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Subcellular localization of Na/K-ATPase isoforms in ventricular myocytes.

ABSTRACT: The sodium/potassium ATPase (NKA) is essential for establishing the normal intracellular [Na+] and [K+] and transmembrane gradients that are essential for many cellular functions, including cardiac electrophysiology and contractility. Different NKA isoforms exhibit differential expression levels, cellular localization, and function in different tissues and species. Prior work has indicated that the NKA-?1 isoform is quantitatively predominant in cardiac myocytes, but that the ?2 isoform is preferentially concentrated in the transverse tubules (TT), possibly at junctions with the sarcoplasmic reticulum (SR) where ?2 may preferentially modulate cardiac contractility. Here we measured subcellular localization of NKA-?1 and ?2 using super-resolution microscopy (STED and STORM) and isoform-selective antibodies in mouse ventricular myocytes. We confirm the preferential localization of NKA-?2 in TT vs. surface sarcolemma, but also show that ?2 is relatively excluded from longitudinal TT elements. In contrast NKA-?1 is relatively uniformly expressed in all three sarcolemmal regions. We also tested the hypothesis that NKA-?2 (vs. ?1) is preferentially concentrated at SR junctional sites near ryanodine receptors (RyR2). The results refute this hypothesis, in that NKA-?1 and ?2 were equally close to RyR2 at the TT, with no preferential NKA isoform localization near RyR2. We conclude that in contrast to relatively uniform NKA-?1 distribution, NKA-?2 is preferentially concentrated in the truly transverse (and not longitudinal) TT elements. However, NKA-?2 does not preferentially cluster at RyR2 junctions, so the TT NKA-?2 concentration may suffice for preferential effects of NKA-?2 inhibition on cardiac contractility.

SUBMITTER: Yuen GK

PROVIDER: S-EPMC5551422 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Subcellular localization of Na/K-ATPase isoforms in ventricular myocytes.

Yuen Garrick K GK Galice Samuel S Bers Donald M DM

Journal of molecular and cellular cardiology 20170603

The sodium/potassium ATPase (NKA) is essential for establishing the normal intracellular [Na<sup>+</sup>] and [K<sup>+</sup>] and transmembrane gradients that are essential for many cellular functions, including cardiac electrophysiology and contractility. Different NKA isoforms exhibit differential expression levels, cellular localization, and function in different tissues and species. Prior work has indicated that the NKA-α1 isoform is quantitatively predominant in cardiac myocytes, but that t ...[more]

PMID: 28587810

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Project description:We measured intracellular Mg2+ concentration ([Mg2+]i) in rat ventricular myocytes using the fluorescent indicator furaptra (25 degrees C). In normally energized cells loaded with Mg2+, the introduction of extracellular Na+ induced a rapid decrease in [Mg2+]i: the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) is thought to represent the rate of Na+-dependent Mg2+ efflux (putative Na+/Mg2+ exchange). To determine whether Mg2+ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na+ gradient, we estimated the initial Delta[Mg2+]i/Deltat after metabolic inhibition. In the absence of extracellular Na+ and Ca2+, treatment of the cells with 1 microM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg2+]i from approximately 0.9 mM to approximately 2.5 mM in a period of 5-8 min (probably because of breakdown of MgATP and release of Mg2+) and cell shortening to approximately 50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg2+]i and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for > or = 90 min. The initial Delta[Mg2+]i/Deltat was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59-85 min), a significant decrease in the initial Delta[Mg2+]i/Deltat (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na+ concentration ([Na+]i) estimated with a Na+ indicator sodium-binding benzofuran isophthalate was, on average, 5.0-10.5 mM during the time required for the initial Delta[Mg2+]i/Deltat measurements, which is well below the [Na+]i level for half inhibition of the Mg2+ efflux (approximately 40 mM). Normalization of intracellular pH using 10 microM nigericin, a H+ ionophore, did not reverse the inhibition of the Mg2+ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (approximately 0.4 mM estimated indirectly in the this study), rather than elevation of [Na+]i or intracellular acidosis, inhibits the Mg2+ efflux, suggesting the absolute necessity of ATP for the Na+/Mg2+ exchange.

Dataset Information

Subcellular localization of Na/K-ATPase isoforms in ventricular myocytes.

Publications

Subcellular localization of Na/K-ATPase isoforms in ventricular myocytes.

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