In whole-cell recordings from mammalian CNS neurons, AMPA-preferring glutamate receptors exhibit strong desensitization in response to AMPA, glutamate, and quisqualate, but not to kainate or domoate. Such desensitization is reduced by lectins, by the nootropic drug aniracetam, and by diazoxide. None of these compounds strongly modulate responses to kainate and domoate, consistent with the apparent lack of desensitization to these agonists. We now report experiments on hippocampal neurons in which responses to kainate were strongly potentiated by cyclothiazide, a benzothiadiazine diuretic and antihypertensive drug structurally related to diazoxide. Cyclothiazide increased the maximum response to a saturating concentration of kainate by approximately 300% and produced a shift to the left in the kainate dose-response curve. Because cyclothiazide was considerably more effective than aniracetam in reducing desensitization evoked by glutamate, we tested the possibility that potentiation of responses to kainate was due to block of a previously undetected component of desensitization in the response to kainate itself. In outside-out patches responses to rapid perfusion of 3 mM kainate showed 34% desensitization, the onset of which developed with a time constant of 2.2 msec. Desensitization of responses to kainate was abolished by 100 microM cyclothiazide, as was the much stronger desensitization evoked by glutamate and AMPA. Cyclothiazide also slowed the rate of deactivation of responses to kainate recorded after return to agonist-free solution. Current-voltage plots for control responses to kainate exhibited outward rectification that was associated with a reduction in the amount of desensitization on depolarization. Both effects were absent in the presence of cyclothiazide, suggesting that rectification of responses to kainate was due to the voltage dependence of desensitization. The complete block of desensitization produced by cyclothiazide provides a powerful new tool for analysis of allosteric regulatory mechanisms at AMPA-preferring glutamate receptors.

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AMPA receptors play a central role in basal excitatory synaptic transmission as well as synaptic maturation and plasticity. The transmembrane AMPA receptor regulatory protein (TARP) stargazin (␥2) serves multiple roles in trafficking and stabilizing synaptic AMPA receptors and may be incorporated as an auxiliary subunit. We wanted to determine whether stargazin altered channel function of neuronal AMPA receptors. Transfection of cultured hippocampal neurons with stargazin produced two distinct effects on AMPA receptor functional properties: a sixfold reduction in glutamate-evoked desensitization and a twofold increase in the relative size of responses to the partial agonist kainate. Kinetic and dose-response analyses suggest that the effect of stargazin on glutamate desensitization results from an allosteric interaction that destabilizes the desensitized state of the receptor and that potentiation of kainate responses reflects increased efficacy rather than a change in affinity. These functional effects were also observed in human embryonic kidney 293 cells transfected with various heteromeric and homomeric AMPA receptors, with distinct subunit-dependent effects on glutamate desensitization, kainate efficacy, and trafficking. Two regions of stargazin mediate its functional effects: the C-terminal intracellular domain seems to be more important for effects on glutamate-evoked desensitization and receptor trafficking, whereas the first extracellular domain makes a larger contribution to effects on kainate efficacy. These data indicate that TARPs are involved both in trafficking and direct modulation of channel function and, as auxiliary subunits of neuronal AMPA receptors, must be considered in the functional heterogeneity of neuronal AMPA receptors.

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