Activation of the protein phosphatase, calcineurin, during carbachol
exposure decreases the extent of recovery from end-plate desensitization.
Hardwick, Jean C. and Rodney L. Parsons.
Department of Anatomy and Neurobiology, University of Vermont, Burlington,
APStracts 3:0176N, 1996.
SUMMARY AND CONCLUSIONS
1. Our previous studies demonstrated that protein kinase C (PKC) activity is
required for acetylcholine (ACh) sensitivity to recover fully at snake twitch
fiber end-plates following prolonged exposure to carbachol. In the present
studies, we have investigated whether protein phosphatase(s), activated during
carbachol exposure, dephosphorylated critical membrane proteins, which
required rephosphorylation by PKC to maintain end-plate sensitivity. End-plate
sensitivity was assessed from measurements of miniature end-plate currents
(MEPCs) and carbachol-activated currents (EPC CARB ). Conductance of ACh-
activated channels was determined from patch clamp recordings of single
channel currents. 2. Pretreatment of snake muscle preparations with the
protein kinase inhibitor staurosporine (0.5 [mu]M), followed by a 10 min
exposure to 540 [mu]M carbachol, reduced mean MEPC amplitudes to values 30-40%
less than those recorded prior to carbachol exposure. Conversely, at control
end-plates exposed to carbachol, the mean MEPC amplitude was reduced by only
5% when compared to pre-carbachol values. This staurosporine-induced decrease
in ACh sensitivity could be prevented by pretreatment with the protein
phosphatase 2B (calcineurin) inhibitor deltamethrin (0.5 [mu]M) whereas
okadaic acid (5 [mu]M) and calyculin A (0.5 [mu]M), inhibitors of protein
phosphatases 1 and 2A, had no effect. 3. Following a 10 min exposure to 540
[mu]M carbachol, EPC CARB amplitudes (produced by local superfusion with 20
[mu]M carbachol) were significantly smaller at staurosporine-treated end-
plates than at control end-plates. In contrast, the EPC CARB amplitude
recorded from end-plates pretreated with both deltamethrin and staurosporine
was not significantly different from that recorded at control end-plates. 4.
Substitution of 10 mM Mn 2+ for external Ca 2+ during the exposure to 540
[mu]M carbachol prevented the decrease in MEPC amplitude recovery at
staurosporine-treated end-plates. These results suggested that the alteration
in sensitivity at staurosporine-treated end-plates was calcium-dependent. 5.
At control end-plates, a single population of ACh-activated channels (45-50
pS) are observed both prior to, and following a 10 min exposure to 540 [mu]M
carbachol. Conversely, at staurosporine-treated end-plates, following exposure
to carbachol, a second population of small conductance (25-30 pS) ACh-
activated channels are present in addition to the predominant 45-50 pS ACh-
activated channels. In preparations pretreated with both deltamethrin and
staurosporine, following carbachol exposure, there was a significant decrease
in the frequency of small conductance ACh-activated channels. Deltamethrin
treatment alone produced no small conductance channels prior to, or following
a 10 min exposure to 540 [mu]M carbachol. Also, no small conductance ACh-gated
channels were recorded at PKC-inhibited end-plates following carbachol
exposure with either pretreatment with 10 [mu]M cyclosporin A (another
inhibitor of calcineurin) or with the substitution of 10 mM Mn 2+ for Ca 2+
during the 10 min agonist exposure. 6. We propose that during prolonged
exposure to the nicotinic agonist carbachol, calcium influx through ACh-gated
channels elevates the level of ionized calcium at the inner surface of the
postjunctional membrane and that this local rise in intracellular calcium
activates the calcium-dependent phosphatase calcineurin. Dephosphorylation of
some key membrane protein by calcineurin leads to a decrease in the extent of
recovery from desensitization. Under normal conditions, this process is
effectively reversed by PKC activity and end-plate sensitivity recovers fully.
However, when PKC is inhibited, the extent of recovery of end-plate
sensitivity is decreased and, associated with this decrease, is the presence
of small conductance ACh-activated channels not normally recorded at snake
twitch fiber end-plates.
Received 16 April 1996; accepted in final form 12 August 1996.
APS Manuscript Number J313-6.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 19 September 1996