Clausen C (2003). Physiol Rev 83, 1269-1324.
University of Heidelberg (2006) Proc Physiol Soc 4, C4
Oral Communications: Ole B. Nielsen1, William A. Macdonald1, George Stephenson2, Torben Clausen1
1. Institute of Physiology and Biophysics, University of Aarhus, Aarhus, Denmark. 2. Department of Zoology, La Trobe University, Bundoora 3083, VIC, Australia.
Contracting muscles lose K+. This leads to increased [K+]o, which may interfere with fibre excitability. In the T-tubules, the build-up of K+ is exaggerated due to a large ratio between membrane area and volume of the T-tubular lumen (Clausen, 2003). The build-up of [K+]o is counteracted by the activity of the Na+-K+ pumps. In rat muscle, only the α2 isoform of the Na+-K+ pump, which is ouabain sensitive, is present in the T-tubules. To quantify T-tubular Na+-K+ pumps, their content of 3H-ouabain-binding sites was determined using a 3H-ouabain-binding assay (Clausen & Hansen, 1977) combined with mechanical skinning of muscle fibres, which removes the sarcolemma (Lamb & Stephenson, 1990). Extensor digitorum longus muscles isolated from rats post mortem were incubated at 30°C to saturation in buffer with 10-6 M 3H-ouabain before being washed at 0°C and transferred to paraffin oil. Then fibre segments were isolated, mechanically skinned and taken for determination of 3H-ouabain binding. Similarly, intact fibres were isolated. Fibre volume was determined from fibre length and diameter while still under paraffin oil. To express the results relative to cell solids, the relation between fibre volume and volume of cell solids in fibres while still under paraffin oil was calculated from similar experiments where muscles were washed at 0°C in buffer containing 3H2O, making it possible to determine the water content of muscles and fibre segments. In intact fibres, the content of 3H-ouabain-binding sites was 2150±271 pmol (cm cell solids)-3 (mean ± SEM, n=10). Skinning of fibres reduced their content of 3H-ouabain-binding sites to 1123±119 pmol (cm muscle)-3, indicating that 53% of the total muscle content of 3H-ouabain-binding sites was located in the T-tubules. In whole muscle, the content of ouabain-binding sites was 399±21 pmol (g wet wt)-1 corresponding to a content of α2 Na+-K+ pumps of 399 pmol (g wet wt)-1. If 53% of these Na+-K+ pumps are situated in the T-tubules, this corresponds to 207 pmol (g muscle)-1, which gives a maximum capacity for active re-uptake of T-tubular K+ of 3300 nmol (g wet wt)-1 min-1 (Clausen, 2003). Based on a T-tubular volume of 0.014 ml (g wet wt)-1 (Launikonis & Stephenson, 2001) this allows the T-tubular Na+-K+ pumps to clear T-tubular K+ at a maximal rate of ~4 mM s-1, indicating that at end of exercise, the recover of T-tubular K+ can be completed within seconds.
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