Oral Presentation 2014 International Biophysics Congress

Ca2+ uptake by the tubular (t-) system of skeletal muscle fibres (#44)

Tanya R. Cully 1 , Joshua N. Edwards 1 , Thomas R. Shannon 2 , Bradley S. Launikonis 1
  1. The University of Queensland, Brisbane, QLD, Australia
  2. Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL, USA

The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane.  The t-system forms a junction with the terminal cisternae of the sarcoplasmic reticulum (SR) at every sarcomere of skeletal muscle.  At any given moment the [Ca2+] within the small volume bound by the junctional membranes will be critically determined by the leak of Ca2+ through ryanodine receptors and the net Ca2+ handling ability of the t-system.  To assess the Ca2+ uptake and handling ability of the t-system we trapped fluo-5N or rhod-5N in the t-system of mechanically skinned fibres of rat extensor digitorum longus muscle and continuously imaged t-system Ca2+-dependent fluorescence on a confocal microscope.  In situ calibration determined the half signal of fluo-5N and rhod-5N to be 335 and 872 µM, respectively.  Rhod-5N was selected for ongoing experiments.  Chronic depletion of [Ca2+]SR with caffeine reduced [Ca2+]t-sys to 0.1 mM via chronic activation of store-operated Ca2+ entry (SOCE).  We then exposed Ca2+-depleted preparations to 0-800 nM [Ca2+]cyto in 50 mM EGTA.  At [Ca2+]cyto > 100 nM the [Ca2+]t-sys­ reached a plateau at 1.8-1.9 mM after 3-5 s.  At [Ca2+]cyto < 100 nM the [Ca2+]t-sys did not always reach this plateau and showed a biphasic uptake of Ca2+.  At the plateau [Ca2+]t-sys lowering [Ca2+]cyto to < 1 nM did not cause a significant loss of [Ca2+]t-sys.  There was an apparent absence of effect of removing [Na+]cyto on these results.  Ca2+ fluxes across the t-system during uptake and SOCE were determined by multiplying the time derivative of [Ca2+]t-sys by buffering capacity and t-system SA:V.  Mathematical modeling of these results suggests that the plasma membrane CaATPase (PMCA) with its low Km for Ca2+ is the major protein responsible for t-system Ca2+ uptake in the resting muscle, despite the higher transport capacity of the Na-Ca exchanger.