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 α-subunit isoforms are present in the heart. NKA-α1 is predominant, although there is a variable amount of NKA-α2 in adult ventricular myocytes of most species. It has been proposed that NKA-α2 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-α1 vs. NKA-α2 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-α1 (low) and NKA-α2 (high) in rat heart. Ouabain-dependent I_Pump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-α2, K_1/2 = 0.38 ± 0.16 μM) that accounts for 29.5 ± 1.3% of I_Pump and a low-affinity isoform (NKA-α1, K_1/2 = 141 ± 17 μM) that accounts for 70.5% of I_Pump. Detubulation decreased cell capacitance from 164 ± 6 to 120 ± 8 pF and reduced I_Pump 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-α2 accounted for only 18.2 ± 1.1% of I_Pump. Thus, -∼63% of I_Pump generated by NKA-α2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-α2/NKA-α1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-α2 is ∼-4.5 times higher in the T-tubules vs. ESL, whereas NKA-α1 is almost uniformly distributed between the TT and ESL.