Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes

The Na+/K+-ATPase (NKA) is the main route for Na+ extrusion from cardiac myocytes. Different NKA alpha-subunit isoforms are present in the heart. NKA-alpha 1 is predominant, although there is a variable amount of NKA-alpha 2 in adult ventricular myocytes of most species. It has been proposed that NKA-alpha 2 is localized mainly in T-tubules ( TT), where it could regulate local Na+/Ca2+ exchange and thus cardiac myocyte Ca2+. However, there is controversy as to where NKA-alpha 1 vs. NKA-alpha 2 are localized in ventricular myocytes. Here, we assess the TT vs. external sarcolemma (ESL) distribution functionally using formamide-induced detubulation of rat ventricular myocytes, NKA current (I-Pump) measurements and the different ouabain sensitivity of NKA-alpha 1 (low) and NKA-alpha 2 (high) in rat heart. Ouabain-dependent I-Pump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-alpha 2, K-1/2 = 0.38 +/- 0.16 mu M) that accounts for 29.5 +/- 1.3% of IPump and a low-affinity isoform (NKA-alpha 1, K-1/2 = 141 +/- 17 mu M) that accounts for 70.5% of IPump. Detubulation decreased cell capacitance from 164 +/- 6 to 120 +/- 8 pF and reduced IPump density from 1.24 +/- 0.05 to 1.02 +/- 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-alpha 2 accounted for only 18.2 +/- 1.1% of IPump. Thus, similar to 63% of IPump generated by NKA-alpha 2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-alpha 2/ NKA-alpha 1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-alpha 2 is similar to 4.5 times higher in the T-tubules vs. ESL, whereas NKA-alpha 1 is almost uniformly distributed between the TT and ESL.