Differential distribution and regulation of mouse cardiac Na+/K+-ATPase alpha1 and alpha2 subunits in T-tubule and surface sarcolemmal membranes.
OBJECTIVE
Two Na+/K+-ATPase (NKA) alpha-subunit isoforms, alpha1 and alpha2, 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 alpha1 and alpha2 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) (Ialpha1) and 12% alpha2-mediated I(pump) (Ialpha2). alpha1 and alpha2 subunits demonstrate distinct ouabain affinities (105+/-6 and 0.3+/-0.1 micromol/L respectively) but similar affinity for intracellular Na+ (K(1/2)Na+ 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) Ialpha2 contribution (12 to 6%). Total I(pump) density was approximately 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 approximately 70% of Ialpha2 and approximately 37% of Ialpha1. Ialpha1 density was substantially higher than Ialpha2 in SSL (Ialpha1:Ialpha2 = 16:1) but this was markedly reduced in T-tubules (4:1). In addition to differential localisation, isoprenaline (ISO, 1 micromol/L) significantly increased alpha1-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 alpha2-mediated NKA activity.


CONCLUSIONS
These data suggest that NKA alpha1 and alpha2 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.
Berry
Roger G
RG
Cardiac Physiology, King's College London, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK.
Despa
Sanda
S
Fuller
William
W
Bers
Donald M
DM
Shattock
Michael J
MJ
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005559:Formamides; D006352:Heart Ventricles; D007527:Isoenzymes / Q000032:analysis* / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D018485:Muscle Fibers, Skeletal / Q000201:enzymology; D032383:Myocytes, Cardiac / Q000201:enzymology* / Q000648:ultrastructure; D018408:Patch-Clamp Techniques; D012508:Sarcolemma / Q000201:enzymology; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis* / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D005559:Formamides; D007527:Isoenzymes; C031066:formamide; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase; D007545:Isoproterenol