Regulation of endogenous and expressed Na+/K+ pumps in Xenopus oocytes by membrane potential and stimulation of protein kinases

Abstract

Modulation of the current generated by the Na+/K+ pump by membrane potential and protein kinases was investigated in oocytes of Xenopus laevis. In addition to a positive slope region in the current-voltage (I-V) relationship of the Na+/K+ pump, a negative slope region has been described in these cells (Lafaire & Schwarz, 1986) and has been attributed to a voltage-dependent apparent Km value for pump stimulation by external [K+] (Rakowski et al., 1991). To study this feature in more detail, Xenopus oocytes were used for comparative analysis of the negative slope of the I-V relationship of the endogenous Na+/K+ pump and of the Na+/K+ pump of the electric organ of Torpedo californica expressed in the oocytes. The effects of stimulation of protein kinases A and C on the negative slope were also analyzed. To investigate the negative slope over a wide potential range, experiments were performed in Na(+)-free solution and in the presence of high concentrations of Ba2+ and tetraethylammonium, to block all nonpump related K(+)-sensitive currents. Pump currents and pump-mediated fluxes were determined as differences of currents or fluxes in solutions with and without extracellular K+. The voltage dependence of the Km value for stimulation of the Na+/K+ pump by external [K+] shows significant species differences. Over the entire voltage range from -140 to +20 mV, the Km value for the Na+/K+ pump of Torpedo electroplax is substantially higher than for the endogenous pump and exhibits more pronounced voltage dependence. For the Xenopus pump, the voltage dependence can be described by voltage-dependent stimulation by external [K+] and can be interpreted by voltage-dependent K+ binding, assuming that an effective charge between 0.37 and 0.56 of an elementary charge is moved in the electrical field. An analogous evaluation of the voltage dependence of the Torpedo pump requires the assumption of movement of two effective charges of 0.16 and 1.0 of an elementary charge. Application of 1,2-dioctanoyl-sn-glycerol (diC8, 10-50 microM), which is known to stimulate protein kinase C, reduces the maximum activity of the Xenopus pumps in the oocyte membrane by 40% and modulates the voltage dependence of K+ stimulation. For the endogenous Xenopus pump, the apparent effective charge increased from 0.37 to 0.51 of elementary charge and the apparent Km at 0 mV increased from 0.46 to 0.83 mM. For the Torpedo pump, one of the apparent effective charges increased from 1.0 to 2.5 of elementary charge.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of the beta‐stimulation of the Na(+)‐K+ pump current in guinea‐pig ventricular myocytes by a cAMP‐dependent PKA pathway.

“…However, active Na+ transport is directly stimulated by Iso in isolated cardiac myocytes from rabbit heart (Desilets & Baumgarten, 1986). Injection or superfusion of Xenopus oocytes with cAMP, which activates PKA, has a clear stimulatory effect on Ip (Vasilets & Schwarz, 1992).…”

Role of Serine/Threonine Protein Phosphatases in Insulin Regulation of Na+/K+-ATPase Activity in Cultured Rat Skeletal Muscle Cells

“…The supernatants were centrifuged at 100,000 ϫ g for 20 min to pellet the membrane fraction (13). The crude plasma membranes were reconstituted in 200 l of ATPase assay buffer containing 100 mM imidazole, 5 mM MgCl 2 , 100 mM NH 4 Cl, 150 mM NaCl, 1 mM EGTA, 2 mM sodium azide, and a mixture of the protease inhibitors indicated above. Aliquots of membrane preparations were used for the assay of proteins and Na ϩ /K ϩ -ATPase activity.…”

“…This enzyme is composed of a 112-kDa catalytic ␣ subunit, of which there are three isoforms, and a 35-kDa ␤ subunit (two isoforms) responsible for targeting the ␣ subunit to the plasma membrane and maintaining its structural integrity (1)(2). Recent studies on purified enzyme preparations as well as intact cells indicate that Na ϩ /K ϩ -ATPase activity may be regulated acutely by phosphorylation/dephosphorylation reactions (2)(3)(4)(5), whereas the long-term regulation exerted by certain hormones is mediated by changes in gene expression (2,6). Thus, phosphorylation of the catalytic subunit (␣ subunit) of Na ϩ /K ϩ -ATPase by protein kinase A and protein kinase C (PKC) 1 in vitro inhibits enzymatic activity (7).…”

The effect of cardiac glycosides on the Na+ pump current‐voltage relationship of isolated rat and guinea‐pig heart cells.

“…Apart from the cations, only few substances have been reported so far to change the voltage dependence of Ip. According to Vasilets & Schwarz (1992), stimulation of protein kinases varies the Ip-V curve of endogenous Xenopus pumps and of Na+-K+ pumps of Torpedo expressed in Xenopus oocytes. The results to be described below demonstrate that cardiac glycosides, specific inhibitors of the Na+-K+ pump, alter the Ip-V relationship of cardiac cells and neurones.…”