Ryanodine receptor modulation by caffeine challenge modifies Na⁺ current properties in intact murine skeletal muscle...

We investigated effects of the ryanodine receptor (RyR) modulator caffeine on Na⁺ current (I_Na) activation and inactivation in intact loose-patch clamped murine skeletal muscle fibres subject to a double pulse procedure. I_Na activation was examined using 10-ms depolarising, V₁, steps to varying voltages 0-80 mV positive to resting membrane potential. The dependence of the subsequent, I_Na inactivation on V₁ was examined by superimposed, V₂, steps to a fixed depolarising voltage. Current-voltage activation and inactivation curves indicated that adding 0.5 and 2 mM caffeine prior to establishing the patch seal respectively produced decreased (within 1 min) and increased (after ~2 min) peak I_Na followed by its recovery to pretreatment levels (after ~40 and ~30 min respectively). These changes accompanied negative shifts in the voltage dependence of I_Na inactivation (within 10 min) and subsequent superimposed positive activation and inactivation shifts, following 0.5 mM caffeine challenge. In contrast, 2 mM caffeine elicited delayed negative shifts in both activation and inactivation. These effects were abrogated if caffeine was added after establishing the patch seal or with RyR block by 10 μM dantrolene. These effects precisely paralleled previous reports of persistently (~10 min) increased cytosolic [Ca²⁺] with 0.5 mM, and an early peak rapidly succeeded by persistently reduced [Ca²⁺] likely reflecting gradual RyR inactivation with ≥1.0 mM caffeine. The latter findings suggested inhibitory effects of even resting cytosolic [Ca²⁺] on I_Na. They suggest potentially physiologically significant negative feedback regulation of RyR activity on Na_v1.4 properties through increased or decreased local cytosolic [Ca²⁺], Ca²⁺-calmodulin and FKBP12.