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

Abstract

Despa S, Bers DM. Functional analysis of Na ϩ /K ϩ -ATPase isoform distribution in rat ventricular myocytes. Am J Physiol Cell Physiol 293: C321-C327, 2007. First published March 28, 2007; doi:10.1152/ajpcell.00597.2006.-The Na ϩ /K ϩ -ATPase (NKA) is the main route for Na ϩ extrusion from cardiac myocytes. Different NKA ␣-subunit isoforms are present in the heart. NKA-␣1 is predominant, although there is a variable amount of NKA-␣2 in adult ventricular myocytes of most species. It has been proposed that NKA-␣2 is localized mainly in T-tubules (TT), where it could regulate local Na ϩ /Ca 2ϩ exchange and thus cardiac myocyte Ca 2ϩ . However, there is controversy as to where NKA-␣1 vs. NKA-␣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-␣1 (low) and NKA-␣2 (high) in rat heart. Ouabain-dependent I Pump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-␣2, K 1/2 ϭ 0.38 Ϯ 0.16 M) that accounts for 29.5 Ϯ 1.3% of IPump and a low-affinity isoform (NKA-␣1, K 1/2 ϭ 141 Ϯ 17 M) that accounts for 70.5% of I Pump. Detubulation decreased cell capacitance from 164 Ϯ 6 to 120 Ϯ 8 pF and reduced I Pump density from 1.24 Ϯ 0.05 to 1.02 Ϯ 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-␣2 accounted for only 18.2 Ϯ 1.1% of I Pump. Thus, ϳ63% of I Pump generated by NKA-␣2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-␣2/NKA-␣1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-␣2 is ϳ4.5 times higher in the T-tubules vs. ESL, whereas NKA-␣1 is almost uniformly distributed between the TT and ESL. The NKA has two essential subunits: ␣ and ␤. The ␣ subunit (ϳ110 kDa) contains the binding sites for Na ϩ -K ϩ -ATP and cardiac glycosides. The smaller ␤ subunit (ϳ50 kDa) has a role in the processing and in the proper membrane insertion of the pump. There are three ␣ (␣1-␣3) and three ␤ (␤1-␤3) NKA subunit isoforms in the heart, with any ␣-␤ combination resulting in a functional pump. NKA-␣1 is present in the heart of all species studied, whereas the expression of NKA-␣2 and NKA-␣3 differs significantly between species (16). For instance, the adult rodent heart expresses NKA-␣1 and NKA-␣2, whereas dogs and monkeys do not have the NKA-␣2 subunit (11). In humans, all three NKA-␣ isoforms can be detected (22).It has been suggested that different NKA isoforms may function differently in the cell, depending on differential localization in the membrane. Juhaszova and Blaustein (14) showed that in smooth muscle NKA-␣2 is localized at the junctions with the sarcoplasmic reticulum (SR) and regulates local Na ϩ /Ca 2ϩ exchange and [Ca 2ϩ ] i . James et al. (13) showed that mouse hearts with genetically reduced levels of NKA-␣ were hypercontractile as a result of increase...

S-Glutathionylation of the Na,K-ATPase Catalytic α Subunit Is a Determinant of the Enzyme Redox Sensitivity

“…Therefore, GRX coordinates the activity of numerous redox-sensitive proteins adjusting to the changes in the redox microenvironment (Fig. 5, C and D) (35)(36)(37). Spontaneous deglutathionylation catalyzed by GRX and thioredoxins is most likely to be the cause of the gradual loss of the inhibitory effect of ischemia on the Na,K-ATPase in heart tissue homogenate with time, as reported by Fuller et al (12).…”

“…The ␣2 isoform of the catalytic subunit is mainly localized in the T-tubular zones, where it is associated with the Na ϩ /Ca 2ϩ exchanger (36). Thus, S-glutathionylation and the following inhibition of the ␣2␤ isozyme may have a profound effect on the intracellular Ca 2ϩ handling in cardiomyocytes (36,37,39). As the ␣2 isoform has been reported to be particularly prone to oxidation (8), more favorable binding of glutathione to this isoform will protect it from oxidation.…”

Activation of cAMP-dependent Signaling Induces Oxidative Modification of the Cardiac Na+-K+ Pump and Inhibits Its Activity

“…We switched to the ouabain-containing superfusate 8 -10 min after the whole-cell configuration had been established in all experiments. As the effect of ouabain is not reversible within the time frame that stable holding currents can be reliably measured (22,23), the suspension of myocytes was removed at the conclusion of the experimental protocol, and a new aliquot of myocytes was added to the tissue bath in ouabain-free solution. As a consequence, only one myocyte was studied per bath.…”

Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

“…However, things are less clear in the heart. The functional density of NKA-␣2 is significantly higher in the T-tubules (versus external sarcolemma) in cardiac myocytes from both rats (16,17) and mice (18), but their precise localization with respect to the junctions with the sarcoplasmic reticulum is not known. Based on Ca 2ϩ transients from heterozygous NKA-␣1 ϩ/Ϫ and NKA-␣2 ϩ/Ϫ mice, James et al (19) concluded that NKA-␣2 is involved in cardiac myocyte Ca 2ϩ regulation, whereas NKA-␣1 is not.…”