Аннотация
OBJECTIVES: 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 (-dNa(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. CONCLUSION: 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.
- adrenergic
- amp-dependent
- analysis/metabolism
- analysis/metabolism;
- animals;
- atpase,
- beta-agonists,
- c57bl;
- cardiac,
- confocal;
- cyclic
- enzymology/ultrastructure;
- enzymology;
- fibers,
- formamides;
- heart
- inbred
- isoenzymes,
- isoproterenol,
- kinases,
- metabolism;
- mice,
- mice;
- microscopy,
- muscle
- myocytes,
- patch-clamp
- pharmacology;
- protein
- sarcolemma,
- sodium,
- sodium-potassium-exchanging
- techniques;
- ventricles;
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