Zinc Inhibits Miniature GABAergic Currents by Allosteric Modulation of GABA_AReceptor Gating

Abstract

Zinc is abundantly present in the CNS, and after nerve stimulation is thought to be released in sufficient quantity to modulate the synaptic transmission. Although it is known that this divalent cation inhibits the GABAergic synaptic currents, the underlying mechanisms were not fully elucidated. Here we report that zinc reduced the amplitude, slowed the rise time, and accelerated the decay of mIPSCs in cultured hippocampal neurons. The analysis of current responses to rapid GABA applications and model simulations indicated that these effects on mIPSCs are caused by zinc modulation of GABA A receptor gating. In particular, zinc slowed the onset of GABA-evoked currents by decreasing both the binding (k on ) and the transition rate from closed to open state (␤ 2 ). Moreover, slower onset and recovery from desensitization as well as an increased unbinding rate (k off ) were shown to underlie the accelerated deactivation kinetics in the presence of zinc. The nonequilibrium conditions of GABA A receptor activation were found to strongly affect zinc modulation of this receptor. In particular, an extremely fast clearance of synaptic GABA is implicated to be responsible for a stronger zinc effect on mIPSCs than on current responses to exogenous GABA. Finally, the analysis of currents evoked by GABA coapplied with zinc indicated that the interaction between zinc and GABA A receptors was too slow to explain zinc effects in terms of competitive antagonism. In conclusion, our results provide evidence that inhibition of mIPSCs by zinc is attributable to the allosteric modulation of GABA A receptor gating.

Declusterization of GABAA Receptors Affects the Kinetic Properties of GABAergic Currents in Cultured Hippocampal Neurons

“…As already mentioned, the analysis of the current evoked in excised patches by ultrafast application of GABA allows to dissect out the kinetic properties of GABA A receptors under non-equilibrium conditions such as those occurring at the synapse (29,30). It should be stressed that upon patch excision the detached portion of the membrane is no longer interacting with the intracellular structural and functional proteins and/or intracellular factors.…”

“…Current responses to the second pulse reveal the fraction of receptors able to be activated when the second pulse is applied, and therefore they are influenced by the number of receptors that have recovered from the desensitized state. It should be stressed, however, that the recovery of the second peak reflects not only the recovery from desensitization but a more complex process, including multiple re-entries into the open and desensitized states due to the functional coupling between desensitization-resensitization, opening-closing, and unbinding, and therefore it reveals important information about the proportions between respective rate constants (28,30,31).…”

Binding Sites, Singly Bound States, and Conformation Coupling Shape GABA-Evoked Currents

“…It is known that a good fit to experimental data are obtained when opening and desensitizing transitions are allowed directly from the bound closed states (see e.g., Barberis et al 2000;Westbrook 1995, 1997;Li and Pearce 2000;McClellan and Twyman 1999;Mozrzymas et al 1999).…”

“…The estimated peak values of synaptic GABA concentrations range from hundreds of micromoles to several millimoles (Jones and Westbrook 1995;Maconochie et al 1994;Mozrzymas et al1999;Nusser et al 2001;Perrais and Ropert 1999), and it is still not clear whether synaptic GABA reaches saturating concentrations (Frerking and Wilson 1996;Hajos et al 2000;Mody et al 1994;Nusser et al 2001Perrais andRopert 1999). Synaptic transmission is thus a dynamic, nonequilibrium process that depends on changes in synaptic agonist concentration, which in turn affects the time course and pharmacology of IPSCs (Barberis et al 2000;Mozrzymas et al 1999;Nusser et al 2001). For a comprehensive description of IPSC mechanisms, it is necessary to mimic the dynamics of the synaptic agonist transient.…”

“…This becomes possible by using piezoelectric-driven perfusion systems (Franke et al 1987;Jonas 1995), which are capable of applying the agonist in the time scale comparable to that of synaptic neurotransmitter transient. However, large differences in the rise times of responses to saturating [GABA] (Barberis et al 2000;Jones and Westbrook 1995;Mozrzymas et al 1999;Perrais and Ropert 1999) raise the possibility that the application speed may significantly affect the measured currents.…”