SINGLE-CHANNEL CURRENTS FROM DIETHYLPYROCARBONATE-MODIFIED NMDA RECEPTORS IN CULTURED RAT-BRAIN CORTICAL-NEURONS

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying rea gent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outsid e-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) sho wed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alteratio ns in channel gating revealed no changes in the numbers of kinetic sta tes: distributions of open intervals were fitted with three exponentia l components, and four components described the shut intervals, in bot h control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent wit h the single-channel current record. After modification, the proportio n of long shut states was decreased while the time constants were larg ely unaffected. Burst kinetics reflected these effects with an increas e in the average number of openings/burst from 1.5 (control) to 2.2 (D EP), and a decrease in the average interburst interval from 54.1 to 38 .2 ms. These effects were most likely due to histidine modification be cause other reagents (n-acetylimidazole and 2,4,6-trinitrobenzene 1-su lfonic acid) that are specific for residues other than histidine faile d to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effe cts on channel gating, DEP had no effect on average single-channel con ductance or reversal potential under bi-ionic (Na+:Cs+) conditions. In hibition by zinc was also unaffected by DEP. We propose a channel gati ng model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state co nditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.