Simultaneous measurement of Ca2+ influx and reversal potentials in recombinant N-methyl-D-aspartate receptor channels.

The Ca(2+) permeability of N-methyl-D-aspartate receptor (NMDA-R) channels was studied in human embryonic kidney cells transfected with the NR1-NR2A subunit combination. To determine the fractional Ca(2+) current (P(f)), measurements of fura-2-based Ca(2+) influx and whole-cell currents were made in symmetrical monovalent ion concentrations at membrane potentials between -50 mV and the reversal potential. The ratios of Ca(2+) flux over net whole-cell charge at 2, 5, and 10 mM external Ca(2+) concentrations ([Ca](o)) were identical at a membrane potential close to the reversal potential of the monovalent current component. Assuming unity of P(f) at this potential, the percentage of current carried by Ca(2+) was found to be 18.5 +/- 1.3% at 2 mM [Ca](o) and -50 mV. This value, which is higher than the ones reported previously, was confirmed in independent experiments in which a pure flux of Ca(2+) through NMDA-R channels was used to calibrate the Ca(2+) influx signals. The measured values of fractional Ca(2+) currents, which agree with the predictions of the Goldman-Hodgkin-Katz equations, are also compatible with a two-barrier model for ion permeation, in which the differences between the energy barriers for Ca(2+) and monovalent ions are similar on the external and internal membrane sides.     

Cooperative phenomena in the membrane potential of parathyroid cells induced by divalent cations

Membrane potentials of mouse parathyroid cells were measured by means of the intracellular microelectrode method. The membrane potential in external Krebs solution containing 2.5 mM of Ca++ was -23.6 +/- 0.4 mV (mean +/- standard error of mean). The low concentration of Ca++ (1.0 mM) caused hyperpolarization of the membrane potential to -61.7 +/- 0.8 mV. The membrane potential was proportional to the logarithm of the concentration of K ion in the solution of low Ca ion. The concentration of external Na+, C1- and HPO4-- had no effect on the membrane potential. The sigmoidal transition of membrane potentials was induced by the change of Ca ion concentration in the range from 2.5 to 1.0 mM. The change of the membrane potentials in low Ca ion is originated from increase in potassium permeability of the cell membrane. The similar sigmoidal changes of the membrane potentials were observed in the solution containing 4 to 3 mM of Sr ion. The Mg and Ba ion showed smaller effect on the membrane potential. The Goldman equation was extended to divalent ions. Appling the extended membrane potential equation, ratios of the permeability coefficients were obtained as follows: PK/PCa = 0.067 for 2.5 mM Ca++, 0.33 for 1.0 mM Ca++; PK/PSr = 0.08 for 4 mM Sr++ and 0.4 for 3 mM Sr++; PK/PMg = 0.5; PK/PBa = 0.67 for all range of concentration. The Hill constants of Sr ion and Ca ion were 20; the relationship between Sr ion and Ca ion was competitive. The Hill constants of Mg and Ba ion were 1 each. The Hill constant of Ca ion was depend of the temperature; nmax = 20 at 36 degrees C, n = 9 at 27 degrees C, n = 2 at 22 degrees C. The enthalpy of Ca-binding reaction was obtained from the Van't Hoff plot as 0.58 kcal. The activation energies of the K+ permeability increase were obtained from the Arrhenius plots as 3.3 kcal and 4 kcal. The difference, 0.7 kcal, corresponds to the enthalpy change of this reaction, of which value is close to that of the Ca-binding reaction.     

The proton electrochemical gradient in Escherichia coli cells.

The internal pH of Escherichia coli cells was estimated from the distribution of either 5,5-[14C]dimethyl-2,4-oxazolidinedione or [14C]methylamine. EDTA/valinomycin treatment of cells was employed to estimate delta psi from 86Rb+ distribution concomitant with the delta pH for calculation of delta muH. Respiring intact cells maintained an internal pH more alkaline by 0.63-0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The delta pH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN. The delta muH, established by EDTA/valinomycin-treated cells, was constant (122-129 mV) over extracellular potassium concentration of 0.01 mM-1 mM. At the lower potassium concentration delta psi (110-120 mV) was the predominant component, and at the higher concentration delta pH increased to 0.7 units (42 mV). At 150 mM potassium delta muH was reduced to 70 mV mostly due to a delta pH component of 0.89 (53 mV). The interchangeability of the delta muH components is consistent with an electronic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of delta muH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone. The highest delta pH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the delta pH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of delta pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the delta psi was almost constant over the whole range of extracellular pH values (6-8) implying electroneutral proton movement. Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required.     

Permeation of both cations and anions through a single class of ATP-activated ion channels in developing chick skeletal muscle.

Micromolar concentrations of extracellular adenosine 5'-triphosphate (ATP) elicit a rapid excitatory response in developing chick skeletal muscle. Excitation is the result of a simultaneous increase in membrane permeability to sodium, potassium, and chloride ions. In the present study we quantify the selectivity of the ATP response, and provide evidence that a single class of ATP-activated ion channels conducts both cations and anions. Experiments were performed on myoballs using the whole-cell patch-clamp technique. We estimated permeability ratios by measuring the shift in reversal potential when one ion was substituted for another. We found that monovalent cations, divalent cations, and monovalent anions all permeate the membrane during the ATP response, and that there was only moderate selectivity between many of these ions. Calcium was the most permeant ion tested. To determine if ATP activates a single class of channels that conducts both cations and anions, or if ATP activates separate classes of cation and anion channels, we analyzed the fluctuations about the mean current induced by ATP. Ionic conditions were arranged so that the reversal potential for cations was +50 mV and the reversal potential for anions was -50 mV. Under these conditions, if ATP activates a single class of channels, ATP should not evoke an increase in noise at the reversal potential of the ATP current. However, if ATP activates separate classes of cation and anion channels, ATP should evoke a significant increase in noise at the reversal potential of the ATP current. At both +40 and -50 mV ATP elicited a clear increase in noise, but at the reversal potential of the ATP current (-5 mV), no increase in noise above background was seen. These results indicate that there is only a single class of excitatory ATP-activated channels, which do not select by charge. Based on analysis of the noise spectrum, the conductance of individual channels is estimated to be 0.2-0.4 pS.     

Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells

Ion channels, activated upon IgE-Fc epsilon receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc epsilon receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc epsilon receptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 microM Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 +/- 11 mV in the presence of sodium, 125 +/- 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological identification of cell types in cortical collecting duct monolayers.

Double-barrel microelectrodes were used to determine membrane voltages and the intracellular pH (pHi) in primary cultures of cortical collecting duct cells (CCD) grown in the absence of aldosterone. Electrophysiologically, two main cell types were identified. In cell type 1, the apical membrane voltage (Va) was -60 +/- 5 mV. The fractional resistance of the apical membrane (fRa) was 0.40 +/- 0.03, and pHi was 7.21 +/- 0.04. Exposure to 50 mM K+ on the apical side depolarized Va by 21 +/- 4 mV. When Cl- was replaced by cyclamate two types of responses were observed: (a) depolarization of Va by 26 +/- 3 mV while pHi remained unchanged, and (b) no change in Va. In cell type 2, Va was -36 +/- 5 mV, fRa was 0.91 +/- 0.03 and increasing apical [K+] from 5 to 50 mM did not change Va. Two subpopulations were distinguished by the response of pHi to lowering apical [Cl-]. In one of them pHi increased from 6.99 +/- 0.05 to 7.11 +/- 0.07. In the other, pHi was significantly decreased from 7.16 +/- 0.08 to 7.03 +/- 0.07. These results are compatible with the conclusion that about 50% of the impaled cells type 2 have a Cl-/HCO-3 exchanger at the apical membrane. In summary, two different cell types can be identified electrophysiologically in CCD monolayers. Cell type 1 has the electrical characteristics of principal cells. Cell type 2 resembles the intercalated cells. The cell alkalinization observed in approximately 50% of the cells type 2 in response to Cl- removal suggests the presence of an apical Cl-/HCO-3 exchanger. Thus, these cells should be the bicarbonate-secreting cells. The remaining cells should correspond to the acid-secreting cells.     

Potassium transport system of Rhodopseudomonas capsulata

Rhodopseudomonas capsulata required potassium (or rubidium or cesium as analogs of potassium) for growth. These cations were actively accumulated by the cells by a process following Michaelis-Menten saturation kinetics. The monovalent cation transport system had Km's of 0.2 mM K+, 0.5 mM Rb+, and 2.6 mM Cs+. The rates of uptake of substrates by the potassium transport system varied with the age of the culture, although the affinity constant for the substrates remained constant. The maximal velocity of uptake of K+ was lower in aerobically grown cells than in photosynthetically grown cells, although the Km's for K+ and for Rb+ were about the same.     

Internal Na+ and Mg2+ blockade of DRK1 (Kv2.1) potassium channels expressed in Xenopus oocytes. Inward rectification of a delayed rectifier

Delayed rectifier potassium channels were expressed in the membrane of Xenopus oocytes by injection of rat brain DRK1 (Kv2.1) cRNA, and currents were measured in cell-attached and inside-out patch configurations. In intact cells the current-voltage relationship displayed inward going rectification at potentials > +100 mV. Rectification was abolished by excision of membrane patches into solutions containing no Mg2+ or Na+ ions, but was restored by introducing Mg2+ or Na+ ions into the bath solution. At +50 mV, half- maximum blocking concentrations for Mg2+ and Na+ were 4.8 +/- 2.5 mM (n = 6) and 26 +/- 4 mM (n = 3) respectively. Increasing extracellular potassium concentration reduced the degree of rectification of intact cells. It is concluded that inward going rectification resulting from voltage-dependent block by internal cations can be observed with normally outwardly rectifying DRK1 channels.     

Oxonol dyes as monitors of membrane potential: the effect of viruses and toxins on the plasma membrane potential of animal cells in monolayer culture and in suspension

Optical indicators of the cationic, cyanine and anionic oxonol classes were used to evaluate the plasma membrane potential of animal cells in suspension and in monolayer culture. The optical signals were calibrated by using diffusion potentials either of K+ (in the presence of valinomycin) or of H+ (in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FCCP); both classes of dye gave similar values of plasma membrane potential, in the range -40 to -90 mV for different cell types. Addition of haemolytic Sendai virus or Staphylococcus aureus alpha-toxin depolarizes cells and causes them to leak monovalent cations; these effects are antagonized by extracellular Ca2+. Cells infected with vesicular stomatitis or Semliki Forest virus become depolarized during an infectious cycle; infection with other viruses was without affect on plasma membrane potential.     

Autolysis of Bacillus subtilis induced by low temperature

By exposure to a temperature below the membrane phase transition point, Bacillus subtilis 168 lost their permeability control followed by the leakage of intracellular K⁺ and incapability of glucose uptake, resulting in cellular lysis in the following incubation at 37°C in the presence of a high concentration (∼ 100 mM) of monovalent cations. The result confirmed that the concomitance of energy deprivation and the presence of monovalent cations were the factors that caused the lysis observed after low temperature exposure.     

Rubidium reduces potassium permeability and fluid secretion in Malpighian tubules of Locusta migratoria, L

The basal membrane potential (V(b)) of Locusta Malpighian tubule cells in control saline results from its relatively high permeability to potassium. In the presence of 1 mM barium added to the control saline V(b) hyperpolarized from a mean resting potential of -72.1 mV to -90.1 mV. On substituting rubidium for potassium in the control saline, V(b) also hyperpolarized to a value of -91.4 mV. Rubidium was also similarly effective in hyperpolarizing the basal membrane even in the presence of control concentrations of potassium in the bathing medium. Substitution of rubidium for potassium also effected a approximately 50% reduction in the rate of fluid secretion. The action of inhibitors on V(b) in the presence of rubidium showed that V(b) under these conditions probably originated from the bafilomycin-sensitive electrogenic potential generated across the apical membrane by a V-type ATPase. The responses of V(b) to potassium, barium and rubidium and their inhibition of fluid secretion suggest the presence of a substantial rubidium-blockable potassium conductance located on the basal membrane of Locusta Malpighian tubule cells.     

Transmembrane electrical characteristics of cultured human skeletal muscle cells

Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3-4 days, and fusion of myoblasts was observed in 5-10 days. Multinucleated myotubes were impaled under high optical magnification, at 37 degrees C, with conventional glass microelectrodes. The mean resting potential was -44.4 mV +/- 2.4 (n = 399); -33 +/- 2.3 mV at 9 days (n = 10) vs -48 +/- 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M omega (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 +/- 2.1 mV with a mean maximum rate of rise (Vmax) of 72.1 +/- 7.5 V/s. The mean overshoot was 13.9 +/- 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5-100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.     

p-Chloromercuribenzenesulfonic acid stimulation of chloride-dependent sodium and potassium transport in human red blood cells

The organic mercurial p-chloromercuribenzensulfonic acid (PCMBS) reversibly increases fluxes of sodium and potassium across the human red blood cell membrane. We examined the effect of different monovalent anions on cation fluxes stimulated by PCMBS. A substantial portion of the fluxes of both cations was found to have a specific anion requirement for chloride or bromide, and was not observed when chloride was replaced by nitrate, acetate or methylsulfate. The chloride-dependent component of the cation fluxes was only observed when the cells were exposed to PCMBS concentrations of 0.5 mM or greater. Furosemide (1 mM) did not inhibit the PCMBS-stimulated cation fluxes. The observed anion specificity is directly associated with the transport process rather than PCMBS binding to the membrane. A portion of the potassium transport stimulated by PCMBS appears to involve K+-K+ exchange; however, Na+ + K+ cotransport is not stimulated by this sulfhydryl reagent.     

The role of the membrane potential in chondrocyte volume regulation

Many cell types have significant negative resting membrane potentials (RMPs) resulting from the activity of potassium‐selective and chloride‐selective ion channels. In excitable cells, such as neurones, rapid changes in membrane permeability underlie the generation of action potentials. Chondrocytes have less negative RMPs and the role of the RMP is not clear. Here we examine the basis of the chondrocyte RMP and possible physiological benefits. We demonstrate that maintenance of the chondrocyte RMP involves gadolinium‐sensitive cation channels. Pharmacological inhibition of these channels causes the RMP to become more negative (100 µM gadolinium: ΔV_m = ?30 ± 4 mV). Analysis of the gadolinium‐sensitive conductance reveals a high permeability to calcium ions (PCa/PNa ≈80) with little selectivity between monovalent ions; similar to that reported elsewhere for TRPV5. Detection of TRPV5 by PCR and immunohistochemistry and the sensitivity of the RMP to the TRPV5 inhibitor econazole (ΔV_m = ?18 ± 3 mV) suggests that the RMP may be, in part, controlled by TRPV5. We investigated the physiological advantage of the relatively positive RMP using a mathematical model in which membrane stretch activates potassium channels allowing potassium efflux to oppose osmotic water uptake. At very negative RMP potassium efflux is negligible, but at more positive RMP it is sufficient to limit volume increase. In support of our model, cells clamped at ?80 mV and challenged with a reduced osmotic potential swelled approximately twice as much as cells at +10 mV. The positive RMP may be a protective adaptation that allows chondrocytes to respond to the dramatic osmotic changes, with minimal changes in cell volume. J. Cell. Physiol. 226: 2979–2986, 2011. © 2011 Wiley‐Liss, Inc.     

Transmembrane potential of isolated rat alveolar type II cells

Type II cells were isolated from rat lungs by elastase digestion and purified by centrifugal elutriation. The fluorescent dye, Di-S-C3(5), was used as a probe to monitor transmembrane potential (Em) of cells suspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered medium. With this technique, the Em of type II cells was estimated to be -27 +/- 2 mV. This resting Em is very close to the equilibrium potential for chloride (-21 mV), which suggests that chloride is passively distributed in type II cells. The resting Em of type II cells is more dependent on the extracellular concentration of potassium (K+) than on external sodium (Na+); i.e., the membrane depolarizes as external sodium is replaced by potassium, suggesting that in unstimulated type II cells the membrane is more permeable to potassium than to sodium. In addition, the resting potential appears to be due, in part, to the activity of a ouabain-sensitive, Na-K pump, which acts to hyperpolarize type II cells. Addition of a membrane perturbant, phorbol myristate acetate (PMA, 10 micrograms/ml), to a type II cell suspension results in an increase in oxygen consumption and membrane depolarization. Both of these responses are sodium dependent and thus appear to be linked to a PMA-induced increase in sodium permeability.     

Rubidium as a probe for function and transport of potassium in the yeast Candida utilis NCYC 321, grown in chemostat culture

Attempts to grow the yeast Candida utilis in continuous culture, using media in which all the potassium had been replaced by other monovalent cations, revealed that neither lithium, sodium, caesium nor ammonium ions could functionally substitute for potassium. However, potassium could be effectively replaced by rubidium which gave (on a molar basis, and under conditions where cation availability limited growth) the same yield of cells as did potassium.Comparison of potassium- and rubidium-limited cultures showed them to be virtually identical in all the measured parameters, with the single exception of the maximum growth rate value which was considerably decreased in the rubidium-containing culture (0.35 h^-1 as compared with 0.55 h^-1).When, with variously-limited chemostat cultures, both potassium and rubidium were supplied in equimolar amounts, these ions were taken up by the cells in a ratio that varied with both the growth rate and the nature of the growth limitation. With glucose-, phosphate- or magnesium-limited cultures, the molar ratio K⁺:Rb⁺ was 1:0.6 (at D=0.1 h^-1) and 1:0.17 (at D=0.5 h^-1). In contrast, ammonia-limited cultures took up increased amounts of rubidium when growing at a low rate such that the ratio was 1:1.2, at D=0.1 h^-1, though still 1:0.17 at the higher growth rate value (D=0.5 h^-1).From a comparison of glucose- and ammonialimited cultures growing first with an equimolar mixture of potassium and rubidium, and then with rubidium alone, it was noted that the yield on oxygen was significantly decreased when potassium was absent.These results are discussed in relation to the transport and possible functions of monovalent cations in micro-organisms. It was concluded that, on the basis of these experiments, some objections could be raised against estimation of potassium transport rates by means of the tracer ⁸⁶Rb.     

Unusual potassium channels mediate nonadrenergic noncholinergic nerve-mediated inhibition in opossum esophagus

Field stimulation of the circular muscle of the opossum esophagus produces a transient hyperpolarization (inhibitory junction potential, IJP) followed by an "off" depolarization. A similar nonadrenergic, noncholinergic (NANC) response in guinea pig taenia caecum has been shown to be due to an increase in the potassium ion permeability of the smooth muscle cell membrane. Double sucrose gap studies showed a decrease in resistance during the IJP, and a reversal at an estimated membrane potential of about -90 mV (4 mM K+). The reversal potential was dependent on the extracellular potassium concentration, shifting to -75 mV when the potassium in the superfusion medium was increased to 10 mM. The IJP in the opossum esophageal circular smooth muscle is therefore like the IJP of the guinea pig taenia caecum in that it is probably due to a selective increase in potassium ion permeability. Potassium conductance blocking agents, tetraethylammonium chloride (TEA, 20 mM) and 4-aminopyridine (4-AP, 5 mM) both caused a depolarization of the smooth muscle cell membrane, but TEA increased the membrane resistance, whereas 4-AP did not affect the membrane conductance in a consistent way. A decrease in IJP amplitude owing to these agents was not apparent. Apamin (10 microM) did not affect the membrane potential, the membrane resistance, or the IJP. Quinine (0.1 mM) produced effects quantitatively similar to those of TEA. Quinine (1 mM) did abolish the IJP, however, this was likely due to a blockade of impulse transmission of the intramural nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane potentials of BHK (baby hamster kidney) cell line: ionic and metabolic determinants

The transmembrane potential of cells from a continuous cell line (BHK-21) has been investigated by a combination of electrophysiological and flame photometric techniques. The ratio of sodium permeability to potassium permeability (P_Na/P_K) determined from membrane potentials recorded at varying external potassium concentrations was 0.082; from membrane potential measurements and the intracellular sodium and potassium concentrations of cells in 6.8 mM K⁺ media the value was 0.075. The P_Na/P_K ratio was not temperature dependent. Dinitrophenol (1 mM) did not significantly alter the membrane potential of cells incubated for one hour with the inhibitor. However, iodoacetate (1 mM) and sodium fluoride (30 mM) caused a significant depolarization during a one-hour incubation. Measurements of sodium and potassium concentrations during incubation at 4°C showed a decrease in internal potassium and an increase in internal sodium accompanied by a decreased membrane potential. Ion concentrations and membrane potentials were measured in cells recovering at 37°C following 24 hours at 4°C. Membrane potentials in excess of E_K during the first ten minutes of recovery may indicate electrogenic pumping.     

Monovalent selectivity of the cyclic guanosine monophosphate-activated ion channel

Monovalent cation selectivity has been characterized for the 3',5'-cyclic guanosine monophosphate (cGMP)-activated channel in vertebrate photoreceptor outer segment plasma membranes without divalent cations. Macroscopic currents in excised, inside-out patches were activated with saturating concentrations of cGMP (200 microM). Using a bi-ionic protocol with symmetrical 120 mM ion concentrations across the membrane, alkali metal ions and certain organic cations were substituted for sodium on the cytoplasmic face. The relative permeabilities, determined from shifts in the reversal potential (Erev), were NH4 much greater than Na greater than guanidinium greater than K greater than Li greater than Rb greater than Cs (3.34: 1.0: 0.97: 0.93: 0.92: 0.74: 0.50, respectively). Erev's were also measured as a function of [Na], [NH4], and [Cs], and the slope of the relation was -59.8, -52.1, and -49.1 mV/decade, respectively. The slopes for NH4 and Cs differ significantly from the Nernst-Planck prediction of -58.2 mV/decade expected for a single ion channel. Relative permeabilities were also determined for the alkali metal series of ions with 20 mM ionic concentrations on both sides of the membrane. The permeability sequence at 20 mM was unchanged, but the relative permeability for NH4 and Cs deviated significantly from the measurements at 120 mM with 1.46 and 0.75 ratios, respectively. The dependence of Erev on absolute concentrations and the deviation from Nernst-Planck predictions are best explained by multi-ion occupancy of the cGMP-activated channel. Selectivity was also examined by comparing the conductance ratios as a function of potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intra and extracellular surface charges near Ca2+ channels in neurons and neuroblastoma cells.

The properties of low (LVA) and high (HVA) voltage-activated calcium currents were investigated in rat sensory neurons and a murine neuroblastoma cell line exposed to various concentrations of intra- or extracellular monovalent ([c+]i/o) and trivalent ([c3+]i/o) cations. In neurons, when [c+]i was changed from 150 to 20 mM, positive shifts of 18-28 mV were observed in activation curves of both LVA and HVA currents, as well as in LVA inactivation curves. Extracellularly, in divalent-free solutions, [c+]o of 20-50 mM produced medium (12-22 mV) negative shifts of the LVA channel properties. These data were used to estimate, by a "screening" model, a negative surface charge density around neuron's calcium channels of 1/1,000 and 1/1,325 eA-2 at the outside or inside face, respectively. In the presence of physiological concentrations of divalent cations, [c+]o of 20-60 mM caused smaller (4-11 mV) negative shifts of the activation and inactivation curves, which can be explained by assuming a partial neutralization of negative charges by divalent cations. By applying the above procedure to LVA channels of neuroblastoma cells, the ratio of extra- to intracellular surface charge density turned out to be more than tenfold higher than in neurons. Effects produced by [c3+]i/o were not in agreement with expectations based on screening or binding models.