Sodium flux ratio in Na/K pump-channels opened by palytoxin.

Palytoxin binds to Na(+)/K(+) pumps in the plasma membrane of animal cells and opens an electrodiffusive cation pathway through the pumps. We investigated properties of the palytoxin-opened channels by recording macroscopic and microscopic currents in cell bodies of neurons from the giant fiber lobe, and by simultaneously measuring net current and (22)Na(+) efflux in voltage-clamped, internally dialyzed giant axons of the squid Loligo pealei. The conductance of single palytoxin-bound "pump-channels" in outside-out patches was approximately 7 pS in symmetrical 500 mM [Na(+)], comparable to findings in other cells. In these high-[Na(+)], K(+)-free solutions, with 5 mM cytoplasmic [ATP], the K(0.5) for palytoxin action was approximately 70 pM. The pump-channels were approximately 40-50 times less permeable to N-methyl-d-glucamine (NMG(+)) than to Na(+). The reversal potential of palytoxin-elicited current under biionic conditions, with the same concentration of a different permeant cation on each side of the membrane, was independent of the concentration of those ions over the range 55-550 mM. In giant axons, the Ussing flux ratio exponent (n') for Na(+) movements through palytoxin-bound pump-channels, over a 100-400 mM range of external [Na(+)] and 0 to -40 mV range of membrane potentials, averaged 1.05 +/- 0.02 (n = 28). These findings are consistent with occupancy of palytoxin-bound Na(+)/K(+) pump-channels either by a single Na(+) ion or by two Na(+) ions as might be anticipated from other work; idiosyncratic constraints are needed if the two Na(+) ions occupy a single-file pore, but not if they occupy side-by-side binding sites, as observed in related structures, and if only one of the sites is readily accessible from both sides of the membrane.

Temporal and steric analysis of ionic permeation and binding in SERCA via molecular dynamic simulations.

The availability of the crystal structure of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) has allowed atomic-level molecular dynamic (MD) simulations of this membrane transport protein to be done. The biomedical and nanotechnological implications of this work are discussed as well as the methods of performing the simulations and analysis. We have performed nanosecond timescale simulations of SERCA for several of its known conformations in a lipid/water environment. One simulation contained Ca(2+) ions, while others without ions were analyzed by techniques such as steric pathway determination. We discuss details of the resulting putative cytoplasmic and lumenal pathways, along with experimental evidence from the literature to support our conclusions. Finally, we give a brief overview of future research directions, as they pertain to MD simulations and their analysis. The methodology used in this work shows that significant insight into the structure-function relationship of ion-motive transmembrane pumps can be derived by a combination of simulation tools and analysis techniques including MD trajectories, steric analysis and electrostatic potentials.

Effect of extracellular pH on presteady-state and steady-state current mediated by the Na+/K+ pump.

A ouabain sensitive inward current occurs in Xenopus oocytes in Na+ and K(+)-free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pHo) produces a conducting pathway through the Na+/K+ pump that is permeable to H+ and blocked by [Na+]o. An alternative suggestion is that the current is mediated by an electrogenic H(+)-ATPase. Here we investigate the effect of pHo and [Na+]o on both transient and steady-state ouabain-sensitive current. At alkaline or neutral pHo the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pHo, as predicted by a model in which protonation of the Na+/K+ pump reduces the energy barrier between the internal solution and the Na+ occluded state. The model also predicts that acidic pHo increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is approximately 6, suggesting that histidine is involved in induction of the conductance pathway. 22Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pHo suggest that both Na+ and H+ are permeant. The acid-induced inward current is reduced by high [Na+]o, consistent with block by Na+. A least squares analysis predicts that H+ is four orders of magnitude more permeant than Na+, and that block occurs when 3 Na+ ions occupy a low affinity binding site (K(0.5) = 130 +/- 30 m M) with a dielectric coefficient of 0.23 +/- 0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na+ and H+ through the Na+/K+ pump.

Found: Na(+) and K(+) binding sites of the sodium pump.

Homology modeling and valence mapping have been used to predict the location and structure of Na(+) and K(+) binding sites in the Na(+)-K(+)-ATPase on the basis of the known atomic resolution structure of SERCA. Additional sites are predicted that may be associated with intracellular access and extracellular egress pathways for Na(+). The model predictions are in excellent agreement with previous structure-function and electrical studies.

Properties of a sodium channel (Na(x)) activated by strong depolarization of Xenopus oocytes.

Short (<1 sec) duration depolarization of Xenopus laevis oocytes to voltages greater than +40 mV activates a sodium-selective channel (Na(x)) with sodium permeability five to six times greater than the permeability of other monovalent cations examined, including K+, Rb+, Cs+, TMA+, and Choline+. The permeability to Li+ is about equal to that of Na+. This channel was present in all oocytes examined. The kinetics, voltage dependence and pharmacology of Na(x)distinguish it from TTX-sensitive or epithelial sodium channels. It is also different from the sodium channel of Xenopus oocytes activated by prolonged depolarization, which is more highly selective for Na+, requires prolonged depolarization to be activated, and is blocked by Li+. Intracellular Mg2+ reversibly inhibits Na(x), whereas extracellular Mg2+ does not have an inhibitory effect. Intracellular Mg2+ inhibition of Na(x), is voltage dependent, suggesting that Mg2+ binding occurs within the membrane field. Eosin is also a reversible voltage-dependent intracellular inhibitor of Na(x), suggesting that a P-type ATPase may mediate the current. An additional cytoplasmic factor is involved in maintaining Na(x) since the current runs down in internally perfused oocytes and excised membrane patches. The rundown is reversible by reintroduction of the membrane patch into oocyte cytoplasm. The cytoplasmic factor is not ATP, because ATP has no effect on Na(x) current magnitude in either cut-open or inside-out patch preparations. Extracellular Gd3+ is also an inhibitor of Na(x). Na(x) activation follows a sigmoid time course. Its half-maximal activation potential is +100 mV and the effective valence estimated from the steepness of conductance activation is 1.0. Na(x) deactivates monoexponentially upon return to the holding potential (-40 mV). The deactivation rate is voltage dependent, increasing at more negative membrane potentials.

Voltage dependence of the apparent affinity for external Na(+) of the backward-running sodium pump.

The steady-state voltage and [Na(+)](o) dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K(+)-free seawater; ATP- and Na(+)-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated (42)K(+) efflux to reverse pump current, I(pump) (both defined by sensitivity to dihydrodigitoxigenin, H(2)DTG), scaled by Faraday's constant, was -1.5 +/- 0.4 (n = 5; expected ratio for 2 K(+)/3 Na(+) stoichiometry is -2.0). Steady-state reverse pump current-voltage (I(pump)-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various V(m) to H(2)DTG or from the difference between membrane I-V relationships obtained by imposing V(m) staircases in the presence or absence of H(2)DTG. With the second method, we also investigated the influence of [Na(+)](o) (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse I(pump)-V relationship. The reverse I(pump)-V relationship is sigmoid, I(pump) saturating at large negative V(m), and each doubling of [Na(+)](o) causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state (22)Na(+) efflux during electroneutral Na(+)/Na(+) exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100-103). This model predicts valence z = nlambda, where n (1.33 +/- 0.05) is the Hill coefficient of Na binding, and lambda (0.61 +/- 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na(+)/Na(+) exchange modes only with additional assumptions that render them less likely.

Three distinct and sequential steps in the release of sodium ions by the Na+/K+-ATPase.

The Na+/K+ pump, a P-type ion-motive ATPase, exports three sodium ions and then imports two potassium ions in each transport cycle. Ions on one side of the membrane bind to sites within the protein and become temporarily occluded (trapped within the protein) before being released to the other side, but details of these occlusion and de-occlusion transitions remain obscure for all P-type ATPases. If it is deprived of potassium ions, the Na+/K+ pump is restricted to sodium translocation steps, at least one involving charge movement through the membrane's electric fields. Changes in membrane potential alter the rate of such electrogenic reactions and so shift the distribution of enzyme conformations. Here we use high-speed voltage jumps to initiate this redistribution and show that the resulting pre-steady-state charge movements relax in three identifiable phases, apparently reflecting de-occlusion and release of the three sodium ions. Reciprocal relationships among the sizes of these three charge components show that the three sodium ions are de-occluded and released to the extracellular solution one at a time, in a strict order.

Access channel model for the voltage dependence of the forward-running Na+/K+ pump.

The voltage dependence of steady state current produced by the forward mode of operation of the endogenous electrogenic Na+/K+ pump in Na(+)-loaded Xenopus oocytes has been examined using a two-microelectrode voltage clamp technique. Four experimental cases (in a total of 18 different experimental conditions) were explored: variation of external [Na+] ([Na]o) at saturating (10 mM) external [K+] ([K]o), and activation of pump current by various [K]o at 0, 15, and 120 mM [Na]o (tetramethylammonium replacement). Ionic current through K+ channels was blocked by Ba2+ (5 mM) and tetraethylammonium (20 mM), thereby allowing pump-mediated current to be measured by addition or removal of external K+. Control measurements and corrections were made for pump current run-down and holding current drift. Additional controls were done to estimate the magnitude of the inwardly directed pump-mediated current that was present in K(+)-free solution and the residual K(+)-channel current. A pseudo two-state access channel model is described in the Appendix in which only the pseudo first-order rate coefficients for binding of external Na+ and K+ are assumed to be voltage dependent and all transitions between states in the Na+/K+ pump cycle are assumed to be voltage independent. Any three-state or higher order model with only two oppositely directed voltage-dependent rate coefficients can be reduced to an equivalent pseudo two-state model. The steady state current-voltage (I-V) equations derived from the model for each case were simultaneously fit to the I-V data for all four experimental cases and yielded least-squares estimates of the model parameters. The apparent fractional depth of the external access channel for Na+ is 0.486 +/- 0.010; for K+ it is 0.256 +/- 0.009. The Hill coefficient for Na+ is 2.18 +/- 0.06, and the Hill coefficient for K+ (which is dependent on [Na]o) ranges from 0.581 +/- 0.019 to 1.35 +/- 0.034 for 0 and 120 mM [Na]o, respectively. The model provides a reasonable fit to the data and supports the hypothesis that under conditions of saturating internal [Na+], the principal voltage dependence of the Na+/K+ pump cycle is a consequence of the existence of an external high-field access channel in the pump molecule through which Na+ and K+ ions must pass in order to reach their binding sites.

Pre-steady-state transient currents mediated by the Na/K pump in internally perfused Xenopus oocytes.

Pre-steady-state transient currents have been investigated in the vegetal pole of Xenopus oocytes using the open-oocyte vaseline-gap technique of Taglialatela, Toro, and Stefani (Biophysical Journal. 61:78-82, 1992). Voltage pulses 40 ms in duration were made from a holding potential of -40 mV to command potentials over the range -160 to +60 mV in increments of 20 mV. Current records (averaged 20X; sampled every 200 microseconds) in the presence of dihydroouabain (DHO) or absence of external Na+ (Nao) were subtracted from current records obtained under Na/Na exchange conditions, i.e. internally perfused with 50 mM Na+, 5 mM ATP, and 5 mM ADP (K(+)-free) and externally superfused with 100 mM Na+,K(+)-free solution. Transient currents were dependent on intracellular Na+ and nucleotides, and diminished by activation of forward pumping; they were also reduced by 10 micrograms ml-1 of oligomycin B applied to the external solution. These properties of the pre-steady state currents are consistent with the Na/K pump operating in its electroneutral Na/Na exchange mode. The voltage dependence of the DHO- and Nao-sensitive transient currents was analyzed using a pseudo two-state model in which only the rate coefficient for Nao-binding/reocclusion is voltage-dependent (Rakowski, R. F. 1993. J. Gen. Physiol. 101:117-144). The apparent valence of the charge moved during the on (zq-on) and off (zq-off) of the pulse were 0.96 +/- 0.05 and 0.95 +/- 0.05 for Nao-sensitive, and 1.10 +/- 0.07 and 0.85 +/- 0.06 for DHO-sensitive transient currents, respectively. The total amount of charge moved (Qtot) and the mid-point voltage of the charge distribution (Vq) were 230 +/- 15 pC and -56.2 +/- 5.1 mV, and 268 +/- 34 pC and -67.0 +/- 7.6 mV for Nao- and DHO-sensitive transient currents, respectively. The apparent valence (zk) and the voltage at which the forward and backward rates are equal (Vk) obtained from the relaxation rates were 0.80 +/- 0.05 and -129.3 +/- 10.0 mV, and 0.86 +/- 0.10 and -135.1 +/- 9.0 mV for the Nao- and DHO-sensitive pre-steady state currents, respectively. The values of the parameters were not statistically significantly different between the Nao- and DHO-sensitive transient currents. Excluding the first 600 microseconds after the onset of a voltage step which was not temporally resolved, transient currents showed no indication of a rising phase. These results support the idea that charge translocation occurs within an external access channel at a rate that is governed by a voltage-dependent binding/reocclusion process and a voltage-independent deocclusion/unbinding process.

Extracellular access to the Na,K pump: pathway similar to ion channel.

In each normal Na,K pump cycle, first three sodium and then two potassium ions are transported; in both cases, the ions become temporarily occluded in pump conformations that isolate them from internal and external solutions. A major charge movement occurs during sodium translocation and accompanies the deocclusion of sodium ions or their release to the cell exterior, or both. The nature of the charge movement was examined by measurement of the undirectional sodium-22 efflux mediated by Nai-Nao exchange (Nai and Nao are internal and external sodium ions) in voltage-clamped, internally dialyzed squid giant axons in the absence of potassium; in this way the pump activity was restricted to the sodium-translocation pathway. Although electroneutral, the Nai-Nao exchange was nevertheless voltage-sensitive: increasingly negative potentials enhanced its rate along a saturating sigmoid curve. Such voltage dependence demonstrates that the release and rebinding of external sodium is the predominant charge-moving (hence, voltage-sensitive) step, suggesting that extracellular sodium ions must reach their binding sites deep in the pump molecule through a high-field access channel. This implies that part of the pump molecule is functionally analogous to an ion channel.

Charge movement by the Na/K pump in Xenopus oocytes.

Pre-steady-state transient currents (1986. Nakao, M., and D. C. Gadsby. Nature [Lond.]. 323:628-630) mediated by the Na/K pump were measured under conditions for Na/Na exchange (K-free solution) in voltage-clamped Xenopus oocytes. Signal-averaged (eight times) current records obtained in response to voltage clamp steps over the range -160 to +60 mV after the addition of 100 microM dihydroouabain (DHO) or removal of external Na (control) were subtracted from test records obtained before the solution change. A slow component of DHO- or Na-sensitive difference current was consistently observed and its properties were analyzed. The quantity of charge moved was well described as a Boltzmann function of membrane potential with an apparent valence of 1.0. The relaxation rate of the current was fit by the sum of an exponentially voltage-dependent reverse rate coefficient plus a voltage-independent forward rate constant. The quantity of charge moved at the on and off of each voltage pulse was approximately equal except at extreme negative values of membrane potential where the on charge tended to be less than the off. The midpoint voltage of the charge distribution function (Vq) was shifted by -24.8 +/- 1.7 mV by changing the external [Na] in the test condition from 90 to 45 mM and by +14.7 +/- 1.7 mV by changing the test [Na] from 90 to 120 mM. A pseudo three-state model of charge translocation is discussed in which Na+ is bound and occluded at the internal face of the enzyme and is released into an external-facing high field access channel (ion well). The model predicts a shift of the charge distribution function to more hyperpolarized potentials as extracellular [Na] is lowered; however, several features of the data are not predicted by the model.

Activation of the 5-HT1C receptor expressed in Xenopus oocytes by the benzazepines SCH 23390 and SKF 38393.

1. A cloned 5-HT1C receptor expressed in Xenopus laevis oocytes was used to characterize the action of four dopamine D1-selective benzazepines at the 5-HT1C receptor. Additionally, the apparent binding of the D1-selective benzazepines to 5-HT1C receptors was measured in the choroid plexus of the pig. 2. In voltage-clamped oocytes expressing the cloned 5-HT1C receptor, 5-hydroxytryptamine (5-HT) elicited a characteristic inward current response with an EC50 of 13 nM. SCH 23390 acted as a stereoselective agonist (or partial agonist) with an EC50 of about 550 nM. SKF 38393 (1 microM-1 mM), SKF 77434 (100 microM), and SKF 82958 (100 microM) also acted as agonists (or partial agonists) at the cloned 5-HT1C receptor. SKF 38393 was not stereoselective at the 5-HT1C receptor. 3. The response to SCH 23390 activated slowly and, although the response contained many oscillations characteristic of the activation of the phosphatidylinositol signal transduction system, SCH 23390 rarely elicited the rapid spike-like response seen routinely in response to 5-HT. However, the responses to SKF 38393, SKF 77434, and SKF 82958 were identical in appearance to the response to 5-HT, except that the responses to the benzazepines were smaller. These comparisons were made by applying both a benzazepine and 5-HT to each individual oocyte expressing the cloned 5-HT1C receptor. 4. Consistent with the responses measured in oocytes, SCH 23390 bound stereoselectively to 5-HT1C receptors in the choroid plexus of the pig (Ki = 6.3 nM), and SKF 38393 bound non-stereoselectively with lower affinity (Ki = 2.0-2.2 microM).5. It is concluded that while these benzazepines demonstrate selectivity for the dopamine D1 receptor, they also can act as agonists or partial agonists at the 5-HT1c receptor in situ and as expressed in Xenopus oocytes. The oocyte expression system is useful for studies of the functional pharmacology of these 5-HTic receptors. Information about the pharmacological actions and variations in stereoselectivity among dopamine and 5-HT receptors should be of interest in modelling the interactions of ligands with these G-protein coupled receptors, and in the testing of such models through receptor mutagenesis.

A negative slope in the current-voltage relationship of the Na+/K+ pump in Xenopus oocytes produced by reduction of external [K+].

To investigate the voltage dependence of the Na+/K+ pump, current-voltage relations were determined in prophase-arrested oocytes of Xenopus laevis. All solutions contained 5 mM Ba2+ and 20 mM tetraethylammonium (TEA) to block K+ channels. If, in addition, the Na+/K+ pump is blocked by ouabain, K(+)-sensitive currents no larger than 50 nA/cm2 remain. Reductions in steady-state current (on the order of 700 nA/cm2) produced by 50 microM ouabain or dihydro-ouabain or by K+ removal, therefore, primarily represent current generated by the Na+/K+ pump. In Na(+)-free solution containing 5 mM K+, Na+/K+ pump current is relatively voltage independent over the potential range from -160 to +40 mV. If external [K+] is reduced below 0.5 mM, negative slopes are observed over this entire voltage range. Similar results are seen in Na(+)- and Ca(2+)-free solutions in the presence of 2 mM Ni2+, an experimental condition designed to prevent Na+/Ca2+ exchange. The occurrence of a negative slope can be explained by the voltage dependence of the apparent affinity for activation of the Na+/K+ pump by external K+, consistent with the existence of an external ion well for K+ binding. In 90 mM Na+, 5 mM K+ solution, Na+/K+ pump current-voltage curves at negative membrane potentials have a positive slope and can be described by a monotonically increasing sigmoidal function. At an extracellular [K+] of 1.3 mM, a negative slope was observed at positive potentials. These findings suggest that in addition to a voltage-dependent step associated with Na+ translocation, a second voltage-dependent step that is dependent on external [K+], possibly external K+ binding, participates in the overall reaction mechanism of the Na+/K+ pump.

Stoichiometry and voltage dependence of the sodium pump in voltage-clamped, internally dialyzed squid giant axon.

The stoichiometry and voltage dependence of the Na/K pump were studied in internally dialyzed, voltage-clamped squid giant axons by simultaneously measuring, at various membrane potentials, the changes in Na efflux (delta phi Na) and holding current (delta I) induced by dihydrodigitoxigenin (H2DTG). H2DTG stops the Na/K pump without directly affecting other current pathways: (a) it causes no delta I when the pump lacks Na, K, Mg, or ATP, and (b) ouabain causes no delta I or delta phi Na in the presence of saturating H2DTG. External K (Ko) activates Na efflux with Michaelis-Menten kinetics (Km = 0.45 +/- 0.06 mM [SEM]) in Na-free seawater (SW), but with sigmoid kinetics in approximately 400 mM Na SW (Hill coefficient = 1.53 +/- 0.08, K1/2 = 3.92 +/- 0.29 mM). H2DTG inhibits less strongly (Ki = 6.1 +/- 0.3 microM) in 1 or 10 mM K Na-free SW than in 10 mM K, 390 mM Na SW (1.8 +/- 0.2 microM). Dialysis with 5 mM each ATP, phosphoenolpyruvate, and phosphoarginine reduced Na/Na exchange to at most 2% of the H2DTG-sensitive Na efflux. H2DTG sensitive but nonpump current caused by periaxonal K accumulation upon stopping the pump, was minimized by the K channel blockers 3,4-diaminopyridine (1 mM), tetraethylammonium (approximately 200 mM), and phenylpropyltriethylammonium (20-25 mM) whose adequacy was tested by varying [K]o (0-10 mM) with H2DTG present. Two ancillary clamp circuits suppressed stray current from the axon ends. Current and flux measured from the center pool derive from the same membrane area since, over the voltage range -60 to +20 mV, tetrodotoxin-sensitive current and Na efflux into Na-free SW, under K-free conditions, were equal. The stoichiometry and voltage dependence of pump Na/K exchange were examined at near-saturating [ATP], [K]o and [Na]i in both Na-free and 390 mM Na SW. The H2DTG-sensitive F delta phi Na/delta I ratio (F is Faraday's constant) of paired measurements corrected for membrane area match, was 2.86 +/- 0.09 (n = 8) at 0 mV and 3.05 +/- 0.13 (n = 6) at -60 to -90 mV in Na-free SW, and 2.72 +/- 0.09 (n = 7) at 0 mV and 2.91 +/- 0.21 (n = 4) at -60 mV in 390 mM Na SW. Its overall mean value was 2.87 +/- 0.07 (n = 25), which was not significantly different from the 3.0 expected of a 3 Na/2 K pump.(ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous measurement of changes in current and tracer flux in voltage-clamped squid giant axon.

A method is described for the simultaneous measurement of changes in membrane current and unidirectional radiotracer flux in internally dialyzed voltage-clamped squid giant axons. The small currents that are produced by electrogenic transport processes or steady-state ionic currents can be resolved using this method. Because the use of grounded guard electrodes in the end pools is not, by itself, an adequate means of eliminating end-effects, two ancillary end pool clamp circuits are described to eliminate extraneous current flow from the ends of the axon. The end pool voltage-clamp circuits serve to minimize net current flow between the end pools and center pool, and employ stable, low-impedance calomel electrodes to monitor the potentials of the end and center pools. The adequacy of the method is demonstrated by experiments in which unidirectional 22Na efflux and current, flowing through tetrodotoxin (TTX)-sensitive Na channels into Na-free seawater, under K-free conditions, are shown to be equal. The equality of unidirectional TTX-sensitive flux and current is maintained over the entire range of membrane potentials examined (-60 to +20 mV). The method has been applied to a series of experiments in which the voltage dependence and stoichiometry of the Na/K pump have been measured (Rakowski et al., 1989), and can be applied in general to the simultaneous measurement of changes in current and flux of other electrogenic transport processes, and of currents through ionic channels that open under steady-state conditions.