The activation mechanism of alpha 1 homomeric glycine receptors

The glycine receptor mediates fast synaptic inhibition in the spinal cord and brainstem. Its activation mechanism is not known, despite the physiological importance of this receptor and the fact that it can serve as a prototype for other homopentameric channels. We analyzed single-channel recordings from rat recombinant alpha1 glycine receptors by fitting different mechanisms simultaneously to sets of sequences of openings at four glycine concentrations (10-1000 muM). The adequacy of the mechanism and the rate constants thus fitted was judged by examining how well these described the observed dwell-time distributions, open-shut correlation, and single-channel P-open dose-response curve. We found that gating efficacy increased as more glycine molecules bind to the channel, but maximum efficacy was reached when only three (of five) potential binding sites are occupied. Successive binding steps are not identical, implying that binding sites can interact while the channel is shut. These interactions can be interpreted in the light of the topology of the binding sites within a homopentamer

Human alpha 3 beta 4 Neuronal Nicotinic Receptors Show Different Stoichiometry if They Are Expressed in Xenopus Oocytes or Mammalian HEK293 Cells

Background: The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the alpha 3 and beta 4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the alpha 5 and/or beta 2 subunits. We compared the properties of human alpha 3 beta 4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and alpha:beta subunit ratio.Methodology/Principal Findings: Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the alpha subunit, as reported for alpha 4 beta 2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9' of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike alpha 4 beta 2 channels, for alpha 3 beta 4 receptors the putative two-alpha form is the predominant one in oocytes (at 1:1 alpha:beta cRNA ratio). This two-alpha form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-alpha form is the predominant one in HEK cells transfected with a 1:1 alpha:beta DNA ratio or in oocytes at 9:1 alpha:beta RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-alpha form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-alpha form gave rise to short bursts (14 ms) of high conductance (39 pS).Conclusions/Significance: Like other neuronal nicotinic receptors, the alpha 3 beta 4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected