MECHANISM FOR THE CHANNEL-OPENING REACTION OF STRYCHNINE-SENSITIVE GLYCINE RECEPTORS ON CULTURED EMBRYONIC MOUSE SPINAL-CORD CELLS

The strychnine-sensitive glycine receptor, a member of a superfamily of proteins involved in chemical reactions that regulate signal transmission between cells of the nervous system, forms an anion-specific transmembrane channel in response to glycine binding. A rapid-reaction technique, a cell-flow method with a 10-ms time resolution, was adapted for measurements with cultured embryonic mouse spinal cord cells containing glycine receptors. Whole-cell current responses resulting from the opening of glycine receptor channels were measured at pH 7.4, 22-24 degrees C, and transmembrane voltages of -40 and -75 mV. Two different receptor forms, A(alpha) and A(beta) were detected. At saturating glycine concentrations, an average of 70% of the whole-cell current amplitude was associated with form A(alpha) and 30% with A(beta). The constants pertaining to the minimum mechanisms that account for the concentration of the two open-channel receptor forms over a 100-fold range of glycine concentration were determined by cell-flow measurements of the current amplitudes and of the falling (desensitizing) rate of the current. The dissociation constant of the site controlling channel opening was 220 mu M On the basis of three binding sites for A(alpha) and 380 mu M on the basis of two binding sites for A(beta). The channel-opening equilibrium constant, Phi(-1), was 170 for A(alpha) and 110 for A(beta). The rate coefficients for desensitization, alpha and beta, associated with these two forms have maximum values of 0.7 and 0.1 s(-1), respectively. The rates at which the receptors recovered from desensitization were also measured, using a double-flow mixing device, and were found to be 0.06 s(-1) for A(alpha) and 0.02 s(-1) for A(beta). In the presence of 100 mu M glycine, the apparent dissociation constant for the inhibitor picrotoxinin from receptor form A(alpha) was 80 mu M, and that from A(beta) was 460 mu M. This suggests that A(beta) contains beta-subunits (58 kD), because this subunit confers picrotoxinin insensitivity to glycine receptors (Pribilla, I., et al. (1992) EMBO J. 11, 4305). In the case of one receptor form (A(alpha)), the chemical mechanism and its constants led to two measurements that could be assessed by an independent method, the single-channel current-recording technique: (i) the fraction of receptor channels open at a given glycine concentration (<(AL(n))over bar>)(0) and (ii) the rate coefficient for desensitization. In the presence of 200 mu M glycine and at a transmembrane voltage of -75 mV, the chemical kinetic measurement and the single-channel current-recording technique gave values for alpha of 0.8 and 0.6 s(-1), respectively. The value of (<(AL(n))over bar>)(0) appears to be the same at -40 and -75 mV. The (<(AL(n))over bar>)(0) value obtained in presence of 200 mu M glycine is 0.9 when measured by the cell-flow technique at -40 mV. The electrophysiological technique gave a value of 0.7 at -75 mV and showed that receptor form A(alpha) has a channel conductance of 48 pS.