Physiological Role of Na/K Pump Isozymes in Rat Hearts

The Na/K pump is an enzyme that transports 3 sodium-out and 2 potassium-in from the cell, using the energy of one ATP molecule. The pump is a member of the P-type ATPase family and is formed by association of one of four α-subunit isoforms (α1-α4) and one of three β-subunit isoforms (β1-β3). It is thought that cell membranes in the heart express the ubiquitous α1 pumps, α2 pumps in atria and ventricles, and α3 pumps in the conduction system. The reasons for isozyme multiplicity, as well as the location of the isozymes in venticular myocytes are not well understood. We used immunofluorescent microscopy to address isozyme location in enzymatically isolated ventricular myocytes from 5 day old-2 month old rats. Immunofluorescence was performed with standard methods. Myocytes were allowed to attach to coverslips, fixed with paraformaldehyde, permeabilized and blocked with BSA. Primary antibodies for α1, α2 or α3(Gift from Dr.T Pressley) were applied for 1 hr at room temperature and left overnight at 4 ºC. A fluorescent-conjugated secondary antibody (Alexa 488, Invitrogen) was applied for 1 hr. Finally, cells were mounted onto slides with Prolong Gold (Invitrogen). The slides were viewed with fluorescent and confocal microscopes. Immunofluorescence results indicate that α1 and α2 isoforms are well expressed in the plasma membrane of all ventricular myocytes with different distribution. Surprisingly, we found that immunostaining with the α3 isoform is seen in some myocytes, with a scarce distribution that appears to be predominantly intracellular.