Properties of sodium pumps in internally perfused barnacle muscle fibers

To study the properties of the Na extrusion mechanism, giant muscle fibers from barnacle (Balanus nubilus) were internally perfused with solutions containing tracer 22Na. In fibers perfused with solutions containing adenosine 5'-triphosphate (ATP) and 30 mM Na, the Na efflux into 10 mM K seawater was approximately 25-30 pmol/cm2.s; 70% of this efflux was blocked by 50-100 microM ouabain, and approximately 30% was blocked by removal of external K. The ouabain-sensitive and K-dependent Na effluxes were abolished by depletion of internal ATP and were sigmoid-shaped functions of the internal Na concentration ([Na]i), with half-maxima at [Na]i approximately or equal to 20 mM. These sigmoid functions fit the Hill equation with Hill coefficients of approximately 3.5. Ouabain depolarized ATP-fueled fibers by 1.5-2 mV ([Na]i greater than or equal to 30 mM) but had very little effect on the membrane potential of ATP-depleted fibers; ATP depletion itself caused a 2-2.5- mV depolarization. When fueled fibers were treated with 3,4- diaminopyridine or Ba2+ (to reduce the K conductance and increase membrane resistance), application of ouabain produced a 4-5 mV depolarization. These results indicate that an electrogenic, ATP- dependent Na-K exchange pump is functional in internally perfused fibers; the internal perfusion technique provides a convenient method for performing transport studies that require good intracellular solute control.