Technical Reference #1657

1657.

Effects of Inserting Fluorescent Proteins into the Alpha1s II-III Loop: Insights into Excitation-Conctraction Coupling Roger A. Bannister; Symeon Papadopoulos; Claudia S. Haarmann; and Kurt G. Beam, University of Colorado, The Journal of General Physiology, 134(1657), (2009)
Link To Paper

Abstract:
In skeletal muscle intermolecular communication between the 14-dihydropyridine receptor (DHPR) and RYR1 isbidirectional: orthograde coupling (skeletal excitation–contraction coupling) is observed as depolarization-inducedCa2+ release via RYR1 and r

Materials & Methods:
Expression of cDNA All procedures involving mice were approved by the University of Colorado Denver Institutional Animal Care and Use Committee. Primary cultures of phenotypically normal (+/+ or +/mdg) or dysgenic (mdg/mdg) myotubes were prepared from newborn mice as described previously (Beam and Franzini-Armstrong 1997). For electrophysiological experiments myoblasts were plated into 35-mm plastic culture dishes (Falcon) coated with entactin–collagen IV–laminin (Millipore). Myoblasts destined for immunocytochemistry were plated into 35-mm culture dishes with entactin–collagen IV–laminin–coated glass coverslip bottoms (MatTek). Cultures were grown for 6–7 d in a humidified 37°C incubator with 5% CO2 in Dulbecco’s modified Eagle’s medium (Mediatech) supplemented with 10% fetal bovine serum/10% horse serum (Hyclone Laboratories). This medium was then replaced with differentiation medium (Dulbecco’s modified Eagle’s medium supplemented with 2% horse serum). 2–4 d after the shift to differentiation medium single nuclei were microinjected with cDNA. For one-piece constructs (1S(671-CFP-YFP-686) 1S(726-CFP-YFP-727) 1S(726-YFP-727) 1S(760-YFP-761) or 1S(785-YFP-786)) myotubes to be used in electrophysiological experiments were injected with 100 ng/μl cDNA and myotubes to be immunostained were injected with 60 ng/μl cDNA. For electrophysiology on two-piece constructs the injection solution contained 60 ng/μl 1S(671) hemichannel cDNA and 100 ng/μl (686)1S hemichannel cDNA. Only myotubes exhibiting YFP fluorescence were used in experiments.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
living