Na,K-ATPase in the myocardium: molecular principles, functional and clinical aspects (original) (raw)
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Two functional Na+/K+-ATPase isoforms in the left ventricle of guinea pig heart
European Journal of Biochemistry, 1991
Guinea pig left ventricular muscle contains two distinct molecular forms of the N a + /K+-ATPase catalytic a subunit. Sarcolemmal vesicles highly enriched in Na+/K+-ATPase were isolated by a new procedure that yielded specific activities of 60 -100 pmol Pi . h-' . mg-'. SDSjPAGE of isolated sarcolemma after reduction and alkylation of the sulfhydryl groups and identification on immunoblots with specific anti-(a subunit) antibodies indicated the presence of two major polypeptides of 100 kDa and 103 kDa, respectively. The two a subunits were functional: the dose/response curves of Na+/K+-ATPase activity with ouabain, dihydroouabain and digitoxigenin were biphasic, revealing the presence of high-affinity [concentration of drug causing 50% inhibition (IC50) = 10 nM] and low-affinity (ICs0 = 2 pM) forms with proportional contributions of 55% and 45%, respectively. The involvement of the high-affinity form in the positive inotropic effect of digitalis and of the low-affinity sites in both inotropy and toxicity are consistent with the literature data on rodents.
The Cellular Basis of Cardiovascular Function in Health and Disease, 1997
In order to understand the functional significance of Na,K-ATPase subunits as well as their isoenzymes, a precise subcellular localization of these in the myocyte is a crucial prerequisite. Cytochemical, immunofluorescence, preembedding immunogold and horse radish peroxidase-diaminobenzidine methods, demonstrated α 1 isoenzyme immunoreactivity on the sarcolemma, T-tubules and the subsarcolemmal cisterns of the adult cardiac myocytes. Cytochemically, ouabain resistant Na,K-ATPase precipitate was localized only in the subsarcolemmal cisterns and junctional sarcoplasmic reticulum. For α 2 isoenzyme, immunoreactivity was demonstrated on the sarcolemma as well as in all areas of the myocytes in particularly a close proximation to the sarcoplasmic reticulum and microsomes. For α 3 isoenzyme, only a weak insignificant signal was noted on the sarcolemma, intercalated disc and sarcoplasm. It is suggested that cytochemical ouabain resistant precipitate present in subsarcolemmal cisterns and junctional sarcoplasmic reticulum represent α 1 isoenzyme of Na,K-ATPase. A differential as well as unique localization of α subunit isoenzymes of Na,K-ATPase in specific structures of cardiac myocytes may suggest importance in physiological function at these sites. (Mol Cell Biochem 176: [107][108][109][110][111][112] 1997)
Molecular Cell, 1999
muscle. The ␣ subunit of the Na,K-ATPase also contains 3 Department of Molecular and Cellular Physiology the receptor site for the cardiac glycoside class of com-4 Division of Cardiology pounds (such as ouabain and digoxin), which inhibit University of Cincinnati College of Medicine the Na,K-ATPase, increase cardiac contractility, and Cincinnati, Ohio 45267 are used in the treatment of congestive heart failure (Thomas et al., 1990).
Turnover rates of the canine cardiac Na,K-ATPases
FEBS Letters, 1993
Two functional isoforms a (a,) and a' (a,) of the Na.K-ATPase catalytic subumt coexist m canine cardiac myocytes [J. Biol. Chem. (1987) 262, 8941-89431 The m vitro turnover rates of ATP hydrolysis have been determined in sarcolemma preparations by comparing ["Hlouabain-binding and Na,K-ATPase activity at various doses of ouabam (0.3-300 nM). The correlation between the occupancy of the ouabain-binding sites and the degree of Na.K-ATPase mhibition was not linear. The results showed that the form of low-affinity for ouabam (& = 300-700 nM) exhibited a lower turnover rate (88 + 10 vs. 147 ? 15 molecules of ATP hydrolyzed per second per ouabain-binding site) than the high affinity form (K, = I-8 nM). Thus our results indicate this specific isoform kmetic difference could contribute to differences in the cardiac cellular function.
Extracellular Allosteric Na+ Binding to the Na+,K+-ATPase in Cardiac Myocytes
Biophysical Journal, 2013
Whole-cell patch-clamp measurements of the current, I p , produced by the Na þ ,K þ-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in I p over the extracellular Na þ concentration range 0-50 mM. This is not predicted by the classical Albers-Post scheme of the Na þ ,K þ-ATPase mechanism, where extracellular Na þ should act as a competitive inhibitor of extracellular K þ binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K þ ions into the cytoplasm. The increase in I p is consistent with Na þ binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K þ to the cytoplasm, E2(K þ) 2 / E1 þ 2K þ. At normal physiological concentrations of extracellular Na þ of 140 mM, it is to be expected that binding of Na þ to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme's ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the aand b-subunits of the enzyme.
Immunodetection and enzymatic characterization of the α3-isoform of Na,K-ATPase in dog heart
FEBS Letters, 1994
The expression of the canine a 2 and 3 subunit isoenxymes of NA,K-ATPase has been investigated in plasma membranes isolated from dog heart, brain and kidney by immunoblotting, employing polyclonal anti rat fusion protein, and enzymological techniques. Western blot analysis revealed with purified dog membrane Na,K-ATPase preparations, one immunoreactive signal with rat specific cc3 antisera in cardiac tissues, and two immunoreactive signals with rat a, and cc3 antisera in cerebral tissues. These findings suggested the specific expression of a3 polypeptide in dog heart (99 kDa), whereas dog brain expressed the a 2 and 3 polypeptides. The stained bands were superimposed. The antibody to rat brain a, fusion protein did not cross-react with dog antigens whatever the three tissues tested. Expression of the a,-subunit isoform in dog heart membranes was consistent with a high affinity digitoxigenin-sensitive class of Na,K-ATPase (IC,, = 7 + 2 nM). A single component with low affinity to digitoxigenin (IC&, = 110 f 10 nM) characterized the a, kidney form. The mixture of a, and a3 isoforms in dog brain exhibited an apparent affinity for digitoxigenin (I& = 17 + 5 nM) lower than the heart. The sodium dependences of the high affinity digitoxigenin sites were for the cardiac a,form (& = 10 ? 1.9 mM) and for the cerebral a2 and a3 mixture (&,, 19.6 ? 4.9 mM). The sensitivities for Na' of the low affinity sites (a,) were: 6.7 f 1.4 mM, 6.3 f 1.2 mM and 11.6 + 2.9 mM in heart, brain and kidney respectively. This is the first report of the catalytic characteristics of the a3 subunit isoenzyme in canine cardiac plasma membranes.
Na(+) /Ca(2+) exchange and Na(+) /K(+) -ATPase in the heart
The Journal of physiology, 2015
This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na(+) /Ca(2+) exchange (NCX) and Na(+) /K(+) -ATPase (NKA). While the relevance of Ca(2+) homeostasis in cardiac function has been extensively investigated, the role of Na(+) regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na(+) content have multiple effects on the heart by influencing intracellular Ca(2+) and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na(+) homeostasis. Among the proteins that accomplish this task are t...