Abstract
Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.
Highlights
Calcium is a universal intracellular signal responsible for regulating many cellular processes including cell proliferation, hormonal secretion, and muscle contraction [1, 2]
excitation-coupled Ca2ϩ entry (ECCE) is triggered by membrane depolarization, whereas SOCE is inhibited by membrane depolarization [17]. ␣1S-dihydropyridine receptor (DHPR) serves as the voltage sensor for triggering the activation of ECCE, and the conformational state of ryanodine receptor type 1 (RyR1) dramatically influences the behavior of ECCE in response to membrane depolarization
The calcium responses observed in the absence of extracellular calcium for C4958S (*, p Ͻ 0.001) were significantly different to that observed in the presence of calcium. c, the functional responses of wild-type RyR1 (n ϭ 11 cells), C4958SRyR1 (n ϭ 14 cells), and C4961S-RyR1 (n ϭ 13 cells) to varying concentrations of Kϩ in the presence of 2 mM extracellular Ca2ϩ
Summary
CDNA Cloning—Cys4958 and Cys4961 were mutated to serine using primer extension-driven site-directed mutagenesis in a ClaI[14,203]XbaI (vector) fragment of RyR1 using a QuikChange (Stratagene, CA) kit, and sequenced in both directions to confirm the mutation and the absence of any other random mutations. Ca2ϩ and Mn2ϩ Imaging—Differentiated 1B5 or primary myotubes were loaded with the Ca2ϩ-sensitive dye Fura-2-AM (5 M) at 37 °C for 20 min in imaging buffer (in mM) 125 NaCl, 5 KCl, 2 CaCl2, 1.2 MgSO4, 6 dextrose, and 25 HEPES, pH 7.4 supplemented with 0.05% bovine serum albumin. A, changes in Fluo fluorescence in intact, primary myotubes expressing wild-type RyR1 (white circles) or C4958S-RyR1 (black circles), and in uninjected dyspedic (gray circles) in response to bath application of 30 M CPA. B, average, normalized peak fluorescence increases during exposure to 30 M CPA for C4958SRyR1 (C-S, black bars; n ϭ 8), wild-type RyR1 (white bars; n ϭ 6), and dyspedic myotubes (Dyp, gray bars; n ϭ 7). After a final rinse step with TTBS, enhanced chemiluminescence techniques (PerkinElmer Life Science Products) were used to visualize the immunoblots
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
