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.

SUMMARY1. Responses to N-methyl-D-aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using whole-cell patchclamp recording. A fast perfusion system, with an exchange time constant of less than 10 ms, was used to study modulation of NMDA receptor desensitization by glycine.2. The onset of NMDA receptor desensitization was well fitted by a singleexponential function; with 30 nM-glycine the time constant was 250 ms, corresponding to a rate of 4 s-1. The rate of onset of desensitization became faster with increasing glycine concentration, with a slope of 0-87 x 107 M-1 s-1. Recovery from desensitization, studied with a twin-pulse technique, was also well fitted by a singleexponential function; with 30 nM-glycine the time constant of recovery was 1-95 s-'. The rate of recovery from desensitization became faster with increasing glycine concentration, with a slope of 0-76 x 107 M-1 s-1. These results are consistent with a model in which the effect of glycine occurs via an increase in the rate constant for recovery from desensitization, with little effect on the rate constant for onset of desensitization. Over the range 30-300 nM-glycine, the ratio of the rate constants calculated for recovery and onset of desensitization was a good predictor of the degree of desensitization recorded at equilibrium.3. Concentration jump experiments with glycine were performed with 100 /LM-NMDA present continuously, and for a single binding site model gave estimates of the association (1l1 x 107 m-l s-') and dissociation (3-1 s-1) rate constants for interaction of glycine with the NMDA receptor. In the presence of NMDA, concentration jumps from 3 ,tM-glycine to lower concentrations gave relaxations which became slower with decreasing glycine concentration over the range 1 uM-30 nM. A similar slowing of desensitization occurred when the glycine concentration was altered over the same range. 3l. BENVVEXISTE AXD OTHERS 4. Glycine analogues of lower affinity produced desensitization with faster kinetics. D-Alanine, 150 nm, produced desensitization with a time constant of 175 ms, faster than recorded with an equipotent concentration of glycine (50 nm, time constant 259 ms). Responses of similar peak amplitude, recorded with 60 JM-Lalanine, and 500 /M-D,L-homoserine, did not produce strong desensitization, consistent with desensitization too rapid to resolve in our experiments. Concentration jump experiments with L-alanine and D,L-homoserine confirmed that the dissociation rate constants for these analogues (42 and 53 s-) were much faster than those obtained for glycine and D-alanine (341 and 4-9 s-').5. Numerical simulations were developed to test possible kinetic schemes for NMDA receptor activation and desensitizatiohn. Together with the experimental data they suggest that transitions to the open state do not occur unless glycine has first bound to a closed state of the NMDA receptor. Potentiation of NMDA responses by glycine results from the absolute requirement for glycine in promoting tra...

The vanilloid receptor [transient receptor potential (TRP)V1, also known as VR1] is a member of the TRP channel family. These receptors share a significant sequence homology, a similar predicted structure with six transmembrane-spanning domains (S1-S6), a pore-forming region between S5 and S6, and the cytoplasmically oriented C- and N-terminal regions. Although structural/functional studies have identified some of the key amino acids influencing the gating of the TRPV1 ion channel, the possible contributions of terminal regions to vanilloid receptor function remain elusive. In the present study, C-terminal truncations of rat TRPV1 have been constructed to characterize the contribution of the cytoplasmic C-terminal region to TRPV1 function and to delineate the minimum amount of C tail necessary to form a functional channel. The truncation of 31 residues was sufficient to induce changes in functional properties of TRPV1 channel. More pronounced effects of C-terminal truncation were seen in mutants lacking the final 72 aa. These changes were characterized by a decline of capsaicin-, pH-, and heat-sensitivity; progressive reduction of the activation thermal threshold (from 41.5 to 28.6 degrees C); and slowing of the activation rate of heat-evoked membrane currents (Q10 from 25.6 to 4.7). The voltage-induced currents of the truncated mutants exhibited a slower onset, markedly reduced outward rectification, and significantly smaller peak tail current amplitudes. Truncation of the entire TRPV1 C-terminal domain (155 residues) resulted in a nonfunctional channel. These results indicate that the cytoplasmic COOH-terminal domain strongly influences the TRPV1 channel activity, and that the distal half of this structural domain confers specific thermal sensitivity.