6] Isolation of triads from skeletal muscle

Abstract

This chapter describes methodology for the isolation of morphologically intact triad structures. Excitation-contraction coupling in skeletal muscle is initiated at the neuromuscular junction. The action potential then proceeds longitudinally along the length of the fiber and transversely to within the fiber by way of invaginations from the plasmalemma, referred to as transverse tubules. The latter are junctionally associated with the terminal cisternae of sarcoplasmic reticulum via "feet" structures. By an unknown mechanism, Ca 2+ is then released from the terminal cisternae, elevating the myoplasmic calcium concentration, and—thereby, triggering muscle contraction. The structure formed by the junctional association of the transverse tubule with the terminal cisternae is referred to as the "triad," and the junction is the triad junction. The methodology is presented in two sections: (1) Procedures for triad isolation, and (2) characterization of the isolated triads. A method of sample preparation for electron microscopy was developed to estimate the purity and morphological integrity of our subcellular fractions. Dextran is added as a nonosmophilic spacer to the prefixed sample and filtration is used in the preparation for thin sectioning.