Ionic diffusion in voltage-clamped isolated cardiac myocytes. Implications for Na,K-pump studies.

The whole-cell voltage-clamp technique employing electrolyte-filled micro-pipette suction electrodes is widely used to investigate questions requiring an electrophysiological approach. With this technique, the ionic composition of the cytosol is assumed to be strongly influenced (as result of diffusion) by the ionic composition of the solution contained in the electrode. If this assumption is valid for isolated cardiac myocytes, the technique would be particularly powerful for studying the dependence of their Na,K-pump on the intracellular Na+]. However, the relationship between the concentrations of ions in the solution filling the electrode and those in the cytosol has not been established. The relationship was investigated to determine in particular whether the Na+] at the intracellular cation ligand binding sites for the Na-pump ( Na+]ps) can be set and clamped by Na+] in the pipette electrode ( Na+]pip). If Na+]pip can set and clamp Na+]ps, this would provide a means for defining the dependence of the Na,K-pump on intracellular Na+]. The relationship between Na+]pip and Na+]ps was analyzed using two approaches. First, a mathematical model of three-dimensional ionic diffusion within a whole-cell patch-clamped myocyte was developed and the effects of experimental parameters on mean Na+]ps were investigated. When typical experimental values were simulated, the time course to achieve steady state mean Na+]ps was found to be most sensitive to variations in electrode pore size, cell length and the Na+ pumping rate, but at steady state, mean Na+]ps varies from Na+]pip by 5% or less depending on pump rate. Second, to provide experimental support for the validity of the simulations, isolated ventricular myocytes were voltage-clamped and the reversal potential for the Na current was determined in order to estimate steady state intracellular Na+]. The results of the mathematical and experimental analyses suggest that steady state Na+]ps can be regulated by the Na+] in suction pipette electrodes. These findings, while also having a broader significance, indicate for isolated cardiac myocytes that whole-cell suction micro-electrodes can provide a means to assess the dependence of the Na,K-pump on Na+]ps.