Detubulation abolishes membrane potential stabilization in amphibian skeletal muscle

Area of Science:

• Muscle physiology
• Cellular electrophysiology
• Membrane transport

Background:

• Resting membrane potential (Em) stabilization in amphibian skeletal muscle during hyperosmotic challenge was previously attributed to high P Cl/P K and elevated [Cl-]i via cotransporters.
• Diuretic-sensitive cotransporters, including NCC and NKCC, were implicated in maintaining intracellular chloride concentrations above equilibrium.

Purpose of the Study:

• To localize the site of cation-chloride cotransporter activity responsible for Em splinting.
• To investigate the role of transverse tubules (T-tubules) in this phenomenon.

Main Methods:

• Established detubulation procedures were applied to amphibian muscle fibers.
• Confocal microscopy visualized lissamine rhodamine fluorescence to confirm T-tubular detachment.
• Fibre volume changes were measured using confocal scanning methods.
• Em was recorded under varying extracellular osmolarity and ionic conditions, including cotransporter blockade.

Main Results:

• Detubulation successfully abolished T-tubular access and action potential afterdepolarization.
• Detubulated fibers lost Em splinting, with Em varying directly with extracellular osmolarity.
• Em changes in detubulated fibers aligned with predictions assuming conserved intracellular ion content and K+ Nernst potential dominance.
• Cation-chloride cotransporter blockade (via ion deprivation or drugs like bumetanide and chlorothiazide) yielded similar results to detubulation.

Conclusions:

• The transverse tubular system, not the surface sarcolemma, is the primary site of cation-chloride cotransporter activity or its regulation in amphibian skeletal muscle.
• This finding contrasts with previous assumptions and highlights the functional importance of T-tubules in maintaining membrane potential homeostasis during osmotic stress.