The localization of the MM isozyme of creatine phosphokinase on the surface membrane of myocardial cells and its functional coupling to ouabain-inhibited (Na+,K+)-ATPase

A rat heart plasma membrane preparation isolated in a sucrose medium and some of its enzymatic properties have been investigated.It has been shown that a rat heart plasma membrane fraction contains high creatine phosphokinase activity which can not be diminished by repeated washing with sucrose solution. Creatine phosphokinase extracted from a plasma membrane fraction with potassium chloride and 0.01% deoxycholate solution is electrophoretically identical to MM isoenzyme of creatine phosphokinase. Under the conditions where (Na⁺,K⁺)-ATPase is activated by addition of Na⁺,K⁺ and MgATP, creatine phosphokinase of plasma membrane fraction is able to maintain a low ADP concentration in the medium if creatine phosphate is present. The rate of creatine release is dependent upon MgATP concentration in accordance with the kinetic parameters of the (Na⁺,K⁺)-ATPase and is significantly inhibited by ouabain (0.5 mM). The rate of creatine release is also dependent on creatine phosphate concentration in conformance with the kinetic parameters of MM isozyme of creatine phosphokinase,It is concluded that in intact heart cells the plasma membrane creatine phosphokinase may ensure effective utilization of creatine phosphate for immediate rephosphorylation of ADP produced in the (Na⁺,K⁺)-ATPase reaction.     

Transport of potassium inChara australis: II. Kinetics of a symport with sodium

Summary An electrogenic K⁺–Na⁺ symport with a high affinity for K⁺ has been found inChara (Smith & Walker, 1989). Under voltage-clamp conditions, the symport shows up as a change in membrane current upon adding either K⁺ or Na⁺ to the bathing medium in the presence of the other. Estimation of kinetic parameters for this transport has been difficult when using intact cells, since K⁺–Na⁺ current changes show a rapid falling off with time at K⁺ concentrations above 50 m. Cytoplasm-enriched cell fragments are used to overcome this difficulty since they do not show the rapid falling off of current change seen with intact cells. Current-voltage curves for the membrane in the absence or presence of either K⁺ or Na⁺ are obtained, yielding difference current-voltage curves which isolate the symport currents from other transport processes. The kinetic parameters describing this transport are found to be voltage dependent, withK _m for K⁺ ranging from 30 down to 2 m as membrane potential varies from –140 to –400 mV, andK _m for Na⁺ ranging between 470 and 700 m over a membrane potential range of –140 to –310 mV.Two different models for this transport system have been investigated. One of these involves the simultaneous transport of both the driver and substrate ions across the membrane, while the other allows for the possibility of the two ions being transported consecutively in two distinct reaction steps. The experimental results are shown to be consistent with either of these cotransport models, but they do suggest that binding of K⁺ occurs before that of Na⁺, and that movement of charge across the membrane (the voltage-dependent step) occurs when the transport protein has neither K⁺ nor Na⁺ bound to it.     

Na+ regulation and (Na++ K+)ATPase activity in the euryhaline fiddler crab Uca minax (Le conte)

• 1.1. Specific activity and kinetic characteristics of the (Na⁺ + K⁺)ATPase have been investigated in the gill epithelium of the hyper-hypoosmoregulator crab Uca minax.
• 2.2. (Na⁺ +K⁺)ATPase activity is shown to be at least three times higher in the posterior gills.
• 3.3. The kinetic study supports the hypothesis of the existence of two different (Na⁺ + K⁺)ATPases: the enzyme activity in the posterior gills could be involved in the transepithelial transport of Na⁺ while the activity of the anterior gills could be responsible for the intracellular regulation of Na⁺ and K⁺.
• 4.4. Significant and specific changes in (Na⁺ +K⁺)ATPase activity occur upon acclimation to media of various salinities.

     

The leak mode of type II Na+-Pi cotransporters

Na⁺-coupled phosphate cotransporters of the SLC34 gene family catalyze the movement of inorganic phosphate (P_i) across epithelia by using the free energy of the downhill electrochemical Na⁺ gradient across the luminal membrane. Electrogenic (NaPi-IIa/b) and electroneutral (NaPi-IIc) isoforms prefer divalent P_i and show strict Na⁺:P_i stoichiometries of 3:1 and 2:1, respectively. For electrogenic cotransport, one charge is translocated per transport cycle. When NaPi-IIa or NaPi-IIb are expressed in Xenopus oocytes, application of the Pi transport inhibitor phosphonoformic acid (PFA) blocks a leak current that is not detectable in the electroneutral isoform. In this review, we present the experimental evidence that this transport-independent leak originates from a Na⁺-dependent uniport carrier mode intrinsic to NaPi-IIa/b isoforms. Our findings, based on the characteristics of the PFA-inhibitable leak measured from wild-type and mutant constructs, can be incorporated into an alternating access class model in which the leak and cotransport modes are mutually exclusive and share common kinetic partial reactions.     

Time-Dependent Molecular Memory in Single Voltage-Gated Sodium Channel

Excitability in neurons is associated with firing of action potentials and requires the opening of voltage-gated sodium channels with membrane depolarization. Sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, can have profound implications on the biophysical properties of voltage-gated ion channels. Therefore, we sought to characterize the effect of sustained membrane depolarization on single voltage-gated Na⁺ channels. Single-channel activity was recorded in the cell-attached patch-clamp mode from the rNa_v1.2α channels expressed in CHO cells. Classical statistical analysis revealed complex nonlinear changes in channel dwell times and unitary conductance of single Na⁺ channels as a function of conditioning membrane depolarization. Signal processing tools like weighted wavelet Z (WWZ) and discrete Fourier transform analyses attributed a “pseudo-oscillatory” nature to the observed nonlinear variation in the kinetic parameters. Modeling studies using the hidden Markov model (HMM) illustrated significant changes in kinetic states and underlying state transition rate constants upon conditioning depolarization. Our results suggest that sustained membrane depolarization induces novel nonlinear properties in voltage-gated Na⁺ channels. Prolonged membrane depolarization also induced a “molecular memory” phenomenon, characterized by clusters of dwell time events and strong autocorrelation in the dwell time series similar to that reported recently for single enzyme molecules. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Voltage-gated Na⁺ channel with the observed time-dependent nonlinear properties and the molecular memory phenomenon may determine the functional state of the channel and, in turn, the excitability of a neuron.     

Involvement of the alpha-subunit N-terminus in the mechanism of the Na+,K+-ATPase

Previous studies have shown that cytoplasmic K⁺ release and the associated E2 → E1 conformational change of the Na⁺,K⁺-ATPase is a major rate-determining step of the enzyme's ion pumping cycle and hence a prime site of acute regulatory intervention. From the ionic strength dependence of the enzyme's distribution between the E2 and E1 states, it has also been found that E2 is stabilized by an electrostatic attraction. Any disruption of this electrostatic attraction would, thus, have profound effects on the rate of ion pumping. The aim of this paper is to identify the location of this interaction. Using enhanced-sampling molecular dynamics simulations with a predicted N-terminal structure added to the X-ray crystal structure of the Na⁺,K⁺-ATPase, a previously postulated salt bridge between Lys32 and Glu233 (rat sequence numbering) of the enzyme's α-subunit can be excluded. The residues never approach closely enough to form a salt bridge. In contrast, strong interactions with anionic lipid head groups were seen. To investigate the possibility of a protein-lipid interaction experimentally, the surface charge density of Na⁺,K⁺-ATPase-containing membrane fragments was estimated from zeta potential measurements to be 0.019 (± 0.001) C m⁻². This is in good agreement with the charge density previously determined to be responsible for stabilization of the E2 state of 0.023 (± 0.009) C m⁻² and the membrane charge density estimated here from published electron-microscopic images of 0.018C m⁻². The results are, therefore, consistent with an interaction of the Na⁺,K⁺-ATPase α-subunit N-terminus with negatively-charged lipid head groups of the neighbouring cytoplasmic membrane surface as the origin of the electrostatic interaction stabilising the E2 state.     

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na⁺-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na⁺-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H⁺ gradient (intravesicular pH<extravesicular pH) and H⁺ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na⁺-independent transport of l-arginine in brush border membrane vesicles which differs from Na⁺-independent uptake of neutral and acidic amino acids. Na⁺-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na⁺-independent transport of dibasic amino acids across the renal brush border membrane.     

The Na+/H+ Exchanger Present in Trout Erythrocyte Membranes is Not Responsible for the Amiloride-insensitive Na+/Li+ Exchange Activity

The protein responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane has not been cloned or isolated. It has been suggested that a Na⁺/H⁺ exchanger could be responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane. Previously, we reported that in the trout erythrocyte, the Li⁺/H⁺ exchange activity (mediated by the Na⁺/H⁺ exchanger βNHE) and the Na⁺/Li⁺ exchange activity respond differently to cAMP, DMA (dimethyl-amiloride) and O₂. We concluded that the DMA insensitive Na⁺/Li⁺ exchange activity originates from a different protein. To further examine these findings, we measured Li⁺ efflux in fibroblasts expressing the βNHE as the only Na⁺/H⁺ exchanger. Moreover, the internal pH of these cells was monitored with a fluorescent probe. Our findings indicate that acidification of fibroblasts expressing the Na⁺/H⁺ exchanger βNHE, induces a Na⁺ stimulated Li⁺ efflux activity in trout erythrocytes. This exchange activity, however, is DMA sensitive and therefore differs from the DMA insensitive Na⁺/Li⁺ exchange activity. In these fibroblasts no significant DMA insensitive Na⁺/Li⁺ exchange activity was found. These results support the hypothesis that the trout erythrocyte Na⁺/Li⁺ exchange activity is not mediated by the Na⁺/H⁺ exchanger (βNHE) present in these membranes. Received: 6 December 1996/Revised: 11 August 1997     

The role of potassium and chloride ions on the Na+/acidic amino acid cotransport system in rat intestinal brush-border membrane vesicles

The Na⁺/l-glutamate (l-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K⁺ and Cl^?. The presence of 100 mM K⁺ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K⁺ concentration and that in the absence of any sodium gradient, an outward K⁺ gradient was unable to influence the Na⁺/acidic amino acid transport system. It was also found that Cl^? could specifically activate the Na⁺-dependent l-glutamate (l-aspartate) uptake either in the presence or in the absence of K⁺. Also the effect of Cl^? was observed only in the presence of an inward Na⁺ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na⁺ gradient and in the presence of chloride gradient. l-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K⁺ or Cl^? did not show any translocation of net charge.     

Effect of caffeine,theophylline and nicotine on d-glucose and folate transport in rat jejunal brush border membrane vesicles

• 1.1. Nicotine at 10 mM, but not caffeine or theophylline, reduced by 20% the overshoot of the Na⁺-dependent d-glucose transport in ratjejunal brush border membrane vesicles.
• 2.2. Since nicotine did not affect the transport of Na⁺, its inhibition on Na⁺-dependent d-glucose transport must be due to a direct effect upon the d-glucose transport system.
• 3.3. Folate transport in these membrane vesicles was found to a be a free diffusion process at pH 7.4.
• 4.4. Neither caffeine, theophylline nor nicotine has any effect on folate transport.

     

Transport in halobacterium halobium: Light-induced cation-gradients,amino acid transport kinetics,and properties of transport carriers

Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na⁺. Measurements of ²²Na flux, exterior pH change, and membrane potential, ΔΨ (with the dye 3,3′-dipentyloxadicarbocyanine) indicate that the means of Na⁺ transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H⁺/Na⁺ > 1). The resulting large chemical gradient for Na⁺ (outside > inside), as well as the membrane potential, will drive the transport of 18 amino acids. The 19th, glutamate, is unique in that its accumulation is indifferent to ΔΨ: this amino acid is transported only when a chemical gradient for Na⁺ is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H⁺ collapses within 1 min, while the large Na⁺ gradient and glutamate transporting activity persists for 10–15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na⁺, arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V_max and K_m comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na⁺, in an electrically neutral fashion, so that only the chemical component of the Na⁺ gradient is a driving force. The transport of all amino acids but glutamate is bidirectional. Actively driven efflux can be obtained with reversed Na⁺ gradients (inside > outside), and passive efflux is considerably enhanced by intravesicle Na⁺. These results suggest that the transport carriers are functionally symmetrical. On the other hand, noncompetitive inhibition of transport by cysteine (a specific inhibitor of several of the carriers) is only obtained from the vesicle exterior and only for influx: these results suggest that in some respects the carriers are asymmetrical. A protein fraction which binds glutamate has been found in cholate-solubilized H. halobium membranes, with an apparent molecular weight of 50,000. When this fraction (but not the others eluted from an Agarose column) is reconstituted with soybean lipids to yield lipoprotein vesicles, facilitated transport activity is regained. Neither binding nor reconstituted transport depend on the presence of Na⁺. The kinetics of the transport and of the competitive inhibition by glutamate analogs suggest that the protein fraction responsible is derived from the intact transport system.     

Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone     

ANALYSIS OF SODIUM TRANSPORT IN THE AMPHIBIAN OOCYTE BY EXTRACTIVE AND RADIOAUTOGRAPHIC TECHNIQUES

The transport of Na⁺ in mature Eurycea oocytes was studied by quantitative radioautography of ²²Na⁺ using techniques suitable for localization of diffusible solutes, together with conventional extractive techniques. Intracellular Na⁺ consisted of three kinetic fractions: a cytoplasmic fast fraction of about 8.5 µeq/ml H₂O; a cytoplasmic slow fraction of about 58.7 µeq/ml H₂O; and a nuclear fast fraction of about 11.1 µeq/ml H₂O. A nuclear slow fraction, if it exists, does not exceed 5% of the cytoplasmic. The fast fractions represent freely diffusible Na⁺ in the two compartments; the nuclear solvent space is 1.3 times the cytoplasmic. The flux of both fast fractions is determined by the permeability of the cortical membrane, with neither the nuclear membrane nor diffusion in the cytoplasm detectably slowing the flux. The cytoplasmic slow fraction is interpreted to represent Na⁺ bound to nondiffusible constituents which are excluded from the nucleus; these may be yolk platelets, although the widespread observation of Na⁺ binding in other cells, and the high Na⁺/K⁺ selectivity, argues against simple ion-binding to the yolk phosphoprotein.     

Potassium activation of Na+-dependent leucine transport in brush-border membrane vesicles from rat jejunum

Na⁺-dependent leucine uptake was greater in potassium loaded brush-border membrane vesicles compared with controls. This effect was not mediated by an electrical potential difference, since it was still present in voltage-clamped conditions. Inhibition experiments indicate the same Na⁺-dependent leucine transport activity in the presence or in the absence of potassium. The affinity of sodium for the cotransporter was identical at 10 or 100 mM potassium. Leucine kinetics at different potassium concentrations showed a maximum 2.4-fold increase in V_max, while K_m was unaffected. The secondary plots of the kinetic results were not linear. This kinetic behaviour suggests that K⁺ acts as a non-essential activator of Na⁺-dependent leucine cotransport. A charge compensation of sodium-leucine influx is most probably a component of the potassium effect in the presence of valinomycin.     

Membrane potential and sodium flux in neuroblastoma X glioma hybrid cells: Effects of amiloride and serum

The Na⁺ uptake into neuroblastoma x glioma hybrid cells was measured in Hepes-buffered EMEM containing 10% calf serum and 5 mM ouabain in the presence and absence of amiloride (1.0 mM). Amiloride was found to markedly inhibit net Na⁺ influx (by approximately 50%). Examination of the effect of amiloride on net Na⁺ influx in the absence of calf serum revealed that a significant amiloride-sensitive Na⁺ influx remains even under serum-deprived conditions, although the degree of amiloride inhibition (35%) is substantially lower than that found in the presence of serum. The amiloride-insensitive portion of Na⁺ influx was found to be independent of serum effects. Estimation of resting membrane potential was made by measurement of the steady state distribution of the lipophilic cation, TPP⁺, in the presence and absence of amiloride. A large, immediate increase in TPP⁺ uptake, indicative of a membrane hyperpolarization, was seen upon addition of amiloride. Determination of the effect of amiloride on resting membrane potential of serum-deprived cells showed that cells are hyperpolarized to a greater extent in the presence than in the absense of amiloride, and that serum exerts a depolarizing effect on the cells. Thus, serum-stimulation of Na⁺ influx results in a depolarization of resting membrane potential, while amiloride inhibition of Na⁺ influx causes a hyperpolarization. These data strongly suggest that NG108-15 cells possess an electrogenic Na⁺ influx pathway that is sensitive to amiloride inhibition and enhanced by serum.     

Two types of modified cardiac Na+ channels after cytosolic interventions at the α-subunit capable of removing Na+ inactivation

Failure of inactivation is the typical response of voltage-gated Na⁺ channels to the cytosolic presence of proteolytic enzymes, protein reagents such as N-bromoacetamide (NBA) or iodate, and antibodies directed against the linker between domains III and IV of the α-subunit. The present patch clamp experiments with cardiac Na⁺ channels aimed to test the hypothesis that these interventions may provoke the occurrence of non-inactivating Na⁺ channels with distinct kinetic properties. A site-directed polyclonal antibody (anti-SLP2, target sequence 1481–1496 of the cardiac Na⁺ channel α-subunit) eliminated fast Na⁺ inactivation to induce burst activity which was accompanied by the occurrence of two open states. A deactivation process terminated channel activity during membrane depolarization proceeding with time constants of close to 40 ms (at –40 mV). NBA-modified and iodate-modified Na⁺ channels were kinetically indistinguishable from the anti-SLP2-modified type since they likewise deactivate and, thus, attain an only moderate P_o of close to 20%. This is fundamentally different from the behaviour of enzymatically-modified Na⁺ channels: after cytosolic proteolysis with α-chymotrypsin, trypsin or pronase, mean P_o during membrane depolarization amounted to approximately 40% because deactivation operated extremely slowly and less efficiently (time constants 100–200 ms at –40 mV, as a minimum) or was virtually non-operating. In-vitro cleavage of the synthetic linker sequence 1481–1496 confirmed that this part of the α-subunit provides a substrate for these peptidases or reactants for NBA but cannot be chemically modified by iodate. This iodate resistance indicates that iodate-modified Na⁺ channels are based on a structural alteration of still another region which is also involved in Na⁺ inactivation, besides the linker between domains III and IV of the α-subunit. Endogenous peptidases such as calpain did not affect Na⁺ inactivation. This stresses the stochastic nature of a kinetic peculiarity of cardiac Na⁺ channels, mode-switching to a non-inactivating mode. Received: 25 May 1996 / Accepted: 12 September 1996     

Cationic interactions with Na+-H+ exchange and passive Na+ flux in cardiac sarcolemmal vesicles

Na⁺-H⁺ exchange and passive Na⁺ flux were investigated in cardiac sarcolemmal vesicles as a function of changing the ionic composition of the reaction media. The inclusion of EGTA in the reaction medium resulted in a potent stumulation of Na⁺ uptake by Na⁺-H⁺ exchange. It was found that millimolar concentrations of Mg²⁺ and Li⁺ were capable of inhibiting Na⁺-H⁺ exchange by 80%. One mechanism by which these ions may inhibit intravesicular Na⁺ accumulation by Na⁺-H⁺ exchange is via an increase in Na⁺ efflux. An examination of Na⁺ efflux kinetics from vesicles pre-loaded with Na⁺ revealed that Na⁺, Ca²⁺, Mg²⁺ and Li⁺ could stimulate Na⁺ efflux. Na⁺-H⁺ exchange was potently inhibited by an organic divalent cation, dimenthonium, which screens membrane surface charge. This would suggest that Na⁺-H⁺ exchange occurs in the diffuse double layer region of cardiac sarcolemma and this phenomenon is distinctly different from other Na⁺ transport processes. The results in this study indicate that in addition to a stimulation of Na⁺ efflux, the inhibitory effects of Mg²⁺, Ca²⁺ and Li⁺ on Na⁺-H⁺ exchange may also involve a charge dependent screening of Na⁺ interactions with the membrane.     

Modeling squid axon Na+ channel by a nucleation and growth kinetic mechanism

A kinetic model accounting for all salient features of the Na⁺ channel of the squid giant axon is provided. The model furnishes explanations for the Cole-Moore-like effect, the rising phase of the ON gating current and the slow ‘intermediate component’ of its decaying phase, as well as the gating charge immobilization. Experimental ON ionic currents are semi-quantitatively simulated by the use of only three free parameters, upon assuming that the Na⁺ channel opening proceeds along with the stepwise aggregation of its four domains, while they are moving their gating charge outward under depolarizing conditions. The inactivation phase of the ON ionic current is interpreted by a progressive electrostatic attraction between the positively charged ‘hinged lid’ containing the hydrophobic IFM triad and its receptor inside the channel pore, as the stepwise outward movement of the S4 segments of the Na⁺ channel progressively increases the negative charge attracting the triad to its receptor. The Na⁺ channel closing is assumed to proceed by repolarization-induced disaggregation of its domains, accompanied by inward movement of their gating charge. The phenomenon of ‘gating charge immobilization’ can be explained by assuming that gradual structural changes of the receptor over the time course of depolarization strengthen the interaction between the IFM triad and its receptor, causing a slow release of the gating charge during the subsequent repolarization.     

Built for speed

Many of us were taught in high school biology that the action potential waveform in nerves and other excitable tissues was generated by an initial rapid influx of external Na⁺ ions across the plasma membrane, followed by an outward movement of intracellular K⁺ ions. The former event, mediated by voltage-gated Na⁺ channels, is responsible for the fast depolarizing upstroke of the action potential, while voltage-gated K+ channels are responsible for the subsequent repolarizing phase, which largely controls action potential duration. Although Hodgkin and Huxley described the fundamental importance of this sequential activation process more than 60 y ago, the molecular and structural details underlying the faster activation of voltage-gated Na⁺ (Nav) vs. K⁺ (Kv) channels have yet to be fully resolved.     

Activity and Stoichiometry of Na+:HCO− 3 Cotransport in Immortalized Renal Proximal Tubule Cells

The proximal tubule Na⁺-HCO⁻ ₃ cotransporter is located in the basolateral plasma membrane and moves Na⁺, HCO⁻ ₃, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO⁻ ₃ anions are transported for each Na⁺ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through the cotransporter was achieved by applying Na⁺ or HCO⁻ ₃ concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl⁻ independent; its conductance ranged between 0.12 and 0.23 mS/cm² and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence of Na⁺ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a cotransport ratio of 3HCO⁻ ₃:1Na⁺. Received: 19 January 1996/Revised: 24 April 1996