Physiological properties of Glycinergic synapses with defined subunit compositions

Abstract

Glycine receptors (GlyRs) mediate fast inhibitory neurotransmission in the spinal cord and brainstem. Four GlyR subunits (a1-3, b) have been identified in humans, and their differential anatomical distributions result in a diversity of synaptic isoforms with unique physiological and pharmacological properties. However, despite their importance in controlling neuronal function, little is known about these properties. To address this, we developed an lartificialr synapse system which allows to control over the synaptic GlyR subunit composition. We induced the formation of recombinant synapses between cultured spinal neurons and HEK293 cells expressing GlyR subunits of interest plus the synapse-promoting molecule, neuroligin-2A. In the heterosynapses thus formed, recombinant a1b and a3b GlyRs mediated fast decaying inhibitory postsynaptic currents (IPSCs) whereas a2b GlyRs mediated slow decaying IPSCs. These results are consistent with the fragmentary information available from native synapses and single channel kinetic studies. As b subunit incorporation is considered essential for localizing GlyRs at the synapse, we were surprised that a1-3 homomers supported robust IPSCs with b subunit incorporation accelerating IPSC rise and decay times in a2b and a3b heteromers only. Finally, heterosynapses incorporating a1D80Ab and a1A52Sb GlyRs exhibited accelerated IPSC decay rates closely resembling those recorded in native synapses from mutant mice homozygous for these mutations, providing an additional validation of our technique. As our model system successfully recapitulates the effects of known GlyR disease mutations, we employed it to investigate the effects of new GlyR disease mutations. Hyperekplexia is a human neuromotor disorder caused by mutations that impair glycinergic neurotransmission. We investigated the mechanism by which gain-of-function GlyR mutations cause hyperekplexia. We identified two new gain-of-function mutations (I43F, W170S) and characterised these along with the known gain-of-function mutations (Q226E, V280M, R414H) to identify how they cause hyperekplexia. Using lartificialr synapses, we show that all mutations prolong the decay of IPSCs and induce spontaneous activation. As these effects may deplete the chloride electrochemical gradient, hyperekplexia could potentially result from reduced glycinergic inhibitory efficacy. However, we consider this unlikely as it should also lead to pain sensitization and a hyperekplexia phenotype that correlates with mutation severity, neither of which is observed in patients. We also rule out small increases in IPSC decay times (as caused by W170S and R414H) as a possible mechanism given that the clinically-important drug, tropisetron, increases glycinergic IPSC decay times without causing motor side effects. A recent study concluded that an elevated intracellular chloride concentration late during development ablates a1b glycinergic synapses but spares GABAergic synapses. As this mechanism satisfies all our considerations, we propose it is primarily responsible for the gain-of-function hyperekplexia phenotype. GlyRs containing a2 subunits exert excitatory effects in immature neurons, and thereby modulate neuronal migration and synapse formation. The a2 R350L mutation has been identified in a human patient with autism spectrum disorder (ASD). This mutation was found to slow both the channel closing rate and the IPSC decay time. As mutation of R350 to Ala, Lys or Ile did not affect the IPSC decay time, we propose the Leu mutation specifically affects a molecular interaction responsible for the prolonged IPSC time courses. However, the link between the R350L and ASD remains to be elucidated. Zn2+ is concentrated into presynaptic vesicles at central synapses and is released into the synaptic cleft by nerve terminal stimulation. There is strong evidence that synaptically released Zn2+ modulates glutamatergic neurotransmission, although there is debate concerning its peak concentration in the cleft. GlyRs are potentiated by low nanomolar Zn2+ and inhibited by micromolar Zn2+. A mutation that ablates Zn2+ potentiation of GlyRs results in a hyperekplexia phenotype suggesting that Zn2+ physiologically modulates glycinergic neurotransmission. There is, however, little evidence that Zn2+ is stored presynaptically at glycinergic terminals and it is possible that the modulation is mediated by tonically bound Zn2+. We sought to estimate the peak Zn2+ concentration in the glycinergic synaptic cleft as a means of evaluating whether it is likely to be synaptically released. We generated lartificialr synapses expressing a1b GlyRs with defined Zn2+ sensitivities. By comparing the effect of Zn2+ chelation on glycinergic IPSCs with the effects of defined Zn2+ plus glycine concentrations applied rapidly to recombinant GlyRs in outside-out patches under simulated synaptic activation conditions, we inferred that synaptic Zn2+ rises to at least 1 mM following a single presynaptic stimulation. Moreover, using the fast high-affinity chelator, ZX1, we found no evidence for tonic Zn2+ bound constitutively to high affinity GlyR sites. We conclude that diffusible Zn2+ reaches 1 mM or higher and is therefore likely to be phasically released at glycinergic synapses. Taken together, these results provide significant new insights into normal and abnormal glycinergic neurotransmission and the physiological modulatory mechanisms of glycinergic IPSCs.