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 potential (E(Cl)) and the consequent role of this conductance in determining the effect of the gamma-aminobutyric acid-A (GABA(A)) receptor-mediated 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 rectifying, with significant conductance only at membrane potentials more negative than E(Cl). For all tested neuronal Cl- concentrations, the activation of G(Cl(V)) could be described by a Boltzman equation with an average 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-dependent inactivation of G(Cl(V)). 3. G(Cl(V)) was modulated by intracellular divalent cations. When magnesium was omitted from the electrode solution, the inward rectification of G(Cl(V)) was unchanged, but the maximum amplitude of G(Cl(V)) increased by a factor of 1.7. G(Cl(V)) was blocked by bath application of 100 mu M zinc (Zn2+), but not when 1-6 mM 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 blocked 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, suggesting that under normal conditions Ga-Cl(V) is blocked by an endogenous divalent 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 Cl-). When G(Cl(V)) was blocked with PdBu, there was no change in the RMP with Cl- loading. When electroneutral Cl- transport was blocked, voltage-clamp experiments using electrode Cl- concentrations of 4-131 mM demonstrated that E(Cl) changed in parallel with the holding potential, but not when G(Cl(V)) was blocked by PdBu. 7. When pyramidal neurons were maximally loaded with Cl-, the Cl- driving force was 15 mV, and GABA(A) receptor-mediated spontaneous and evoked synaptic activity never 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, and GABA(A) receptor activation always triggered action potentials. 8. G(Cl(V)) is a large, noninactivating, inwardly rectifying Cl- conductance that operates independently of electroneutral Cl- transport to stabilize the relationship between E(Cl) and the resting membrane potential. Down-regulation of G(Cl(V)) is thus a necessary condition for excitatory GABA(A) receptor-mediated postsynaptic Cl- flux.