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-alpha1 is predominant, although there is a variable amount of NKA-alpha2 in adult ventricular myocytes of most species. It has been proposed that NKA-alpha2 is localized mainly in T-tubules (TT), where it could regulate local Na(+)/Ca(2+) exchange and thus cardiac myocyte Ca(2+). However, there is controversy as to where NKA-alpha1 vs. NKA-alpha2 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-alpha1 (low) and NKA-alpha2 (high) in rat heart. Ouabain-dependent I(Pump) inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-alpha2, K(1/2) = 0.38 +/- 0.16 microM) that accounts for 29.5 +/- 1.3% of I(Pump) and a low-affinity isoform (NKA-alpha1, K(1/2) = 141 +/- 17 microM) 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-alpha2 accounted for only 18.2 +/- 1.1% of I(Pump). Thus, approximately 63% of I(Pump) generated by NKA-alpha2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-alpha2/NKA-alpha1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-alpha2 is approximately 4.5 times higher in the T-tubules vs. ESL, whereas NKA-alpha1 is almost uniformly distributed between the TT and ESL.
Despa
Sanda
S
Dept. of Physiology, Loyola University Chicago, Stritch School of Medicine, 2160 South First Ave., Maywood, IL 60153, USA.
Bers
Donald M
DM
D000818:Animals; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005559:Formamides / Q000494:pharmacology; D006352:Heart Ventricles / Q000166:cytology / Q000201:enzymology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D008564:Membrane Potentials; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D012508:Sarcolemma / Q000187:drug effects / Q000201:enzymology*; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013997:Time Factors
D004791:Enzyme Inhibitors; D005559:Formamides; D007527:Isoenzymes; C031066:formamide; D010042:Ouabain; D012964:Sodium; C499075:Atp1a1 protein, rat; C498532:Atp1a2 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase