Expression of ion channels during differentiation of a human skeletal muscle cell line

An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4-40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins alpha-actinin, alpha-sarcomeric actin, myosin and titin were present in early stages. Receptors for gamma toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9-1.0 pmol mg-1 protein) during stage 1 (0-6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors.