Sf. Traynelis et P. Wahl, CONTROL OF RAT GLUR6 GLUTAMATE-RECEPTOR OPEN PROBABILITY BY PROTEIN-KINASE-A AND CALCINEURIN, Journal of physiology, 503(3), 1997, pp. 513-531
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).