Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers

Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K+ and 1.6 for Na+:Li+. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCl concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found.     

Expression of amiloride-sensitive Na+ channels of hen lower intestine in Xenopus oocytes: electrophysiological studies on the dependence of varying NaCl intake.

Epithelial Na+ channels were incorporated into the plasma membrane of Xenopus laevis oocytes after micro-injection of RNA from hen lower intestinal epithelium (colon and coprodeum). The animals were fed either a normal poultry food which contained NaCl (HS), or a similar food devoid of NaCl (LS). Oocytes were monitored for the expression of amiloride-sensitive sodium channels by measuring membrane potentials and currents. Oocytes injected with poly(A)+RNA prepared from HS animals or non-injected control oocytes showed no detectable sodium currents, whereas oocytes injected with LS-poly(A)+RNA had large amiloride-blockable sodium currents. These currents were almost completely saturated by sodium concentrations of 20 mM with a Km of about 2.6 mM sodium. Amiloride (10 microM) inhibits the expressed sodium channels entirely and examination of dose response relationships yielded a half-maximal inhibition concentration (Ki) of 120 nM amiloride. I-V difference curves in the presence or absence of sodium or amiloride (10 microM) indicate a potential dependence of the sodium transport which can be described by the Goldman equation. When Na+ is replaced by K+, no amiloride response was detected indicating a high selectivity for Na+ over K+. These results provide strong evidence that intestinal Na+ channels are regulated by dietary salt intake on the RNA level.     

Molecular organization of glutamate-sensitive chemoexcitable membranes of nerve cells. Function of glutamate-binding proteins of the central nervous system when incorporated into liposomes

The functioning of the glutamate-binding protein of rat brain cortex synaptic membranes was studied by its incorporation into liposomes. The optimal conditions for the receptor protein incorporation were established and the kinetics of 22Na+ and 86Rb+ incorporation into the liposomes in the presence of L-glutamate were analyzed. Modelling of the CNS glutamate receptor functions was found to be dependent on the lipid composition and amount of the incorporated membrane protein. The selective transport of 22Na+ into the liposomes was stimulated in the presence of 10(-4) M glutamate. Addition of monoclonal antibodies against glutamate-binding proteins blocked the incorporation of Na+ into the liposomes. The experimental results are suggestive of the nativity of the liposome-incorporated membrane protein, which is capable of binding glutamate and regulating selective transport of Na+. It was assumed that the glutamate receptor macromolecule represents an integral complex made up of several low molecular weight subunits of glucoprotein nature that form a selective ionic channel.     

Effect of amiloride on the activity of membrane-bound and soluble Na+-,K+-ATPase preparations

Amiloride (8 X 10(-4), an inhibitor of sodium channels of nonexcited membranes, inhibits the activity of Na+,K+-ATPase in the kidney cortex homogenate as well as that of the partially purified membrane-bound and lubrol-soluble Na+,K+-ATPase preparations from the cattle brain. Inhibition of Na+,K+-ATPase from different organs of various animals by amiloride, a blocker of sodium channels, indicates similarity of the molecular organization of the Na+-recognizing component both of sodium channels and sodium centres of Na+,K+-ATPase.     

IS3 peptide-formed ion channels in rat skeletal muscle cell membranes

A 22-mer peptide, identical to the primary sequence of domain I segment 3 (IS3) of rat brain sodium channel I, was synthesized. With the patch clamp cell-attached technique, single channel currents could be recorded from the patches of cultured rat myotube membranes when the patches were held at hyperpolarized potentials and the electrode solution contained NaCl and 1 microM IS3, indicating that IS3 incorporated into the membranes and formed ion channels. The single channel conductances of IS3 channels were distributed heterogeneously, but mainly in the range of 10-25 pS. There was a tendency that the mean open time and open probability of IS3 channels increased and the mean close time decreased with the increasing of hyperpolarized membrane potentials. IS3 channels are highly selective for Na+ and Li+ but not for Cl- and K+, similar to the authentic Na+ channels.     

Calcium channels in smooth muscle cells

In smooth muscle cells, the electrophysiological properties of potential-dependent calcium channels are similar to those described in other excitable cells. The calcium current is dependent on the extracellular calcium concentration; it is insensitive to external sodium removal and tetrodotoxin application. Other ions (Ba2+, Sr2+, Na+) can flow through the calcium channel. This channel is blocked by Mn2+, Co2+, Cd2+ and by organic inhibitors. The inactivation mechanism is mediated by both the membrane potential and the calcium influx. Ca2+ ions can also penetrate into the cell through receptor-operated channels. These channels show a low ionic selectivity and are generally less sensitive to organic Ca-blockers than the potential-dependent calcium channels. The finding of specific channel inhibitors as well as the study of the biochemical pathways between receptor activation and channel opening are prerequisites to further characterization of receptor-operated channels.     

Effect of changing temperature on the ionic permeation through the cyclic GMP-gated channel from vertebrate photoreceptors.

Native cGMP-gated channels were studied in rod outer segments of the larval tiger salamander Ambystoma tigrinum. The alpha subunit of the cGMP-gated channel from bovine rods, here referred to as the wild type (w.t.), and mutant channels were heterologously expressed in Xenopus laevis oocytes. These channels were studied in excised membrane patches in the inside-out configuration and were activated by the addition of 100 or 500 microM cGMP. The effect of temperature on the ionic permeation was studied. The macroscopic current flowing through the native channel at +100 mV had an activation energy of 35.8, 30, 31.8, 34.5, 41.3, and 22.4 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The macroscopic current flowing through the w.t. channel at +100 mV had an activation energy of 45.2, 38.2, 37.5, 47.3, 49.4, and 38.9 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The activation energy of the macroscopic current flowing through the native and w.t. channels did not vary significantly when the ionic concentration of the permeant ion was changed between 2.5 and 110 mM. The activation energy of the single-channel current of the w.t. channel at +100 mV was 40.4 and 33 kJ mol-1 for Na+ and NH4+, respectively. The reversal potential of biionic solutions changed significantly with temperature. These results can be used to obtain an estimate of the enthalpic and entropic contributions to the barrier of the Gibbs free energy experienced by an ion during its permeation through the open channel. These estimates indicate that the ionic permeation and selectivity of the cGMP-gated channel are controlled both by enthalpic and entropic factors and that the selectivity of the native channel for Li+ over Na+ is primarily caused by entropic effects.     

Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes

A purified (Na+ + K+)-ATPase large subunit obtained from microsomes by water-alcohol extraction was incorporated into a bilayer lipid membrane. The protein formed in the membrane conductance channels which were sensitive to ouabain and selective for monovalent cations. ATP activated these channels in the presence of sodium and potassium ions. When sodium ions were eliminated ATP did not change the conductance of the modified membrane whereas p-nitrophenyl phosphate increased it. The (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membrane possessed an ATPase activity. The presence of a potential on the membrane was a necessary condition for the enzyme incorporated into a bilayer lipid membrane to show high ATPase activity. Increasing the potential above 100 mV resulted in the closing of conductance channels.     

Acetyl phosphate can act as a substrate for Na+ transport by (Na+ + K+)-ATPase

Experiments using liposomes with (Na+ + K+)-ATPase incorporated showed that in the presence of extravesicular Mg2+, acetyl phosphate was able to stimulate Na+ uptake when the liposomes contained Na+ or choline and were K+-free; this acetyl phosphate-dependent Na+ transport was similar to the ATP-dependent transport observed with 0.003 mM or 3 mM ATP. When the intravesicular solution contained K+, there was an ATP-dependent Na+ uptake which was large with 3 mM ATP and small (about the size seen in K+-free liposomes) with 0.003 mM ATP; in this case, although acetyl phosphate produced a slight activation of Na+ transport, the effect was not statistically significant. All ATP and acetyl phosphate-stimulated Na+ transport disappeared in the absence of extravesicular Mg2+ or in the presence of ouabain in the intravesicular solution. These results are consistent with the hypothesis that, at the concentration used, acetyl phosphate can replace ATP in the catalytic but not in the regulatory site of the (Na+ + K+)-ATPase and active Na+ transport system. This suggests that as far as the early stages of the pump cycle are concerned the role of ATP is simply to phosphorylate.     

On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.     

Simultaneous measurements of calcium mobilization and afferent nerve activity in electroreceptor organs of anesthetized Kryptopterus bicirrhis

The transduction pathway of ampullary electroreceptor organs involves ionic currents. It has been shown that calcium, as well as sodium and potassium play important parts in this process. In this study we examine the stimulus-evoked changes in the Fura-2 ratio in electroreceptor cells. Furthermore, we recorded stimulus-evoked Fura-2 ratio changes while Na+ and K+ channels were blocked by amiloride and TEA. Simultaneously, extracellular recordings of the afferent spike activity were made. The results show the presence of stimulus evoked fluctuations in the Fura-2 ratio. These fluctuations can be abolished by the application of Cd2+, TEA, and amiloride. The stimulus-evoked activity of the afferent nerve was decreased due to application of these drugs. We conclude that the transduction current is carried by Na+, K+, and probably Ca2+. This fits the existing model on transduction in electroreceptors.     

Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block

Batrachotoxin-modified, voltage-dependent sodium channels from canine forebrain were incorporated into planar lipid bilayers. Single-channel conductances were studied for [Na+] ranging between 0.02 and 3.5 M. Typically, the single-channel currents exhibited a simple two-state behavior, with transitions between closed and fully open states. Two other conductance states were observed: a subconductance state, usually seen at [NaCl] greater than or equal to 0.5 M, and a flickery state, usually seen at [NaCl] less than or equal to 0.5 M. The flickery state became more frequent as [NaCl] was decreased below 0.5 M. The K+/Na+ permeability ratio was approximately 0.16 in 0.5 and 2.5 M salt, independent of the Na+ mole fraction, which indicates that there are no interactions among permeant ions in the channels. Impermeant and permeant blocking ions (tetraethylammonium, Ca++, Zn++, and K+) have different effects when added to the extracellular and intracellular solutions, which indicates that the channel is asymmetrical and has at least two cation-binding sites. The conductance vs. [Na+] relation saturated at high concentrations, but could not be described by a Langmuir isotherm, as the conductance at low [NaCl] is higher than predicted from the data at [NaCl] greater than or equal to 1.0 M. At low [NaCl] (less than or equal to 0.1 M), increasing the ionic strength by additions of impermeant monovalent and divalent cations reduced the conductance, as if the magnitude of negative electrostatic potentials at the channel entrances were reduced. The conductances were comparable for channels in bilayers that carry a net negative charge and bilayers that carry no net charge. Together, these results lead to the conclusion that negative charges on the channel protein near the channel entrances increase the conductance, while lipid surface charges are less important.     

Membrane contact, fusion, and hexagonal (HII) transitions in phosphatidylethanolamine liposomes

The behavior of phosphatidylethanolamine (PE) liposomes has been studied as a function of temperature, pH, ionic strength, lipid concentration, liposome size, and divalent cation concentration by differential scanning calorimetry (DSC), by light scattering, by assays measuring liposomal lipid mixing, contents mixing, and contents leakage, and by a new fluorometric assay for hexagonal (HII) transitions. Liposomes were either small or large unilamellar, or multilamellar. Stable (impermeable, nonaggregating) liposomes of egg PE (EPE) could be formed in isotonic saline (NaCl) only at high pH (greater than 8) or at lower pH in the presence of low ionic strength saline (less than 50 mOsm). Bilayer to hexagonal (HII) phase transitions and gel to liquid-crystalline transitions of centrifuged multilamellar liposomes were both detectable by DSC only at pH 7.4 and below. The HII transition temperature increased, and the transition enthalpy decreased, as the pH was raised above 7.4, and it disappeared above pH 8.3 where PE is sufficiently negatively charged. HII transitions could be detected at high pH following the addition of Ca2+ or Mg2+. No changes in light scattering and no lipid mixing, mixing of contents, or leakage of contents were noted for EPE liposomes under nonaggregating conditions (pH 9.2 and 100 mM Na+ or pH 7.4 and 5 mM Na+) as the temperature was raised through the HII transition region. However, when aggregation of the liposomes was induced by addition of Ca2+ or Mg2+, or by increasing [Na+], it produced sharp increases in light scattering and in leakage of contents and also changes in fluorescent probe behavior in the region of the HII transition temperature (TH). Lipid mixing and contents mixing were also observed below TH under conditions where liposomes were induced to aggregate, but without any appreciable leakage of contents. We conclude that HII transitions do not occur in liposomes under conditions where intermembrane contacts do not take place. Moreover, fusion of PE liposomes at a temperature below TH can be triggered by H+, Na+, Ca2+, or Mg2+ or by centrifugation under conditions that induce membrane contact. There was no evidence for the participation of HII transitions in these fusion events.     

Incorporation of Na+ - Ca2+ antiporter and of (Na+ + K+)-ATPase into liposomes and demonstration of their non-identity

(Na+ + K+)-ATPase was isolated from the grey matter of brain and incorporated into liposomes. Most of the reconstituted enzyme was oriented 'inside-out' with respect to its in vivo orientation and externally added ATP promoted Na+ uptake that was inhibitable by internally trapped ouabain. Using the same proteoliposomes, an Na+ - Ca2+ exchange system was observed as indicated by the following pieces of evidence. (1) The Na+ gradient provided the only readily apparent driving force for acceleration of Ca2+ accumulation into proteoliposomes. (2) The antiporter was specific for Ca2+, high Mg2+ excess did not inhibit Ca2+ antiport. (3) The Na+ efflux was dependent on the extravesicular Ca2+ concentration. (4) The Na+ efflux was not inhibited by tetrodotoxin. The demonstrated Na+ - Ca2+ exchange could not be related to (Na+ + K+)-ATPase protein, since it was not purified with (Na+ + K+)-ATPase, as followed from transport studies with liposomes containing (Na+ + K+)-ATPase of different specific activity. The results strongly indicate that plasma membranes isolated from the grey matter of brain contain an Na+ - Ca2+ exchange system and that the proteoliposomes are suitable for further purification of the carrier molecule.     

Cation selectivity characteristics of the reconstituted voltage-dependent sodium channel purified from rat skeletal muscle sarcolemma

In this report, the alkali metal cation selectivity of the purified, voltage-dependent sodium channel from rat skeletal muscle is described. Isolated sodium channel protein (980-2840 pmol of saxitoxin binding/mg of protein) was reconstituted into egg phosphatidylcholine vesicles, and channels were subsequently activated by either batrachotoxin (5 X 10(-6) M) or veratridine (5 X 10(-4) M). Activation of the reconstituted sodium channel by batrachotoxin permitted rapid specific influx of cations into channel-containing vesicles. Quenched flow kinetic techniques were adapted to allow resolution of the kinetics of cation movement. Uptake rates for 42K+, 86Rb+, and 137Cs+ were measured directly and half-times for equilibration at 18 degrees C were determined to be 350 ms, 2.5 s, and 10 s, respectively, in this vesicle population. 22Na+ equilibration occurred within the mimimum quenching time of the apparatus (90 ms) but an upper limit of 50 ms at 18 degrees C could be assigned to its half-time. Based on this upper estimate for Na+, cation selectivity ratios of the batrachotoxin-activated channel were Na+ (1):K+ (0.14):Rb+ (0.02):Cs+ (0.005). Toxin-stimulated influx could be blocked by saxitoxin with a Ki of approximately 5 X 10(-9) M at 18 degrees C. Rates of cation movement through veratridine-activated channels were much slower, with half-times of 1.0, 1.2, 2.0, and 2.6 min at 36 degrees C for Na+, K+, Rb+, and Cs+, respectively. The temperature dependences of batrachotoxin and veratridine-stimulated cation uptake were markedly different. The activation energies for 86Rb+ and 137Cs+ movement into batrachotoxin-activated vesicles were 7.6 and 6.1 kcal/mol, respectively, while comparable measurements for these two cations in veratridine-activated vesicles yielded activation energies of 31 kcal/mol. Measurements of cation exchange with batrachotoxin-activated channels may reflect characteristics of an open sodium channel while the process of channel opening itself may be rate-limiting when veratridine is used for activation.     

Sodium: a regulator of glucose uptake in virus-transformed and nontransformed cells.

Observations of cells transformed by the Bryan strain of Rous sarcoma virus (RSV-BH) suggested that the intracellular concentrations of sodium ion (Na+) may play a critical role in cellular metabolism. In an attempt to manipulate intracellular Na+, chick embryo cells were exposed to graded concentrations of Na+ in the cellular growth medium, and the effects on capacity for glucose uptake was examined. After incubation for six hours, the incorporation rate of 2-deoxyglucose (used as a substitute for glucose) was proportional to the external Na+ concentration over the range, 100 mM to 200 mM. Cells transformed by RSV-BH were less responsive than nontransformed cells to differences in Na+ at low concentrations. The changes were specifically dependent upon Na+, since K+, Li+, or choline + were ineffective as substitutes, and increasing the ionic strength above that of 120 mM Na+ was effective only when Na+ was the added cation.     

Action potentials dependent on monovalent cations in developing mouse embryos

Action potentials were examined using intracellular recording techniques to study the ionic mechanisms of excitability in oocytes and embryos of the mouse from the 1-cell through to the 16-cell stages of development. At all stages examined, action potentials dependent on monovalent cations (Na+ or Li+) were observed under Ca2+-free conditions, and the maximum rate of rise (MRR) of the Na action potential was larger than that of the Li action potential at a given concentration of monovalent cations. Both the Na and Li action potentials were insensitive to tetrodotoxin, and they were blocked by inorganic (Co2+, Cd2+, Mn2+, La3+) and organic (diltiazem) Ca antagonists. These properties were exactly the same as those of the Ca channels present in the membranes of the mouse embryos. In addition, competition was observed between permeant monovalent and divalent cations: the overshoot and MRR of the Na or Li action potentials were reduced in the presence of Ca2+. These results suggest that Na+ or Li+ go through the Ca channels when the external Ca2+ concentration was very low, and that the Ca channels are more permeable to Na+ than to Li+. Separate Na channels could not be detected or induced at any stages of development.     

Partial purification and reconstitution of the Na+-D-glucose cotransport protein from pig renal proximal tubules

Brush border membranes from renal proximal tubules were solubilized with deoxycholate, and the proteins were incorporated into liposomes formed from cholesterol and phosphatidylserine by a freeze-thaw procedure. In the proteoliposomes Na+-D-glucose cotransport was demonstrated by showing that the D-glucose concentration in the liposomes increased far above the equilibrium value if a Na+ gradient was applied. The initial D-glucose uptake rate, stimulated by an inside directed gradient of 89 mM Na+, was 4 pmol/mg of protein-1 s-1. High affinity phlorizin binding could not be measured. After two precipitation steps with the solubilized membrane proteins, a protein fraction was obtained in which significantly high affinity phlorizin binding was detected. After reconstitution, proteoliposomes were formed in which more than 70% of the protein was represented by two polypeptides with molecular weights of 94,000 and 52,000. An initial Na+ gradient-dependent D-glucose uptake rate of 118 pmol/mg of protein-1 s-1 was obtained. In these liposomes, the D-glucose uptake rate could be inhibited by phlorizin (Ki = 0.3 microM), and 55-pmol phlorizin-binding sites per mg of protein (KD = 0.5 microM) were measured. In different liposomal preparations a correlation between Na+ gradient-dependent D-glucose uptake rate and the amount of 52,000 molecular weight polypeptide was observed.     

Interactions of amiloride and small monovalent cations with the epithelial sodium channel. Inferences about the nature of the channel pore.

The voltage dependence of amiloride-induced inhibition of current flow through apical membrane sodium channels in toad urinary bladder was studied at different ionic conditions. The "inert" salt N-methyl-D-glucamine HCl (NMDG HCl) affected neither the apparent inhibition constant (Kl) for the amiloride-induced current inhibition nor the apparent fraction of the transmembrane voltage that falls between the mucosal solution and the amiloride-binding site (delta). When NMDG+ was replaced with Na+, Kl increased, reflecting amiloride-Na+ competition, whereas delta was unchanged. Similar results were obtained with another permeant cation, Li+. When NMDG+ was replaced by K+, an impermeant but channel-blocking cation, Kl increased whereas delta decreased. Similar results were obtained using another impermeant, channel-blocking cation guanidinium. The results are interpreted on the premise that Na+ and K+ compete with amiloride by binding to cation binding sites within the channel lumen such that ion occupancy of these sites vary with voltage. Occupancy by K+, which cannot traverse the channel, will increase as the mucosal solution becomes positive, relative to the serosal solution. Occupancy by Na+, which can traverse the channel, is comparatively voltage independent. Ion movement through the channels was simulated using discrete-state kinetic models. Two types of models could describe the shape of the current-voltage relationship and the voltage dependence of the amiloride-induced channel block. One model had a single ion-binding site with a broad energy barrier at the inner (cytoplasmic) side of the site. The other model had two binding sites separated from each other and from the aqueous solutions by sharp energy barriers.     

A bar model for the pump and channel function of the reconstituted Na+,K+-ATPase

Purified Na+,K+-ATPase is treated with trypsin. The altered enzyme is then reconstituted into liposomes and the change in active and passive Na+,K+-fluxes is recorded. Trypsin treatment transforms the slow passive Na+,K+-fluxes into leaks. The leak formation is correlated with the degree of proteolysis and the associated decrease in Na+,K+-ATPase activity. The active Na+,K+-transport capacity decreases in parallel with the passive transport. It is thus proposed that the Na+,K+-ATPase molecule primarily contains unspecific transmembrane tunnels that are rendered ion-selective by transverse bars of specific length (bar model).