Na⁺-K⁺ pumps in the transverse tubular system of skeletal muscle fibers preferentially use ATP from glycolysis

The Na(+)-K(+) pumps in the transverse tubular (T) system of a muscle fiber play a vital role keeping K(+) concentration in the T-system sufficiently low during activity to prevent chronic depolarization and consequent loss of excitability. These Na(+)-K(+) pumps are located in the triad junction, the key transduction zone controlling excitation-contraction (EC) coupling, a region rich in glycolytic enzymes and likely having high localized ATP usage and limited substrate diffusion. This study examined whether Na(+)-K(+) pump function is dependent on ATP derived via the glycolytic pathway locally within the triad region. Single fibers from rat fast-twitch muscle were mechanically skinned, sealing off the T-system but retaining normal EC coupling. Intracellular composition was set by the bathing solution and action potentials (APs) triggered in the T-system, eliciting intracellular Ca(2+) release and twitch and tetanic force responses. Conditions were selected such that increased Na(+)-K(+) pump function could be detected from the consequent increase in T-system polarization and resultant faster rate of AP repriming. Na(+)-K(+) pump function was not adequately supported by maintaining cytoplasmic ATP concentration at its normal resting level ( approximately 8 mM), even with 10 or 40 mM creatine phosphate present. Addition of as little as 1 mM phospho(enol)pyruvate resulted in a marked increase in Na(+)-K(+) pump function, supported by endogenous pyruvate kinase bound within the triad. These results demonstrate that the triad junction is a highly restricted microenvironment, where glycolytic resynthesis of ATP is critical to meet the high demand of the Na(+)-K(+) pump and maintain muscle excitability.