Krashia_2011_J.Gen.Physiol_137_197

The alpha2 glycine receptor (GlyR) subunit, abundant in embryonic neurons, is replaced by alpha1 in the adult nervous system. The single-channel activity of homomeric alpha2 channels differs from that of alpha1-containing GlyRs, as even at the lowest glycine concentration (20 microM), openings occurred in long (>300-ms) groups with high open probability (P(open); 0.96; cell-attached recordings, HEK-expressed channels). Shut-time intervals within groups of openings were dominated by short shuttings of 5-10 micros. The lack of concentration dependence in the groups of openings suggests that they represent single activations, separated by very long shut times at low concentrations. Several putative mechanisms were fitted by maximizing the likelihood of the entire sequence of open and shut times, with exact missed-events allowance (program hjcfit). Records obtained at several glycine concentrations were fitted simultaneously. The adequacy of the different schemes was judged by the accuracy with which they predicted not only single-channel data but also the time course and concentration dependence of macroscopic responses elicited by rapid glycine applications to outside-out patches. The data were adequately described only with schemes incorporating a reaction intermediate in the activation, and the best was a flip mechanism with two binding sites and one open state. Fits with this mechanism showed that for alpha2 channels, the opening rate constant is very fast, approximately 130,000 s(-1), much as for alpha1beta GlyRs (the receptor in mature synapses), but the estimated true mean open time is 20 times longer (around 3 ms). The efficacy for the flipping step and the binding affinity were lower for alpha2 than for alpha1beta channels, but the overall efficacies were similar. As we previously showed for alpha1 homomeric receptors, in alpha2 glycine channels, maximum P(open) is achieved when fewer than all five of the putative binding sites in the pentamer are occupied by glycine.