Differential distribution and regulation of mouse cardiac Na+/K+-ATPase alpha(1) and alpha(2) subunits in T-tubule and surface sarcolemmal membranes

Objectives: Two Na+/K+-ATPase (NKA) alpha-subunit isoforms, alpha(1) and alpha(2), are expressed in the adult mouse heart. The subcellular distribution of these isoforms in T-tubule and surface sarcolemmal (SSL) membranes and their regulation by cAMP-dependent protein kinase (PKA) is unclear.Methods: We used formamide-induced detubulation of mouse ventricular myocytes to investigate differential functional distribution and regulation by PKA of alpha(1) and alpha(2) in T-tubule versus SSL membranes by measuring NKA current (I-pump) and NKA-mediated Na+ efflux (-d[Na](i)/dt).Results: I-pump is composed of 88% alpha(1)-mediated I-pump (I alpha(1)) and 12% alpha(2)-mediated I-pump (I alpha(2)). alpha(1) and alpha(2) subunits demonstrate distinct ouabain affinities (105 +/- 6 and 0.3 +/- 0.1 mu mol/L respectively) but similar affinity for intracellular Na+ (K1/2Na+ of 16.6 +/- 0.8 and 16.7 +/- 2.6 mmol/L respectively). Detubulation reduced (i) I-pump density (1.42 +/- 0.1 to 1.20 +/- 0.04 pA/pF), (ii) cell capacitance (181 +/- 12 to 127 +/- 17 pF), and (iii) I alpha(2) contribution (12 to 6%). Total I-pump density was similar to 60% higher in T-tubule (1.94 pA/pF, derived) vs. SSL membranes. Although T-tubule membranes represent only 30% of total surface area, they generate similar to 70% of I alpha(2) and similar to 37% of I alpha(1). I alpha(1) density was substantially higher than I alpha(2) in SSL (I alpha(1):I alpha(2) = 16:1) but this was markedly reduced in T-tubules (4:1). In addition to differential localisation, isoprenaline (ISO, 1 mu mol/L) significantly increased alpha(1)-mediated NKA Na+ affinity (from 16.6 +/- 0.8 to 13.3 +/- 1.4 mmol/L) and caused a small increase in maximal NKA Na+ efflux rate. ISO had no effect on alpha(2)-mediated NKA activity.Conclusion: These data suggest that NKA alpha(1) and alpha(2) subunits are differentially localised and regulated by PKA in T-tubule and SSL membranes and may have distinct regulatory roles in cardiac excitation-contraction coupling. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.