Coexpression of a , with putative 83 subunits results in functional Na ' / K ' pumps in Xenopus oocytes

The active Na+/K' pump is composed of an a and a (3 subunit. Until now, three putative isoforms of the .3 subunit have been identified that share sequence similarity. We have expressed the (31 and (33 isoforms of Xenopus laevis Na+/K+-ATPase in Xenopus oocytes to compare functional properties of the Na+/K' pump, including either of these two isoforms. Na+/K' pump current, estimated as K+-induced outward current in voltage-clamped oocytes, was doubled by coexpression of a, subunits with either isoform of the (3subunit compared to expression of a, subunits alone. The kinetics of activation by external K+ and the voltage dependence of the electrogenic activity of the Na+/K' pump were similar with both (3 isoforms, indicating that both (3, and .83 isoforms can support expression at the oocyte surface of an active Na+/K' pump with similar functional properties. The Na+/K' pump (Na+/K+-ATPase) is a ubiquitous transport system in vertebrate plasma membranes, responsible for maintaining the high-K+, low-Na' concentrations of the intracellular milieu. Although all the functional sites (Na'-, K+-, and ATP-binding sites) are located on the larger a subunit, the active enzyme always includes a smaller glycosylated ,( subunit (for review, see ref. 1). One function of this ,3 subunit is to support structural and functional maturation of the a subunit and allow its transport to the cell membrane (for review, see ref. 2). The first identified ( isoform, f31, has been described in several species and is the ( subunit of the Na+/K+-ATPase isolated from kidney as well as other tissues (for review, see ref. 3). In addition, a putative (32 subunit has been identified by sequence similarity to the (1 subunit and is preferentially expressed in the nervous system (4). This isoform is nearly identical with the adhesion molecule on glia cloned from mouse brain (5). The (32 isoform has been copurified with the a2 subunit of rat brain Na+/K+-ATPase (6). More recently, Good et al. (7) have isolated in Xenopus laevis a cDNA encoding a protein showing significant homology with both (1 and (32 isoforms. This cDNA was taken from a group of transcripts expressed during early development and in the adult nervous system (8). Although no other (8 isoform similar to rat (32 or mouse adhesion molecule on glia has been identified in Xenopus, this isoform of the ( subunit ofXenopus Na+/K+-ATPase has been named(83 because of important size and sequence dissimilarities with known mammalian 832 isoforms (7). Structure similarities with the 831 subunit and preliminary evidence showing association with the a subunit (9) suggest that (83 isoform might also support the Na+/K+-ATPase activity ofan a subunit, but a functional role for this protein has not yet been demonstrated. The purpose of our study was to determine whether this putative(33 subunit isoform could form, with an a subunit, a competent and functional Na+/K' pump at the plasma membrane. We took advantage of the electrogenic activity of the Na+/K' pump (10) to study the function of Na+/K' pump as a K+-activated or strophanthidin-sensitive current in voltage-clamped Xenopus oocytes previously injected with cRNAs coding for a and (3 subunits. A similar approach has already been used to study the functional properties of Na+/K+-ATPase by injecting heterologous cRNA (Torpedo cRNA in Xenopus oocytes) (11). MATERIALS AND METHODS Oocyte Ijection with Xenopus a and (8 cRNA. Stage V-VI oocytes were obtained from Xenopus females (African Xenopus Facility, Noordhoek, Republic of South Africa) by removing ovary segments from an anesthetized frog. Oocytes were defoliculated with 0.25% collagenase (type 1A; Sigma) in modified Barth's saline without Ca2+ (12). After overnight incubation in complete modified Barth's solution, oocytes were injected with either 10 ng of a,, or 10 ng of a1 plus 7 ng of (1, or 10 ng of a, plus 7 ng of 833 cRNA (in a total 50-nl volume). cRNA were obtained by in vitro transcription of linearized templates according to Melton et al. (13). For templates, cDNAs from al and 81 subunits cloned from Xenopus kidney-derived A6 cells (14) or from P33 cloned from a Xenopus neurula library (8) were recloned into the plasmid pSD5, which allows for synthesis of capped, full-length, poly(A)+ RNA (15). Na+/K' Pump Current Measurements. Electrophysiological measurements were done 2-3 days after cRNA injection. To increase the Na+/K' pump activity and to decrease variation of this activity, the oocytes were first loaded with Na' by a 2-hr exposure to K+and Ca2+-free solution (16). Whole-cell currents were then measured with the twoelectrode voltage-clamp technique (electrode resistance 1-3 Mfl); a Dagan 8500 clamp instrument (Dagan Instruments, Minneapolis) was used. For determination ofcurrent-voltage (I-V) curves, data were recorded and analyzed with the PClamp system (Axon Instruments, Burlingame, CA). All experiments were done at room temperature (22-250C). Na+/K' pump-generated current was estimated by measuring the outward current produced by adding 10 mM K+ to a K+-free solution, while the membrane potential was set at -50 mV and 5 mM barium was present to block currents flowing through K+ channels. Na+/K+ pump current measurements were restricted to oocytes showing a total membrane conductance <5 uS, measured in K+-free solution. Voltage dependence of the Na+/K+ pump activity was determined by establishing the whole-cell I-V curve in the Abbreviation: I-V, current-voltage. tTo whom reprint requests should be addressed. 8397 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8398 Cell Biology: Horisberger et al. presence of 10 mM K+ before and after addition of 25 ILM strophanthidin. Starting from a holding membrane potential of -60 mV, rectangular voltage pulses (400 ms) of various amplitude were applied every 2 s, and steady-state current was measured at 300 ms after start of the voltage step. The kinetics of activation by external K+ was studied by measuring the current induced by successive addition of 0.3, 1.0, 3.0, and 10 mM K+ (in replacement of Na'). The Na+/K' pump current values obtained in each oocyte were fitted to the Hill equation: I = Imax/[l + (Ki,2/Ko)n], where I is the measured current at each external K+ concentration (K.), Im. is the maximal current, K1l2 is the half-activation constant, and n is the Hill coefficient. Bestfitting parameters were obtained by a nonlinear method with the Simplex algorithm (17). Results are expressed as mean ± SE, and the Student's t test was used to evaluate statistical significance ofdifferences between means. Solutions and Drugs. All solutions had a low-chloride concentration to prevent swelling of depolarized oocytes and to decrease leak currents. The composition of the control solution was as follows: 97mM Na+, 0.0 mM K', 5mM Ba2+, 0.82 mM Mg2+, 0.41 mM Ca2+, 90 mM gluconate, 22.5 mM Cl-, 2.4 mM HCO3-, and 10 mM Mopsj/Mops-H. The sodium loading solution was similar, except that no Ba2+ and no Ca2' were added, and 0.5 mM EGTA was added. The high-K' solution contained 10.0 mM K+ instead of sodium. The pH of all solutions was set to 7.2. Strophanthidin (Sigma) was added from a 100 mM stock solution in dimethyl sulfoxide.