Sarcoplasmic reticular Ca(2+)-ATPase inhibition paradoxically upregulates murine skeletal muscle Na(v)1.4 function

Skeletal muscle Na(+) channels possess Ca(2+)- and calmodulin-binding sites implicated in Nav1.4 current (I(Na)) downregulation following ryanodine receptor (RyR1) activation produced by exchange protein directly activated by cyclic AMP or caffeine challenge, effects abrogated by the RyR1-antagonist dantrolene which itself increased I(Na). These findings were attributed to actions of consequently altered cytosolic Ca(2+), [Ca(2+)](i), on Na(v)1.4. We extend the latter hypothesis employing cyclopiazonic acid (CPA) challenge, which similarly increases [Ca(2+)](i), but through contrastingly inhibiting sarcoplasmic reticular (SR) Ca(2+)-ATPase. Loose patch clamping determined Na(+) current (I(Na)) families in intact native murine gastrocnemius skeletal myocytes, minimising artefactual [Ca(2+)](i) perturbations. A bespoke flow system permitted continuous I(Na) comparisons through graded depolarizing steps in identical stable membrane patches before and following solution change. In contrast to the previous studies modifying RyR1 activity, and imposing control solution changes, CPA (0.1 and 1 µM) produced persistent increases in I(Na) within 1–4 min of introduction. CPA pre-treatment additionally abrogated previously reported reductions in I(Na) produced by 0.5 mM caffeine. Plots of peak current against voltage excursion demonstrated that 1 µM CPA increased maximum I(Na) by ~ 30%. It only slightly decreased half-maximal activating voltages (V(0.5)) and steepness factors (k), by 2 mV and 0.7, in contrast to the V(0.5) and k shifts reported with direct RyR1 modification. These paradoxical findings complement previously reported downregulatory effects on Nav1.4 of RyR1-agonist mediated increases in bulk cytosolic [Ca(2+)]. They implicate possible local tubule-sarcoplasmic triadic domains containing reduced [Ca(2+)](TSR) in the observed upregulation of Nav1.4 function following CPA-induced SR Ca(2+) depletion.