Previously, we showed that ethanol increases synaptic glycine currents, an effect that depends on ethanol concentration and developmental age of the preparation. Glycine receptor (GlyR) subunits undergo a shift from α2/β to α1/β from neonate to juvenile ages, with synaptic glycine currents from neonate hypoglossal motoneurons (HMs) being less sensitive to ethanol than those from juvenile HMs. Here we investigate whether these dose and developmental effects are also present in excised membrane patches containing GlyRs and if ethanol changes response kinetics. We excised outside-out patches from rat HM somata and applied glycine using either a picospritzer or piezo stack translator. Ethanol (100 mM) increased the response to glycine (200 μM) of patches from neonate and juvenile HMs. However, 30 mM ethanol increased the response from only juvenile HM patches. Using a lower concentration of glycine (30 μM) to observe single channel openings, we found that 100 mM ethanol increased the number of GlyRs that open in response to glycine and decreased first latency to channel opening. To investigate GlyR kinetic properties, we rapidly applied 1 mM glycine for 1 ms and found that glycine currents were increased by ethanol (100 mM) at both ages. For patches from juvenile HMs, ethanol consistently decreased response rise-time and increased response decay time. Using kinetic modeling, we determined that ethanol's potentiation of the glycine response arises from an increase in the glycine association (kon) and a decrease in the dissociation (k off) rate constants, resulting in increased glycine affinity of the GlyR.
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