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 α-subunit isoforms are present in the heart. NKA-α1 is predominant, although there is a variable amount of NKA-α2 in adult ventricular myocytes of most species. It has been proposed that NKA-α2 is localized mainly in T-tubules (TT), where it could regulate local Na+/Ca2+ exchange and thus cardiac myocyte Ca2+. However, there is controversy as to where NKA-α1 vs. NKA-α2 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 (IPump) measurements and the different ouabain sensitivity of NKA-α1 (low) and NKA-α2 (high) in rat heart. Ouabain-dependent IPump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-α2, K1/2 = 0.38 ± 0.16 μM) that accounts for 29.5 ± 1.3% of IPump and a low-affinity isoform (NKA-α1, K1/2 = 141 ± 17 μM) that accounts for 70.5% of IPump. Detubulation decreased cell capacitance from 164 ± 6 to 120 ± 8 pF and reduced IPump 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-α2 accounted for only 18.2 ± 1.1% of IPump. Thus, ∼63% of IPump generated by NKA-α2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-α2/NKA-α1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-α2 is ∼4.5 times higher in the T-tubules vs. ESL, whereas NKA-α1 is almost uniformly distributed between the TT and ESL.

Functional expression of putative H+-K+-ATPase from guinea pig distal colon

A guinea pig cDNA encoding the putative colonic H+-K+-ATPase α-subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854 ) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonic H+-K+-ATPase was cotransfected with cDNA for either rabbit gastric H+-K+-ATPase or TorpedoNa+-K+-ATPase β-subunit. In both expressions, Na+-independent, K+-dependent ATPase (K+-ATPase) activity was detected in the membrane fraction of the cells, with a Michaelis-Menten constant for K+ of 0.68 mM. The expressed K+-ATPase activity was inhibited by ouabain, with its IC50 value being 52 μM. However, the activity was resistant to Sch-28080, an inhibitor specific for gastric H+-K+-ATPase. The ATPase was not functionally expressed in the absence of the β-subunits. Therefore, it is concluded that the cDNA encodes the catalytic subunit (α-subunit) of the colonic H+-K+-ATPase. Although the β-subunit of the colonic H+-K+-ATPase has not been identified yet, both gastric H+-K+-ATPase and Na+-K+-ATPase β-subunits were found to act as a surrogate for the colonic β-subunit for the functional expression of the ATPase. The present colonic H+-K+-ATPase first expressed in mammalian cells showed the highest ouabain sensitivity in expressed colonic H+-K+-ATPases so far reported (rat colonic in Xenopus oocytes had an IC 50 = 0.4–1 mM; rat colonic in Sf9 cells had no ouabain sensitivity).

K+ self-exchange by the Na+ pump: regulation by P(i) and metabolic perturbations

We have previously demonstrated that the Na(+)-K+ pump on the basolateral membrane of the rabbit cortical collecting duct can function in the K+/K+ exchange mode. Increasing intracellular phosphate in red blood cells inhibits the Na+ pump and increases K+/K+ exchange. We found that maneuvers designed to increase intracellular phosphate in collecting duct cells caused an increase in K+/K+ exchange. Subjecting the cells to a metabolic insult (cyanide) increased K+/K+ exchange by the pump as judged by its ouabain sensitivity and lack of electrogenic or conductive characteristics. The results demonstrate that the rate of K+/K+ exchange by the Na(+)-K+ pump can be altered by changes in intracellular phosphate over a range that is physiologically or pathologically achievable. The results also suggest a mechanism for inhibition of vectorial Na+ transport during metabolic stress.