Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes

To examine effects of cytosolicNa⁺, K⁺, and Cs⁺ on the voltagedependence of the Na⁺-K⁺ pump, we measuredNa⁺-K⁺ pump current (I_p)of ventricular myocytes voltage-clamped at potentials(V_m) from 100 to +60 mV. Superfusates weredesigned to eliminate voltage dependence at extracellular pump sites.The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na⁺ concentration ([Na]_pip)of 80 mM and a K⁺ concentration from 0 to 80 mM or withsolutions containing Na⁺ in concentrations from 0.1 to 100 mM and K⁺ in a concentration of either 0 or 80 mM. When[Na]_pip was 80 mM, K⁺ in pipette solutionshad a voltage-dependent inhibitory effect on I_pand induced a negative slope of theI_p-V_m relationship. Cs⁺ in pipette solutions had an effect onI_p qualitatively similar to that ofK⁺. Increases in I_p with increasesin [Na]_pip were voltage dependent. The dielectriccoefficient derived from[Na]_pip-I_p relationships at thedifferent test potentials was 0.15 when pipette solutions included 80 mM K⁺ and 0.06 when pipette solutions were K⁺ free.

     

Sodium and Proton Effects on Inward Proton Transport through Na/K Pumps

The Na/K pump hydrolyzes ATP to export three intracellular Na (Na_i) as it imports two extracellular K (K_o) across animal plasma membranes. Within the protein, two ion-binding sites (sites I and II) can reciprocally bind Na or K, but a third site (site III) exclusively binds Na in a voltage-dependent fashion. In the absence of Na_o and K_o, the pump passively imports protons, generating an inward current (I_H). To elucidate the mechanisms of I_H, we used voltage-clamp techniques to investigate the [H]_o, [Na]_o, and voltage dependence of I_H in Na/K pumps from ventricular myocytes and in ouabain-resistant pumps expressed in Xenopus oocytes. Lowering pH_o revealed that H_o both activates I_H (in a voltage-dependent manner) and inhibits it (in a voltage-independent manner) by binding to different sites. Na_o effects depend on pH_o; at pH_o where no H_o inhibition is observed, Na_o inhibits I_H at all concentrations, but when applied at pH_o that inhibits pump-mediated current, low [Na]_o activates I_H and high [Na]_o inhibits it. Our results demonstrate that I_H is a property inherent to Na/K pumps, not linked to the oocyte expression environment, explains differences in the characteristics of I_H previously reported in the literature, and supports a model in which 1), protons leak through site III; 2), binding of two Na or two protons to sites I and II inhibits proton transport; and 3), pumps with mixed Na/proton occupancy of sites I and II remain permeable to protons.     

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cl_i above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Na_i homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Cl_o to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a _infNa ^supi to its control level and the accompanying hyperpolarization could be blocked by 10^–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring K_o (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of a_Na was attenuated by 10^–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Na_i. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.     

Voltage dependence of current through the Na,K-exchange pump ofRana oocytes

Summary We have studied current (I _Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I _Str was measured as the strophanthidin-sensitive current in the presence of Ba^2–, Cd²⁺ and gluconate (in place of external Cl^–). In addition,I _Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK _0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na⁺-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI _Str depended directly onV _m up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na⁺] markedly decreased the voltage sensitivity ofI _p , while producing only a small change in the maximal outwardI _Str. In contrast, decreasing the external [K⁺] from 25 to 2.5mm reducedI _Str at 0 mV without substantially affecting its voltage dependence. At K⁺ concentrations of 1mm, both the absolute value ofI _Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI _Str by [K⁺] _o over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K⁺ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K⁺] _o >1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na⁺ and K⁺ are consistent with results obtained in other tissues and may reflect an ion-well effect.     

Sodium and Proton Effects on Inward Proton Transport through Na/K Pumps

The Na/K pump hydrolyzes ATP to export three intracellular Na (Na_i) as it imports two extracellular K (K_o) across animal plasma membranes. Within the protein, two ion-binding sites (sites I and II) can reciprocally bind Na or K, but a third site (site III) exclusively binds Na in a voltage-dependent fashion. In the absence of Na_o and K_o, the pump passively imports protons, generating an inward current (I_H). To elucidate the mechanisms of I_H, we used voltage-clamp techniques to investigate the [H]_o, [Na]_o, and voltage dependence of I_H in Na/K pumps from ventricular myocytes and in ouabain-resistant pumps expressed in Xenopus oocytes. Lowering pH_o revealed that H_o both activates I_H (in a voltage-dependent manner) and inhibits it (in a voltage-independent manner) by binding to different sites. Na_o effects depend on pH_o; at pH_o where no H_o inhibition is observed, Na_o inhibits I_H at all concentrations, but when applied at pH_o that inhibits pump-mediated current, low [Na]_o activates I_H and high [Na]_o inhibits it. Our results demonstrate that I_H is a property inherent to Na/K pumps, not linked to the oocyte expression environment, explains differences in the characteristics of I_H previously reported in the literature, and supports a model in which 1), protons leak through site III; 2), binding of two Na or two protons to sites I and II inhibits proton transport; and 3), pumps with mixed Na/proton occupancy of sites I and II remain permeable to protons.     

Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes ofXenopus laevis

Summary Electrophysiological experiments were performed to analyze the Na⁺/K⁺-ATPase in full-grown prophase-arrested oocytes ofXenopus laevis. If the Na⁺/K⁺-ATPase is inhibited by dihydroouabain (DHO), the resting potential of the membrane of Na⁺-loaded oocytes may depolarize by nearly 50 mV. This hyperpolarizing contribution to the resting potential depends on the degree of activation of the Na⁺/K⁺-ATPase and varies with intra-cellular Na⁺ activity (a _Na ⁱ ), and extracellular K⁺ (K ₀ ⁺ ) It is concluded that variations ofa _Na ⁱ among different oocytes are primarily responsible for the variations of resting potentials measured in oocytes ofX. laevis. Under voltage-clamp conditions, the DHO-sensitive current also exhibits dependence ona _Na ⁱ that may be described by a Hill equation with a coefficient of 2. This current will be shown to be identical with the electrogenic current generated by the 3Na⁺/2K⁺ pump. The voltage dependence of the pump current was investigated at saturating values ofa _Na ⁱ (33 mmol/liter) and of K ₀ ⁺ (3 mmol/liter) in the range from –200 to +100 mV. The current was found to exhibit a characteristic maximum at about +20 mV. This is taken as evidence that in the physiological range at least two steps within the cycle of the pump are voltage dependent and are oppositely affected by the membrane potential.     

Effect of metabolic depletion on the furosemide-sensitive Na and K fluxes in human red cells

Summary We report in this paper the effect of metabolic depletion on several modes of furosemide-sensitive (FS) Na and K transport in human red blood cells. The reduction of ATP content below 100 mol/liter cells produced a marked decrease in the maximal activation (V _max) of the outward. FS transport of Na and K into choline medium in the presence of ouabain (0.1 mM) and 1 mM MgCl₂. TheK _0.5 for internal Na to activate the FS Na efflux was not altered by metabolic depletion. However, metabolic depletion markedly decreased the K _i for external K (K _o ) to inhibit the FS Na efflux into choline medium (from 25 to 11 mM). Repletion of ATP content by incubation of cells in a substraterich medium recovered control levels ofV _max of the FS Na and K fluxes and of K _i for external K to inhibit FS Na efflux. TheV _max of FS Na and K influxes was also markedly decreased when the ATP content dropped below 100 mol/liter cells. This was mainly due to a decrease in the inward-coupled transport of K and Na (Na _o -stimulated K influx and the K _o -stimulated Na influx). The FS K _i /K _o exchange pathway of the Na–K cotransport, estimated from the FS K influx from choline-20 mM K _o medium into cells containing 22 mmol Na/liter cells, was also reduced by starvation. Starvation did not inhibit the FS Na _i /Na _o exchange pathway, estimated as FS Na influx from a medium containing 130 mM NaCl into cells containing 22 mmol Na/liter cells. The unidirectional FS²²Na efflux and influx were also measured in control and starved cells containing 22 mmol Na/liter cells, incubated in a Na medium (130 mM) at varying external K (0 to 20 mM). In substrate-fed cells, incubated in the absence of external K, FS Na efflux was larger than Na influx. This FS net Na extrusion (400 to 500 mol/liter cells·hr) decreased when external K was increased, approaching zero around 15 mM K _o . In starved cells the net Na extrusion was markedly decreased and it approached zero at lower K _o than in substrate-fed cells. Our results indicate that the FS Na and K fluxes, and their major component, the gradient driven Na–K–Cl cotransport system, are dependent on the metabolic integrity of the cells.     

Voltage-dependent stimulation of the Na(+)-K(+) pump by insulin in rabbit cardiac myocytes

Insulin enhancesNa⁺-K⁺ pump activity in various noncardiactissues. We examined whether insulin exposure in vitro regulates Na⁺-K⁺ pump function in rabbit ventricularmyocytes. Pump current (I_p) was measured using thewhole-cell patch-clamp technique at test potentials(V_ms) from 100 to +60 mV. When theNa⁺ concentration in the patch pipette([Na]_pip) was 10 mM, insulin caused aV_m-dependent increase in I_p.The increase was ~70% when V_m was at nearphysiological diastolic potentials. This effect persisted afterelimination of extracellular voltage-dependent steps and whenK⁺ and K⁺-congeners were excluded from thepatch pipettes. When [Na]_pip was 80 mM, causingnear-maximal pump stimulation, insulin had no effect, suggesting thatit did not cause an increase in membrane pump density. Effects oftyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I inpipette solutions suggested that the insulin-induced increase inI_p involved activation of tyrosine kinase,phosphatidylinositol 3-kinase, and protein phosphatase 1, whereasprotein phosphatase 2A and protein kinase C were not involved.

     

Voltage dependence of Na/K pump current inXenopus oocytes

Summary Stage V and VI (Dumont, J.N., 1972.J. Morphol. 136:153–180) oocytes ofXenopus laevis were treated with collagenase to remove follicular cells and were placed in K-free solution for 2 to 4 days to elevate internal [Na]. Na/K pump activity was studied by restoring the eggs to normal 3mm K Barth's solution and measuring membrane current-voltage (I–V) relationships before and after the addition of 10 m dihydroouabain (DHO) using a two-microelectrode voltage clamp. Two pulse protocols were used to measure membraneI–V relationships, both allowing membrane currents to be determined twice at each of a series of membrane potentials: (i) a down-up-down sequence of 5 mV, 1-sec stair steps and (ii) a similar sequence of 1-sec voltage pulses but with consecutive pulses separated by 4-sec recovery periods at the holding potential (–40 mV). The resulting membraneI–V relationships determined both before and during exposure to DHO showed significant hysteresis between the first and second current measurements at each voltage. DHO difference curves also usually showed hysteresis indicating that DHO caused a change in a component of current that varied with time. Since, by definition, the steady-state Na/K pumpI–V relationship must be free of hysteresis, the presence of hysteresis in DHO differenceI–V curves can be used as a criterion for excluding such data from consideration as a valid measure of the Na/K pumpI–V relationship. DHO differenceI–V relationships that did not show hysteresis were sigmoid functions of membrane potential when measured in normal (90mm) external Na solution. The Na/K pump current magnitude saturated near 0 mV at a value of 1.0–1.5 A cm^–2, without evidence of negative slope conductance for potentials up to +55 mV. The Na/K pump current magnitude in Na-free external solution was approximately voltage independent. Since these forward-going Na/K pumpI–V relationships do not show a region of negative slope over the voltage range –110 to +55 mV, it is not necessary to postulate the existence of more than one voltage-dependent step in the reaction cycle of the forward-going Na/K pump.     

Editorial

The voltage dependence of the rat renal type II Na⁺/P_i cotransporter (NaP_i-2) was investigated by expressing NaP_i-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (P_i) induced inward currents (I_p) in the presence of 96 mM Na⁺ over the potential range −140 ≤ V ≤ +40 mV. With P_i as the variable substrate, the apparent affinity constant (K _m ^Pi) was strongly dependent on Na⁺, increasing sixfold for a twofold reduction in external Na⁺. K _m ^Pi increased with depolarizing voltage and was more sensitive to voltage at reduced Na⁺. The Hill coefficient was close to unity and the predicted maximum I_p (I_pmax) was 40% smaller at 50 mM Na⁺. With Na⁺ as the variable substrate, K _m ^Na was weakly dependent on both P_i and voltage, the Hill coefficient was close to 3 and I_pmax was independent of P_i at −50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na⁺-dependent manner, indicating the existence of uncoupled Na⁺ slippage. Voltage steps induced pre–steady state relaxations typical for Na⁺-coupled cotransporters. NaP_i-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na⁺, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V_0.5) in the range −20 to −50 mV and an apparent valency of ∼0.5 e⁻. V_0.5 became more negative as Na⁺ was reduced. P_i suppressed relaxations in a dose-dependent manner, but had little effect on their voltage dependence. Reducing external pH shifted V_0.5 to depolarizing potentials and suppressed relaxations in the absence of Na⁺, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na⁺ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na⁺ binding step.     

Acetylcholine-Induced Na+ influx in the mouse lacrimal gland acinar cells: Demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements

Summary In the isolated, superfused mouse lacrimal gland, intracellular Na⁺ activities (aNa _i ) of the acinar cells were directly measured with double-barreled Na⁺-selective microelectrodes. In the nonstimulated conditionaNa _i was 6.5±0.5 mM and membrane potential (V _m ) was –38.9±0.4 mV. Addition of 1 mM ouabain or superfusion with a K⁺-free solution slightly depolarized the membrane and caused a gradual increase inaNa _i . Stimulation with acetylcholine (ACh, 1 M) caused a membrane hyperpolarization by about 20 mV and an increase inaNa _i by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNa _i by approximately 50%, without affectingV _m and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH₄Cl or by replacement of Tris⁺-buffer saline solution with HCO ₃ ^– /CO₂-buffered solution increasedaNa _i by a few mM. Superfusion with a Cl^–-free NO ₃ ^– solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNa _i levels, however, it reduced the ACh-induced increase inaNa _i by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNa _i and the ACh-induced increase inaNa _i .The present results suggest the presence of multiple Na⁺ entry mechanisms activated by ACh, namely, Na⁺/H⁺ exchange, Na-K-Cl cotransport and organic substrate-coupled Na⁺ transport mechanisms.     

Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K⁺]_o was increased from 1.2 to 10 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. As [K⁺]_o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K⁺]_o was increased from 60 to 122 mM, an increase in [K⁺]_i and [Cl^–]_i and a decrease in [Na⁺]_i were observed.Inneurons, as [K⁺]_o was increased from 1.2 to 2.8 mM, [Na⁺]_i and [Cl^–]_i were decreased, whereas [K⁺]_i was increased. As [K⁺]_o was increased from 2.8 to 30 mM, [K⁺]_i, [Na⁺]_i and [Cl^–]_i showed no significant change. When [K⁺]_o was increased from 30 to 122 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. Inastrocytes, pH_i increased when [K⁺]_o was increased. Inneurons, there was a biphasic change in pH_i. In lower [K⁺]_o (1.2–2.8 mM) pH_i decreased as [K⁺]_o increased, whereas in higher [K⁺]_o (2.8–122 mM) pH_i was directly related to [K⁺]_o. In bothastrocytes andneurons, changes in [K⁺]_o did not affect the extracellular water content, whereas the intracellular water content increased as the [K⁺]_o increased. Transmembrane potential (E_m) as measured with Tl-204 was inversely related to [K⁺]_o between 1.2 and 90 mM, a ten-fold increase in [K⁺]_o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The E_m values measured with Tl-204 were close to the potassium equilibrium potential (E_k) except those in neurons at lower [K⁺]_o. However, they were not equal to the chloride equilibrium potential (E_Cl) at [K⁺]_o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K⁺]_o produced different changes in [K⁺]_i, [Na⁺]_i, [Cl^–]_i, and pH_i in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K⁺]_o, in such a way to maintain a more suitable environment for neurons.     

[Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea pig ventricular myocytes

Na/K pump current was determined between -140 and +60 mV as steady-state, strophanthidin-sensitive, whole-cell current in guinea pig ventricular myocytes, voltage-clamped and internally dialyzed via wide-tipped pipettes. Solutions were designed to minimize all other components of membrane current. A device for exchanging the solution inside the pipette permitted investigation of Na/K pump current-voltage (I-V) relationships at several levels of pipette [Na] [( Na]pip) in a single cell; the effects of changes in external [Na] [( Na]o) or external [K] [( K]o) were also studied. At 50 mM [Na]pip, 5.4 mM [K]o, and approximately 150 mM [Na]o, Na/K pump current was steeply voltage dependent at negative potentials but was approximately constant at positive potentials. Under those conditions, reduction of [Na]o enhanced pump current at negative potentials but had little effect at positive potentials: at zero [Na]o, pump current was only weakly voltage dependent. At 5.4 mM [K]o and approximately 150 mM [Na]o, reduction of [Na]pip from 50 mM scaled down the sigmoid pump I-V relationship and shifted it slightly to the right (toward more positive potentials). Pump current at 0 mV was activated by [Na]pip according to the Hill equation with best-fit K0.5 approximately equal to 11 mM and Hill coefficient nH approximately equal to 1.4. At zero [Na]o, reduction of [Na]pip seemed to simply scale down the relatively flat pump I-V relationship: Hill fit parameters for pump activation by [Na]pip at 0 mV were K0.5 approximately equal to 10 mM, nH approximately equal to 1.4. At 50 mM [Na]pip and high [Na]o, reduction of [K]o from 5.4 mM scaled down the sigmoid I-V relationship and shifted it slightly to the right: at 0 mV, K0.5 approximately equal to 1.5 mM and nH approximately equal to 1.0. At zero [Na]o, lowering [K]o simply scaled down the flat pump I-V relationships yielding, at 0 mV, K0.5 approximately equal to 0.2 mM, nH approximately equal to 1.1. The voltage-independent activation of Na/K pump current by both intracellular Na ions and extracellular K ions, at zero [Na]o, suggests that neither ion binds within the membrane field. Extracellular Na ions, however, seem to have both a voltage-dependent and a voltage-independent influence on the Na/K pump: they inhibit outward Na/K pump current in a strongly voltage-dependent fashion, with higher apparent affinity at more negative potentials (K0.5 approximately equal to 90 mM at -120 mV, and approximately 170 mM at -80 mV), and they compete with extracellular K ions in a seemingly voltage-independent manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation,dependence on extracellular and intracellular Na+, kinetics,pH dependency and the effect of cell volume and N-ethylmaleimide

Summary The effect of extracellular and intracellular Na⁺ (Na _o ⁺ , Na _i ⁺ ) on ouabain-resistant, furosemide-sensitive (FS) Rb⁺ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na⁺+1 K⁺+2 Cl^–) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na⁺–K⁺) cotransport, and modeii) a Na _o ⁺ -independent 11 K _o ⁺ /K _i ⁺ exchange requiring Na _i ⁺ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb⁺ uptake through Rb _o ⁺ /K _i ⁺ exchange contributes about 25% to total Rb⁺ uptake in 145mm NaCl media containing 5mm RbCl at normal Na _i ⁺ (pH 7.4). Na⁺–K⁺ cotransport into the cells occurs largely additive to K⁺/K⁺ exchange. Inward Na⁺–Rb⁺ cotransport exhibits a substrate inhibition at high Rb _o ⁺ . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K⁺/K⁺ exchange (apparent pK close to pH 7.4). The apparentK _m ^Rb _o ⁺ of Na⁺–K⁺ cotransport is low (2mm) and almost independent of pH, and high for K⁺/K⁺ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na⁺+1 K⁺+2 Cl^–) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl^–-dependent K⁺ transport pathway that is independent of both Na _o ⁺ and Na _i ⁺ . This pathway differs in many properties from the basal, Na _o ⁺ -independent K⁺/K⁺ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na _o ⁺ -independent FS K⁺ transport pathway which most probably is not identical to basal K⁺/K⁺ exchange. K _o ⁺ o ⁺

o ⁺ o ²⁺ reduce furosemide-resistant Rb⁺ inward leakage relative to choline _o ⁺ .     

Effects of internal and external sodium on the sodium current-voltage relationship in thesquid giant axon

Summary The early transient current-voltage relationship was measured in internally perfused voltage clamped squid giant axons with various concentrations of sodium on the two sides of the membrane. In the absence of sodium on either side there is an outward transient current which is blocked by tetrodotoxin and varies with internal potassium concentration. The current increases linearly with voltage for positive potentials. Adding sodium ions internally increases the slope of the current-voltage relationship. Adding sodium ions externally also increases the slope between +10 and +80 mV. Adding sodium to both sides produces the sum of the two effects.The current-voltage relationships were fit by straight lines between +10 and +80 mV. Plotting the extrapolated intercepts with the current axis against the differences in sodium concentrations gave a straight line,I _o =–P(c _o –c _i )F.P, the Fickian permeability, is about 10^–4 cm/sec. Plotting the slopes in three dimensions against the two sodium concentrations gave a planeg=g _o +(aNa _o +bNa _i )F.a is about 10^–6 cm/mV-sec andb about 3×10^–6 cm/mV-sec. Thus the current-voltage relationship for the sodium current is well described byI=–P(c _o –c _i )F+(ac _o +bc _i )FV for positive potentials. This is the linear sum of Fick's Law and Ohm's Law.P/(a+b)=25±1 mV (N=6) and did not vary with the absolute magnitude of the currents. Within experimental error this is equal tokT/e orRT/F.Increasing temperature increasedP, a andb proportionately. Adding external calcium, lithium, or Tris selectively decreasedP anda without changingb. In the absence of sodium, altering internal and external potassium while observing the early transient currents suggests this channel is more asymmetric in its response to potassium than to sodium.     

Interaction of External K, Na, and Cardioactive Steroids with the Na-K Pump of the Human Red Blood Cell

The interaction of extracellular Na (Na_o), K (K_o), and strophanthidin with the Na-K pump of the human red blood cell has been investigated. Inhibition by submaximal concentrations of strophanthidin rapidly reaches a level which does not increase further over a relatively long period of time. Under these circumstances, it is possible to apply a steady-state kinetic analysis to the interaction of Na_o, K_o, and strophanthidin with the pump. In Na-free solutions, strophanthidin increases the apparent K_1/2 of the pump for K_o, but does not change the form of the relation between the reciprocal of the active K influx (ⁱM_K^P–1) and the reciprocal of [K_o] ([K_o]^–1); the relation both in the presence and absence of strophanthidin is adequately described by a straight line. In solutions containing Na, strophanthidin changes the form of the curve describing the relation between ⁱM_K^P–1 vs. [K_o]^–1; the curve becomes more parabolic in solutions containing strophanthidin. The rate of ouabain binding to K-free cells has also been measured; in the absence of K, the rate of binding is unaffected by Na_o. The data are considered in terms of a simple kinetic model. The findings can be explained if it is supposed that at low external K the form of the pump combined with one Na_o is more likely to combine with strophanthidin than is the uncombined form of the pump. The uncombined form of the pump is more likely to combine with K even at very low K_o than with strophanthidin.     

Ion transport and regulation ofTetrahymena pyriformis in isotonic and hypotonic media

Summary The ion and volume regulatory mechanisms ofTetrahymena pyriformis were studied in normal or hypotonic nutrient media and in isotonic inorganic media with different Na/K ratios, in conjunction with the effects of a general metabolic inhibitor (low temperature) and a specific inhibitor (iodoacetate). For K two mechanisms of active influx were found: The first is sensitive to IAc and seems to be the basic mechanism for the maintenance of the K_i/K_o gradient. The second is sensitive to cooling and related to the function of the contractile vacuole; it is also responsible for the high intracellular levels of K. The passive K efflux seems to be a basic factor for volume regulation, together with the contractile vacuole. It increases in hypotonic media and this seems to be related to structural changes of the membranes occurring in hypotonic media. For Na two mechanisms of active transport were also found: One for active Na efflux with highK _m, which is associated with the contractile vacuole and another, for active Na influx with lowK _m, which is inhibited by high levels of intracellular K.The electrochemical potentials of Na and K and the membrane potential (Cl Nernst potential) were also studied in isotonic inorganic media. The membrane is negatively polarized, except if Na_o<5 mM when it becomes positive. In normal conditions, Na is transported outwards actively and leaks passively, while K is transported inwards actively and leaks 56 times more rapidly than Na ions.A model for the overall transport and regulation of ions inTetrahymena is proposed.Abbreviations IAc iodoacetate - PCV packed cell volume - Na _i,K _i,Cl intracellular concentrations ofNa ⁺,K ⁺,Cl ^–, respectively - Na _o,K _o, Cl_o extracellular concentrations of Na⁺, K⁺, Cl^–, respectively - DR distribution ratio - HyN hypotonic nutrient medium - IsN isotonic nutrient medium - HyS IsS hypotonic, and isotonic salt medium, respectively     

Effect of extracellular pH on sodium current in isolated,single rat ventricular cells

Summary Effects of extracellular pH on the sodium current (I _Na) of single rat ventricular cells were examined under conditions of voltage clamp and internal perfusion. In this way, pH _i was controlled while pH _o was changed. The combined suction pipette-microelectrode method was used. The suction pipette passed current and perfused the cell's interior; the microelectrode measured membrane potential. Increasing extracellular H⁺ depressedI _Na and slowed inactivation. The current-voltage curves forI _Na and Slowed inactivation. The current-voltage curves forI _Na were shifted to positive and negative potentials at low and high pH _o , respectively. Similar potential shifts were observed in both the conductance voltage curve and the steadystate inactivation voltage curve (h ). Conduction was also depressed at low pH _o . The shifts were probably due to surface charge effects, while the impaired conduction was probably due to protonation of a site in the Na channel.     

Voltage Gating of Shaker K+ Channels : The Effect of Temperature on Ionic and Gating Currents

Ionic (I_i) and gating currents (I_g) from noninactivating Shaker H4 K⁺ channels were recorded with the cut-open oocyte voltage clamp and macropatch techniques. Steady state and kinetic properties were studied in the temperature range 2–22°C. The time course of I_i elicited by large depolarizations consists of an initial delay followed by an exponential rise with two kinetic components. The main I_i component is highly temperature dependent (Q₁₀ > 4) and mildly voltage dependent, having a valence times the fraction of electric field (z) of 0.2–0.3 e_o. The I_g On response obtained between −60 and 20 mV consists of a rising phase followed by a decay with fast and slow kinetic components. The main I_g component of decay is highly temperature dependent (Q₁₀ > 4) and has a z between 1.6 and 2.8 e_o in the voltage range from −60 to −10 mV, and ∼0.45 e_o at more depolarized potentials. After a pulse to 0 mV, a variable recovery period at −50 mV reactivates the gating charge with a high temperature dependence (Q₁₀ > 4). In contrast, the reactivation occurring between −90 and −50 mV has a Q₁₀ = 1.2. Fluctuation analysis of ionic currents reveals that the open probability decreases 20% between 18 and 8°C and the unitary conductance has a low temperature dependence with a Q₁₀ of 1.44. Plots of conductance and gating charge displacement are displaced to the left along the voltage axis when the temperature is decreased. The temperature data suggests that activation consists of a series of early steps with low enthalpic and negative entropic changes, followed by at least one step with high enthalpic and positive entropic changes, leading to final transition to the open state, which has a negative entropic change.     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier