CONTROL OF RAT GLUR6 GLUTAMATE-RECEPTOR OPEN PROBABILITY BY PROTEIN-KINASE-A AND CALCINEURIN

1. We have used non-stationary variance analysis to examine the single channel conductance and the probability of channel opening at the pea k of the homomeric GluR6 response (P-o,P-peak) to 100-200 ms applicati on (10-90% exchange time, 0.3 ms) of glutamate onto excised membrane p atches from transiently transfected human embryonic kidney cells (HEK 293). 2. Our determinations of both P-o,P-peak and single channel cond uctance of simulated current responses are insensitive to system filte ring, response rise time, desensitization rate and measured variation in our drug perfusion speed. Isolation of stochastic current fluctuati ons using the local mean response waveform minimizes problems associat ed with modest rundown of response amplitude during the experiment. 3. The slope conductance calculated from the weighted mean unitary curre nts for the channels activated in response to glutamate application is 16 pS. Chord conductance between -40 and -80 mV is independent of ago nist concentration. Conversion of the codon for glutamine(621) to argi nine (Q621R) by RNA editing reduces conductance by more than 35-fold t o less than 0.4 pS without changing response time course, desensitizat ion, or P-o,P-peak. 4. P-o,P-peak is high at saturating glutamate conc entrations (0.65 +/- 0.23; mean +/- S.D.) and varies with agonist conc entration. The half-maximally effective glutamate concentration (EC50) determined for P-o,P-peak (0.2 mM; Hill slope = 0.6) is similar to th at determined for the macroscopic peak current amplitude (0.5 mM; Hill slope = 1.0) in response to rapid agonist application. 5. Inclusion o f the purified catalytic subunit of cAMP-dependent protein kinase A (P KA) in the patch pipette increases P-o,P-peak to 0.85 +/- 0.12 and co- transfection of cells with a cDNA encoding the catalytic subunit of PK A (C alpha-PKA) increases P-o,P-peak to 0.94 +/- 0.09. 6. Inclusion of purified calcineurin plus its coactivators 200 nM Ca2+ and calmodulin in the patch pipette decreases P-o,P-peak to 0.48 +/- 0.10. The calci neurin-stimulated decrease of P-o,P-peak in cells co-transfected with C alpha-PKA is blocked by 800 nM deltamethrin, a calcineurin inhibitor . Calmodulin, 200 nM Ca2+ and deltamethrin have no effect on P-o,P-pea k in the absence of calcineurin. As predicted from its effects on P-o, P-peak inclusion of calcineurin in the patch pipette accelerates the r un-down of whole cell GluR6 responses in cells co-transfected with C a lpha-PKA. 7. The effects of both calcineurin and PKA on P-o,P-peak for GluR6 receptors in excised patches occur without any detectable chang es to response time course, desensitization, or chord conductance. 8. We conclude that the binding of glutamate to homomeric GluR6 receptors is associated with a high probability of channel opening, which is un der the control of two signalling systems that are known to be co-loca lized at the neuronal membrane PKA (P-o,P-peak near 1.0) and calcineur in (P,(o,peak) near 0.5).