Functional coupling of skeletal muscle ryanodine receptors and calcineurin demonstrated by lipid bilayer single channel recordings.

I Barrett, YM Zheng, Z Lui, YX Wang. Albany Medical College, Albany, NY, USA

Aim: To assess the effect of calcineurin upon the channel activity of the skeletal muscle type 1 ryanodine receptor (RYR1) using lipid bilayer single channel recordings.

Methods: Skeletal muscle RYR1 was isolated from the hind limbs of adult New Zealand white rabbits, purified and enriched using sucrose gradients, as described previously (1). Animals were housed and maintained according to an approved protocol by the Animal Care and Use Committee of The Wadsworth Center, New York State Department of Health. The RYR1-enriched SR vesicles were fused to planar phospholipid bilayers which separated “cytoplasmic” (cis) and “intraluminal” (trans) recording solutions, comprised of 20mM HEPES and either 500mM or 50mM CsCl, respectively, and balanced at pH 7.4 with KOH. The addition of EGTA then Ca2+ to the cis side of the membrane was used to confirm Ca2+ sensitivity. In the presence of 30 or 100 nM Ca2+ and 2 mM ATP in the cis solution, calcineurin auto-inhibitory peptide (CAIP, 20 μM) was added to inhibit the activity of calcineurin, NiCl2 (0.5 mM) was added to enhance activity of calcineurin, and ruthenium red (30 μM) was used to show that elevated channel activity channel activity was due to activation of ryanodine receptors.

Results: The addition of 20 μM CAIP in the presence of 100 nM Ca2+ reduced the open probability (Po) from nPo = 0.657 to nPo = 0.210 (P = 0.037), with subsequent applications of 0.5 mM NiCl2 eliciting no enhancement of channel activity. As these observations exhibited multiple channel openings at baseline, experiments with 30nM Ca2+ were carried out, exhibiting single channel activity, and 20 μM CAIP also reduced the openings from Po = 0.105 to Po = 0.005 (P = 0.013). As before, inhibition by CAIP resulted in no further channel activity, even after multiple washouts of the cis chamber. In the absence of CAIP, the addition of 0.5 mM NiCl2 in the presence of 100 nM Ca2+ enhanced the Po in three out of four experiments by 4 to 80 times baseline levels, while in the presence of 30nM Ca2+ or less, NiCl2 enhanced Po in 2 out of 3 experiments by 4 to 20 times. The addition of 30 μM ruthenium red reduced Po to below baseline levels in all experiments exhibiting activity of only one channel.

Discussion: These results show evidence for functional coupling of calcineurin and RYR1 due to the enhanced currents elicited by addition of NiCl2 and its inhibition by CAIP, which has recently been demonstrated in confocal microscopic imaging studies of smooth muscle cells (2). In this lipid bilayer model, the inhibition of Ca2+ sensitive currents by CAIP was total, in contrast to studies in live smooth muscle cells, perhaps due to cycling of RYR1 and/or calcineurin that is absent in the bilayer system. This suggests that calcineurin plays a compensatory role in the presence of kinases, like PKCε, that are known to be associated with RYRs.

References:

1. Saito, A. et al. 1984, J Cell Biol 262: 1740-1747

2. Savoia, C. et al. 2014 Am J Physiol Lung Cell Mol Physiol (epub ahead of print)