA and NMDA receptor currents by PKA and PKC, respectively, has also been demonstrated (5-7). In all cases, enhancement was due mainly to an increase in single-channel opening fre- quency (3, 4, 7) and/or mean open duration (4). Alternatively, phosphorylation has been shown to affect the biochemistry of receptor assembly and degradation. Claudio This work was supported by a grant from the Lucille P. Markey Charitable Trust Fund and a United States Public Health Service Grant (P01 NS19163) to R.L.M. T.P.A. is the recipient of a Pharmaceutical Manufacturers Association Foundation Predoctoral Fellowship and a National Institutes of Health Training Grant Fellowship (GM 07767-14). This work is submitted in partial fulfillment of the requirements for the degree of Doctorate of Philosophy (Pharmacology) (T.P.A.). and colleagues (8, 9) have provided evidence supporting a role for cAMP, acting through PKA activation, in up-regulating nAchR expression by increasing the efficiency of receptor as- sembly from its constituent subunits. Increased expression was correlated with phosphorylation of the ‘y subunit (10). Using chick ciliary ganglion neurons, it was shown that cAMP may also regulate the levels of functional nAchRs on the cell surface (11). Additionally, it has been shown that cAMP can stabilize junctional nAchRs, preventing their degradation (12). Initial biochemical analysis of GABAR phosphorylation re- vealed that fi subunits could be phosphorylated in vitro by PKA (13, 14). Subsequent studies of GABAR modulation by PKA phosphorylation have revealed conflicting outcomes. GABA- gated Cl flux in synaptosomes was reported to be decreased by treatment with cAMP analogs and forskolin (15-17). Other experiments showed no effect of activators of PKA on seCl_ flux desensitization (18). Electrophysiological analysis of GA- BAR current modulation by externally applied cAMP analogs initially revealed a decrease in whole-cell current amplitudes at Stanford Univ Med Ctr Lane Med Lib/Periodical Dept/Rm L109 on June 9, 2013 molpharm.aspetjournals.org Downloaded from