THE ROLE OF AN INWARDLY RECTIFYING CHLORIDE CONDUCTANCE IN POSTSYNAPTIC INHIBITION

1. The relationship of the activation of a voltage-sensitive chloride conductance [ G(Cl(V))] to the chloride transmembrane equilibrium pote ntial (E(Cl)) and the consequent role of this conductance in determini ng the effect of the gamma-aminobutyric acid-A (GABA(A)) receptor-medi ated transmembrane chloride (Cl-) flux were investigated with the use of whole-cell recordings in the CA1 and dentate gyrus regions of adult rat hippocampal slice preparations. 2. G(Cl(V)) was inwardly rectifyi ng, with significant conductance only at membrane potentials more nega tive than E(Cl). For all tested neuronal Cl- concentrations, the activ ation of G(Cl(V)) could be described by a Boltzman equation with an av erage half-activation voltage 15 mV negative to E(Cl), a slope factor of 14 mV, and a maximum conductance of 5 nS. There was no time-depende nt inactivation of G(Cl(V)). 3. G(Cl(V)) was modulated by intracellula r divalent cations. When magnesium was omitted from the electrode solu tion, the inward rectification of G(Cl(V)) was unchanged, but the maxi mum amplitude of G(Cl(V)) increased by a factor of 1.7. G(Cl(V)) was b locked by bath application of 100 mu M zinc (Zn2+), but not when 1-6 m M ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid(BAPTA) were present in the electrode solution. 4. G(Cl(V)) was increased by 10 mu M norepinephrine, and by activation of protein kinase A (PKA) with 1 mM 8-bromoadenosine cyclic monophosphate (8-Br cAMP). G(Cl(V)) was blo cked by activation of protein kinase C(PKC) with 10 mu M phorbol 12,13 -dibutyrate (PdBu) or 1-oleoyl-2-acetyl-sn-glycerol (OAG). 5. G(Cl(V)) was present in all tested CA1 pyramidal neurons but no dentate gyrus neurons. In standard extracellular solution, the amplitude of G(Cl(V)) was initially negligible but increased with recording time, suggestin g that under normal conditions Ga-Cl(V) is blocked by an endogenous di valent cation or downregulated by PKC. 6. In current-clamp recordings, the steady-state resting membrane potential(RMP) diminished with Cl- loading, from -73 mV (4 mM electrode Cl-) to -27 mV(131 mM electrode C l-). When G(Cl(V)) was blocked with PdBu, there was no change in the R MP with Cl- loading. When electroneutral Cl- transport was blocked, vo ltage-clamp experiments using electrode Cl- concentrations of 4-131 mM demonstrated that E(Cl) changed in parallel with the holding potentia l, but not when G(Cl(V)) was blocked by PdBu. 7. When pyramidal neuron s were maximally loaded with Cl-, the Cl- driving force was 15 mV, and GABA(A) receptor-mediated spontaneous and evoked synaptic activity ne ver triggered action potentials when G(Cl(V)) was upregulated. When G( Cl(V)) was blocked by PdBu or Zn2+, the Cl- driving force was 48 mV, a nd GABA(A) receptor activation always triggered action potentials. 8. G(Cl(V)) is a large, noninactivating, inwardly rectifying Cl- conducta nce that operates independently of electroneutral Cl- transport to sta bilize the relationship between E(Cl) and the resting membrane potenti al. Down-regulation of G(Cl(V)) is thus a necessary condition for exci tatory GABA(A) receptor-mediated postsynaptic Cl- flux.