TETRAETHYLAMMONIUM AND TETRABUTYLAMMONIUM AS TOOLS TO STUDY NMDA CHANNELS OF NEURONAL MEMBRANE

Neurons isolated from CA1 region of rat hippocampal slices by the 'vib rodissociation' method were voltage-clamped in the wholecell configura tion. Currents through NMDA channels were recorded in the response to the rapid application (solution exchange time, < 30 ms) of 100 M aspar tate in the presence of 2.5 M glycine. It was shown that tetraethylamm onium (TEA) when added to the aspartate solution caused a concentratio n-dependent current decrease, its peak value (I(p)) being always less decreased than the quasistationary level (I(s)) measured at the end of 1-s aspartate application. On the contrary, tetrabutylammonium (TBA) at all concentrations decreased I(p) to a larger extent than J(s), so that at high TBA concentrations the current decay from desensitization almost disappeared. Upon aspartate + blocker application, a transient increase in the current appeared which in the case of TBA always exce eded the corresponding control current value. The block by both TEA an d TBA was highly voltage-dependent. Concentrations of TEA and TBA that had little effect at positive potentials were quite effective at nega tive ones. Estimation of the dependence of I(s) blockade of membrane p otential using the Woodhul's model showed that TBA is a more potential -dependent blocker of NMDA-channels than TEA. The fraction of the tran smembrane potential experienced by TEA - and TBA-binding sites are app roximately 0.9 and approximately 0.7, respectively. At -100 mV concent rations of TEA and TBA required for a 50% reduction of I(s), proved to be 7.7 +/- 0.44 mM (10 cells) and 0.067 +/- 0.006 mM (6 cells). The e ffects of TEA and TBA were also explored for the case of their applica tion during a continuous superfusion of cell by aspartate, i.e. agains t the background of stationary inward current. Under these conditions one could reveal both fast and slow (seconds) components of TEA block and unblock, By contrast, the TBA block lacked these slow components: moreover, upon the end of TBA application a transient increase of the current appeared which then slowly decayed back to the stationary leve l of exchange solution. The data obtained led us to conclude that TEA blockage of open NMDA channels stabilizes their desensitization. In co ntrast, TBA when bound to the open channels prevents desensitization.