Multiple Regions of Junctin and Triadin Interact with Calsequestrin 1 and 2 in Skeletal Muscle Cells

Abstract

confocal microscope. In situ calibration determined the half signal of fluo-5N and rhod-5N to be 335 and 872 mM, respectively. Rhod-5N was selected for ongoing experiments. Chronic depletion of [Ca 2þ ]SR with caffeine reduced [Ca 2þ ]t-sys to 0.1 mM via chronic activation of storeoperated Ca 2þ entry (Launikonis et al 2003, PNAS). We then exposed Ca 2þdepleted preparations to 0-800 nM [Ca 2þ ]cyto in 50 mM EGTA. At [Ca 2þ ] cyto > 100 nM the [Ca 2þ ]t-sys reached a plateau at 1.8-1.9 mM after 3-5 s. At [Ca 2þ ]cyto < 100 nM the [Ca 2þ ]t-sys did not always reach this plateau and showed a biphasic uptake of Ca 2þ . At the plateau [Ca 2þ ]t-sys lowering [Ca 2þ ]cyto to < 1 nM did not cause a significant loss of [Ca 2þ ]t-sys. There was an apparent absence of effect of removing [Na þ ]cyto on these results. Mathematical modeling of these results suggests that the plasma membrane CaATPase (PMCA) with its low Km for Ca 2þ is the major protein responsible for t-system Ca 2þ uptake in the resting muscle, despite the higher transport capacity of the Na-Ca exchanger.