Probing glycine receptor stoichiometry in superficial dorsal horn neurones using the spasmodic mouse

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Abstract
Inhibitory glycine receptors (GlyRs) are pentameric ligand gated ion channels composed of alpha and beta subunits assembled in a 2:3 stoichiometry. The alpha 1/beta heteromer is considered the dominant GlyR isoform at 'native' adult synapses in the spinal cord and brainstem. However, the alpha 3 GlyR subunit is concentrated in the superficial dorsal horn (SDH: laminae I-II), a spinal cord region important for processing nociceptive signals from skin, muscle and viscera. Here we use the spasmodic mouse, which has a naturally occurring mutation (A52S) in the alpha 1 subunit of the GlyR, to examine the effect of the mutation on inhibitory synaptic transmission and homeostatic plasticity, and to probe for the presence of various GlyR subunits in the SDH. We used whole cell recording (at 22-24 degrees C) in lumbar spinal cord slices obtained from ketamine-anaesthetized (100 mg kg-1, i.p.) spasmodic and wild-type mice (mean age P27 and P29, respectively, both sexes). The amplitude and decay time constants of GlyR mediated mIPSCs in spasmodic mice were reduced by 25% and 50%, respectively (42.0 +/- 3.6 pA vs. 31.0 +/- 1.8 pA, P < 0.05 and 7.4 +/- 0.5 ms vs. 5.0 +/- 0.4 ms, P < 0.05; means +/- SEM, n = 34 and 31, respectively). Examination of mIPSC amplitude versus rise time and decay time relationships showed these differences were not due to electrotonic effects. Analysis of GABA(A)ergic mIPSCs and A-type potassium currents revealed altered GlyR mediated neurotransmission was not accompanied by the synaptic or intrinsic homeostatic plasticity previously demonstrated in another GlyR mutant, spastic. Application of glycine to excised outside-out patches from SDH neurones showed glycine sensitivity was reduced more than twofold in spasmodic GlyRs (EC50 = 130 +/- 20 mu m vs. 64 +/- 11 mu m, respectively; n = 8 and 15, respectively). Differential agonist sensitivity and mIPSC decay times were subsequently used to probe for the presence of alpha 1-containing GlyRs in SDH neurones. Glycine sensitivity, based on the response to 1-3 mu m glycine, was reduced in > 75% of neurones tested and decay times were faster in the spasmodic sample. Together, our data suggest most GlyRs and glycinergic synapses in the SDH contain alpha 1 subunits and few are composed exclusively of alpha 3 subunits. Therefore, future efforts to design therapies that target the alpha 3 subunit must consider the potential interaction between alpha 1 and alpha 3 subunits in the GlyR.