Ionic permeability of K, Na, and Cl in crayfish nerve. Regulation by membrane fixed charges and pH.

Teorell's fixed charge theory for membrane ion permeability was utilized to calculate specific ionic permeabilities from measurements of membrane potential, conductance, and specific ionic transference numbers. The results were compared with the passive ionic conductances calculated from the branched equivalent circuit membrane model of Hodgkin Huxley. Ionic permeabilities for potassium, sodium, and chloride of crayfish (Procambarus clarkii) medial giant axons were examined over an external pH range from 3.8 to 11.4. Action potentials were obtained over this pH range. Failures occurred below pH 3.8 during protonation of membrane phospholipid phosphate and carboxyl, and above pH 11.4 from calcium precipitation. In general, chloride permeability increases with membrane protonation, while cation permeability decreases. At pH 7.0, PK = 1.33 X 10(-5), PCl = 1.49 X 10(-6), PNa = 1.92 X 10(-8) cm/s. PK: PCl: PNa = 693:78:1. PCl is zero above pH 10.6 and is opened predominately by protonation of epsilon-amino, and partially by tyrosine and sulfhydryl groups from pH 10.6 to 9. PK is activated in part by ionization of phospholipid phosphate and carboxyl around pH 4, then further by imidazole from pH 5 to 7, and then predominately from pH 7 to 9 by most probably phosphatidic acid. PNa permeability parallels that of potassium from pH 5 to 9.4. Below pH 5 and above pH 9.4, PNa increases while PK decreases. Evidence was obtained that these ions possibly share common passive permeable channels. The data best support the theory of Teorell, that membrane fixed charges regulate permiability and that essentially every membrane ionizable group appears involved in various amounts in ionic permeability control.     

Sodium and potassium fluxes and membrane potential of human neutrophils: evidence for an electrogenic sodium pump

Sodium and potassium ion contents and fluxes of isolated resting human peripheral polymorphonuclear leukocytes were measured. In cells kept at 37 degrees C, [Na]i was 25 mM and [K]i was 120 mM; both ions were completely exchangeable with extracellular isotopes. One-way Na and K fluxes, measured with 22Na and 42K, were all approximately 0.9 meq/liter cell water . min. Ouabain had no effect on Na influx or K efflux, but inhibited 95 +/- 7% of Na efflux and 63% of K influx. Cells kept at 0 degree C gained sodium in exchange for potassium ([Na]i nearly tripled in 3 h); upon rewarming, ouabain-sensitive K influx into such cells was strongly enhanced. External K stimulated Na efflux (Km approximately 1.5 mM in 140-mM Na medium). The PNa/PK permeability ratio, estimated from ouabain insensitive fluxes, was 0.10. Valinomycin (1 microM) approximately doubled PK. Membrane potential (Vm) was estimated using the potentiometric indicator diS-C3(5); calibration was based on the assumption of constant-field behavior. External K, but not Cl, affected Vm. Ouabain caused a depolarization whose magnitude dependent on [Na]i. Sodium-depleted cells became hyperpolarized when exposed to the neutral exchange carrier monensin; this hyperpolarization was abolished by ouabain. We conclude that the sodium pump of human peripheral neutrophils is electrogenic, and that the size of the pump-induced hyperpolarization is consistent with the membrane conductance (3.7-4.0 microseconds/cm2) computed from the individual K and Na conductances.     

Activation of K+ and Cl− channels in MDCK cells during volume regulation in hypotonic media

Single-channel patch-clamp experiments were performed on MDCK cells in order to characterize the ionic channels participating in regulatory volume decrease (RVD). Subconfluent layers of cultured cells were exposed to a hypotonic medium (150 mOsm), and the membrane currents at the single-channel level were measured in cell-attached experiments. The results indicate that MDCK cells respond to a hypotonic swelling by activating several different ionic conductances. In particular, a potassium and a chloride channel appeared in the recordings more frequently than other channels, and this allowed a more detailed study of their properties in the inside-out configuration of the patch-clamp technique. The potassium channel had a linear I/V curve with a unitary conductance of 24 +/- 4 pS in symmetrical K+ concentrations (145 mM). It was highly selective for K+ ions vs. Na+ ions: PNa/PK less than 0.04. The time course of its open probability (P0) showed that the cells responded to the hypotonic shock with a rapid activation of this channel. This state of high activity was maintained during the first minute of hypotonicity. The chloride channel participating in RVD was an outward-rectifying channel: outward slope conductance of 63.3 +/- 4.7 pS and inward slope conductance of 26.1 +/- 4.9 pS. It was permeable to both Cl- and NO3- and its maximal activation after the hypotonic shock was reached after several seconds (between 30 and 100 sec). The activity of this anionic channel did not depend on cytoplasmic calcium concentration. Quinine acted as a rapid blocker of both channels when applied to the cytoplasmic side of the membrane. In both cases, 1 mM quinine reversibly reduced single-channel current amplitudes by 20 to 30%. These results indicate that MDCK cells responded to a hypotonic swelling by an early activation of highly selective potassium conductances and a delayed activation of anionic conductances. These data are in good agreement with the changes of membrane potential measured during RVD.     

Sodium channel selectivity. Dependence on internal permeant ion concentration

The selectivity of sodium channels in squid axon membranes was investigated with widely varying concentrations of internal ions. The selectivity ratio, PNa/PK, determined from reversal potentials decreases from 12.8 to 5.7 to 3.5 as the concentration of internal potassium is reduced from 530 to 180 to 50 mM, respectively. The internal KF perfusion medium can be diluted by tetramethylammonium (TMA), Tris, or sucrose solutions with the same decrease in PNa/PK. The changes in the selectivity ratio depend upon internal permeant ion concentration rather than ionic strength, membrane potential, or chloride permeability. Lowering the internal concentration of cesium, rubidium, guanidnium, or ammonium also reduces PNa/Pion. The selective sequence of the sodium channel is: Na greater than guanidinium greater than ammonium greater than K greater than Rb greater than Cs.     

Ionizable groups and conductances of the rod photoreceptor membrane

The ionizable groups and conductances of the rod plasma membrane were studied by measuring membrane potential and input impedance with micropipettes that were placed in the rod outer segments. Reduction of the pH from 8.0 to 6.8 or from 7.8 to 7.3 resulted in membrane depolarization in the dark from 8.0 to 6.8 or from 7.8 to 7.3 resulted in membrane depolarization in the dark (by 2- 3 mV) and an increased size of the light response (also by 2-3 mV). The dark depolarization was accompanied by and increased resting input impedance (by 11-35 Mω). When the pH was decreased in a perfusate in which Cl(-) was replaced by isethionate, the membrane depolarized. When the pH was decreased in a perfusate in which Na(+) was replaced by choline, an increase of input impedance was observed (11-50 Mω) even though a depolarization did not occur. These results are consistent with the interpretation that the effects of decreased extracellular pH result mainly from a decrease in rod membrane K(+) conductance that is presumably cause by protonation of ionizable groups having a pK(a) between 7.3 and 7.8. Furthermore, from these results and results obtained by using CO(2) and NH(3) to affect specifically the internal pH of the cell, it seems unlikely that altered cytoplasmic [H(+)] is a cytoplasmic messenger for excitation of the rod. When the rods were exposed to perfusate in which Na(+) was replaced by choline, the resting (dark) input impedance increased (by 26 Mω +/- 5 Mω SE), and the light-induced changes in input impedance became undetectable. Replacement of Cl(-) by isethionate had no detectable effect on either the resting input impedance or the light-induced changes in input impedance. These results confirm previous findings that the primary effect of light is to decrease the membrane conductance to Na(+) and show that, if any other changes in conductance occur, they depend upon the change in Na(+) conductance. The results are consistent with the following relative resting conductances of the rod membrane: G(Na(+)) similar to G(K(+)) more than 2-5 G(Cl(-)).     

Atrioventricular block in low potassium and K dependence of the nodal resting potential

A progressive conduction block leading to atrioventricular dissociation develops in perfused rabbit hearts within 20-30 min of exposure to Krebs containing 0.5 mM potassium (low K). A decrease in potassium permeability resulting in membrane depolarization (as seen in Purkinje fibers) could be responsible for the loss of excitability in nodal cells. We investigated the K dependence of the resting potential and the long-term effects of low K perfusion on the resting and action potentials of nodal cells in rabbit hearts. The resting potential of atrial, atrionodal, and nodal cells varied by 52, 41, and 34 mV per decade of change in Ko within the range of 5-50 mM K. Hyperpolarization of the resting membrane, a progressive decline in action potential amplitude, and a decrease in maximum rate of rise were observed in nodal fibers when exposed to low K. Loss of propagated activity occurred in the middle node within 20-30 min while the cells remained hyperpolarized. There was no evidence of electrogenic Na extrusion and it seems that the low nodal resting potential results from a high resting PNa/PK permeability ratio. The early decrease in rate of rise in low K probably reflects an increase in K-dependent outward currents, whereas the progressive deterioration and final loss of conducted electrical activity may result from an accumulation of internal Na and Ca overload produced by low K inhibition of the Na pump.     

A large conductance Cl- channel revealed by patch-recordings in human fibroblasts

A Cl- channel with large single-unit conductance and characteristic voltage-dependent inactivation was studied on cultured human fibroblasts. The channel was activated only after excision and lasting depolarization of the membrane patch. In inside-out configuration and in symmetrical 135 mM NaCl, the conductance was 300 pS. The channel was usually open at the membrane potentials between -20 to +20 mV, while more negative or positive voltages closed the channel. The time course of this apparent inactivation process was dependent on increasing potential. Recovery from inactivation was made possible by returning the membrane potential to 0 mV. The channel was selective to Cl- over Na+ with a PCl/PNa of 6. The order of permeability among anions was: I greater than Br = Cl greater than isethionate greater than F greater than glutamate. The channel was blocked by internal application of a derivative of the diphenylamine-2-carboxilate (Blocker 144) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.     

The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

Electrophysiology of flounder intestinal mucosa. I. Conductance properties of the cellular and paracellular pathways

We evaluated the conductances for ion flow across the cellular and paracellular pathways of flounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels. An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (psi a) and, at [K]m less than 40 mM, the K dependence of psi a was abolished by 1-2 mM mucosal Ba. The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (psi b) with reduced serosal Cl concentrations, [Cl]s. psi b was unaffected by changes in [K]s or [Na]s. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (Gp) normally accounts for 96% of transepithelial conductance (Gt). The high Gp attenuates the contribution of the cellular pathway to psi t while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes. Thus, psi a and psi b (approximately 60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membrane. Equivalent circuit analysis suggests that apical conductance (Ga approximately equal to 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb approximately equal to 1.5 mS/cm2) would account for only 50% of net Cl absorption. This, together with our failure to detect a basolateral K conductance, suggests that Cl absorption across this barrier involves KCl co-transport.     

Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma

Membrane potentials and conductances, and intracellular ionic activities were studied in isolated perfused collecting tubules of K+-adapted Amphiuma. Intracellular Na+ (aNai) and K+ (aKi) activities were measured, using liquid ion-exchanger double-barreled microelectrodes. Apical and basolateral membrane conductances were estimated by cable analysis. The effects of inhibition of the apical conductance by amiloride (10(-5) M) and of inhibition of the basolateral Na-K pump by either a low K+ (0.1 mM) bath or by ouabain (10(-4) M) were studied. Under control conditions, aNai was 8.4 +/- 1.9 mM and aKi 56 +/- 3 mM. With luminal amiloride, aNai decreased to 2.2 +/- 0.4 mM and aKi increased to 66 +/- 3 mM. Ouabain produced an increase of aNai to 44 +/- 4 mM, and a decrease of aKi to 22 +/- 6, and similar changes were observed when the tubule was exposed to a low K+ bath solution. During pump inhibition, there was a progressive decrease of the K+-selective basolateral membrane conductance and of the Na+ permeability of the apical membrane. A similar inhibition of both membrane conductances was observed after pump inhibition by low K+ solution. Upon reintroduction of K+, a basolateral membrane hyperpolarization of -23 +/- 4 mV was observed, indicating an immediate reactivation of the electrogenic Na-K pump. However, the recovery of the membrane conductances occurred over a slower time course. These data imply that both membrane conductances are regulated according to the intracellular ionic composition, but that the basolateral K+ conductance is not directly linked to the pump activity.     

Transmembrane K+, Na+, and Cl- permeability and conductance changes in the frog sartorius fibres following ouabain and zero [K+]o treatment

Changes in the K+, Na+, and Cl- permeabilities (P) and conductances (g) of the intact frog sartorius fibre membrane following ouabain or zero [K+]o treatment were calculated from intrafibre activity and whole muscle electrolyte changes. Conventional equations relating ionic fluxes to resting potential (E), ionic gradient potential, and internal and external ionic activities were used. Both treatments produced a three- to five-fold increase in PNa and gNa. In addition, ouabain produced a fivefold increase in PK (and gK) and a small decrease in PCl (and gCl), whereas zero [K+]o produced a 60% reduction in PK, a 90% reduction in gK, and a threefold increase in PCl (and gCl). When the two treatments were combined, the P and g changes were paradoxical, suggesting that the ouabain-induced increase in gK and the zero [K+]o-induced decrease in gK were occurring but in different channels (or carriers). During ouabain treatment, E reflects mainly the transmembrane K+ gradient potential; during zero [K+]o treatment, E reflects mainly the Cl- gradient potential. Despite channel (or carrier) specificity, it appears that all three ionic permeabilities are altered during the perturbations.     

Model of ion transport regulation in chloride-secreting airway epithelial cells. Integrated description of electrical, chemical, and fluorescence measurements.

An electrokinetic model was developed to calculate the time course of electrical parameters, ion fluxes, and intracellular ion activities for experiments performed in airway epithelial cells. Model variables included cell [Na], [K], [Cl], volume, and membrane potentials. The model contained apical membrane Cl, Na, and K conductances, basolateral membrane K conductance, Na/K/2 Cl and Na/Cl symport, and 3 Na/2 K ATPase, and a paracellular conductance. Transporter permeabilities and ion saturabilities were determined from reported ion flux data and membrane potentials in intact canine trachea. Without additional assumptions, the model predicted accurately the measured short-circuit current (Isc), cellular conductances, voltage-divider ratios, open-circuit potentials, and the time course of cell ion composition in ion substitution experiments. The model was used to examine quantitatively: (a) the effect of transport inhibitors on Isc and membrane potentials, (b) the dual role of apical Cl and basolateral K conductance in cell secretion, (c) whether the basolateral symporter requires K, and (d) the regulation of apical Cl conductance by cAMP and Ca-dependent signaling pathways. Model predictions gave improved understanding of the interrelations among transporting systems and in many cases gave surprising predictions that were not obvious without a detailed model. The model developed here has direct application to secretory or absorptive epithelial cells in the kidney thick ascending limb, cornea, sweat duct, and intestine in normal and pathophysiological states such as cystic fibrosis and cholera.     

Potassium channels in primary cultures of seawater fish gill cells. I. Stretch-activated K(+) channels

Previous studies using the patch-clamp technique demonstrated the presence of a small conductance Cl(-) channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A K(+) channel of 123 +/- 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for K(+) over Na(+) and Ca(2+). The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 microM), and gadolinium (500 microM). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on K(+) channel activity is consistent with the hypothesis that stretch-activated K(+) channels could mediate an increase in K(+) conductance during cell swelling.     

Is the K permeability of the resting membrane controlled by the excitable K channel?

To test whether or not the potassium permeability of the resting membrane is controlled by the excitable K channels (delayed rectifier), we examined changes in the Na and K permeability ratio, PNa/PK, of the squid axon before and after the excitable K channels were blocked. The blockage of the K channels was accomplished by three independent methods: internal application of tetraethylammonium, internal application of 4-aminopyridine plus Cs, and prolong internal perfusion of NaF solution. The permeability ratio was determined using two different methods: the conventional electrophysiological method and a new method based on the measurements of the hyperpolarizing effect of Na removal. We found that blocking the K channels did not cause a proportional decrease in the K permeability of the resting membrane, suggesting that the semipermeable property of the resting membrane is not determined by the excitable K channels.     

Substates of cardiac sodium channels are due to both decrease in conductance and changes in selectivity.

Currents through DPI 201-106 modified single cardiac sodium channels in guinea pig ventricular cells were measured using the patch clamp technique in the cell-free configuration to control the sodium concentrations on both sides of the patch membrane. Current-voltage relationships of the single channels were obtained by application of linear voltage ramps from -140 to 100 mV. With 10 mmol/l Na+ at the inner surface of the patch, openings of sodium channels with conductances of 17 pS (selectivity ratios PK/PNa = 0.083 and PK/PNa = 0.58) and 12 pS (selectivity ratios PK/PNa = 0.084 and PK/PNa = 1.832) were obtained. With 30 mmol/l internal sodium, conductances of 20, 10, and 7 pS and selectivity ratios of 0.084, 0.386, and 0.543, respectively, could be measured. It is concluded that substates of sodium channel currents are due to changes in single channel conductance as well as in selectivity, or to changes of both independently of each other which accounts for the variability of conductance levels of cardiac Na channels.     

A patch-clamp study of histamine-secreting cells

The ionic conductances in rat basophilic leukemia cells (RBL-2H3) and rat peritoneal mast cells were investigated using the patch-clamp technique. These two cell types were found to have different electrophysiological properties in the resting state. The only significant conductance of RBL-2H3 cells was a K+-selective inward rectifier. The single channel conductance at room temperature increased from 2-3 pS at 2.8 mM external K+ to 26 pS at 130 mM K+. This conductance, which appeared to determine the resting potential, could be blocked by Na+ and Ba2+ in a voltage-dependent manner. Rat peritoneal mast cells had a whole-cell conductance of only 10-30 pS, and the resting potential was close to zero. Sometimes discrete openings of channels were observed in the whole-cell configuration. When the Ca2+ concentration on the cytoplasmic side of the membrane was elevated, two types of channels with poor ion specificity appeared. A cation channel, observed at a Ca2+ concentration of approximately 1 microM, had a unit conductance of 30 pS. The other channel, activated at several hundred micromolar Ca2+, was anion selective and had a unit conductance of approximately 380 pS in normal Ringer solution and a bell-shaped voltage dependence. Antigenic stimulation did not cause significant changes in the ionic conductances in either cell type, which suggests that these cells use a mechanism different from ionic currents in stimulus-secretion coupling.     

Ion conduction in substates of the batrachotoxin-modified Na+ channel from toad skeletal muscle.

Batrachotoxin-modified Na+ channels from toad muscle were inserted into planar lipid bilayers composed of neutral phospholipids. Single-channel conductances were measured for [Na+] ranging between 0.4 mM and 3 M. When membrane preparations were made in the absence of protease inhibitors, two open conductance states were identified: a fully open state (16.6 pS in 200 mM symmetrical NaCl) and a substate that was 71% of the full conductance. The substate was predominant at [Na+] > 65 mM, whereas the presence of the fully open state was predominant at [Na+] < 15 mM. Addition of protease inhibitors during membrane preparation stabilized the fully open state over the full range of [Na+] studied. In symmetrical Na+ solutions and in biionic conditions, the ratio of amplitudes remained constant and the two open states exhibited the same permeability ratios of PLi/PNa and PCs/PNa. The current-voltage relations for both states showed inward rectification only at [Na+] < 10 mM, suggesting the presence of asymmetric negative charge densities at both channel entrances, with higher charge density in the external side. An energy barrier profile that includes double ion occupancy and asymmetric charge densities at the channel entrances was required to fit the conductance-[Na+] relations and to account for the rectification seen at low [Na+]. Energy barrier profiles differing only in the energy peaks can give account of the differences between both conductance states. Estimation of the surface charge density at the channel entrances is very dependent on the ion occupancy used and the range of [Na+] tested. Independent evidence for the existence of a charged external vestibule was obtained at low external [Na+] by identical reduction of the outward current induced by micromolar additions of Mg2+ and Ba2+.     

Identification of a stretch-activated monovalent cation channel from teleost urinary bladder cells.

The urinary bladder of euryhaline teleost is an important osmoregulatory organ which absorbs Na+, Cl-, and water from urine. Using patch clamp technique, single stretch-activated channels, which were permeable to K+ and Na+ (PNa/PK approximately 0.75) and had conductances of 55 and 116 pS, were studied. In excised, inside-out patches which were voltage-clamped in the physiological range of membrane potential, the single-channel open probability (Po) was low (approximately 0.02), and increased to a maximum of 0.9 with applied pipette suction. Single-channel conductance also increased with suction. The channels showed adaptation to applied suction and relaxed to a steady-state activity about 20 seconds after application of suction. The Po increased up to 0.9 with strong membrane depolarization (Vm = 0 to +80 mV); however, there was little dependence of Po on membrane potential in the physiological range. The kinetic data suggest that there is one conducting state and at least two non-conducting states of the channel. The open-time constant increased with suction but remained unchanged with membrane potential (Vm = -70 to +60 mV). The mean closed-time of the channel decreased with suction and membrane depolarization. These results demonstrate the presence of a non-selective monovalent cation channel which may be involved in cell volume regulation in the goby urinary bladder. Additionally, this channel may function as an enhancer of Na+ influx and K+ efflux across the bladder cell as part of transepithelial ion transport if it is located in apical membrane.     

Electrical properties of the cellular transepithelial pathway in Necturus gallbladder. II. Ionic permeability of the apical cell membrane.

Microelectrode techniques were employed to study the ionic permeability of the apical cell membrane of Necturus gallbladder epithelium. Results obtained from continuous records in single cells, and from several cellular impalements shortly after a change in solution, were similar and indicate that both the apical membrane equivalent electromotive force (Va) and electrical resistance (Ra) strongly depend on external [K]. Cl substitutions produced smaller effects, while the effects of Na substitutions with N-methyl-D-glucamine on both Va and Ra were minimal. These results indicate that the permeability sequence of the apical membrane is PKgreater thanPClgreater than PNa. From the calculated absolute value of PNa it is possible to estimate the diffusional Na flux from the mucosal solution into the cells (from the cell potential and an assumed intracellular Na concentration). The calculated flux is roughly three orders of magnitude smaller than the measured net transepithelial flux in this tissue and in gallbladders of other species. Thus, only a minimal portion of Na entry can be attributed to independent diffusion. From estimations of the electrochemical potential gradient across the apical membrane, Cl transport at that site must be active. At the serosal cell membrane, Na transport takes place against both chemical and electrical potentials, while a significant portion of the Cl flux can be passive, if this membrane has a significant Cl conductance. The changes in shunt electromotive force and in transepithelial potential after mucosal substitutions were very similar, indicating that transepithelial bi-ionic potentials yield appropriate results on the properties of shunt pathway.     

Ion transport and permeability in the mouse egg

Sodium, potassium, and chloride unidirectional fluxes have been studied in the mature mouse egg. Their relationship to cell membrane potential and conductance has been investigated. Unidirectional Na efflux is composed of a ouabain sensitive component, presumably representing an active Na efflux, an external Na-dependent component and a diffusional component. The data indicate that the external Na-dependent component represents a Na:Na exchange mechanism. There also exists an ouabain-sensitive component of K influx. The stoichiometry of the ouabain-sensitive fluxes is approx. 2.7:1 (Na to K). From the diffusional components of Na and K flux, the membrane permeability to these cations has been estimated. P_Na and P_K are 1.2 × 10⁻⁷ cm sec⁻¹ and 0.8 × 10⁻⁷ cm sec⁻¹ respectively. These permeabilities, in conjunction with the internal exchangeable fractions of Na and K and the external concentrations, predict an egg membrane potential of −11 mV (inside negative). Microelectrode measurements yield an egg membrane potential of −14 ± 0.4 mV, indicating that the cell membrane potential is predominantly a result of the Na and K permeabilities and distributions. Internal exchangeable Cl is 67 ± 3 mM in standard medium, as determined from ³⁶Cl distribution. The chloride equilibrium potential is therefore −15 mV, which is not significantly different from the egg membrane potential. This suggests that Cl distributes passively across the egg membrane, reflecting the egg membrane potential. Hyperpolarization of the egg membrane potential to −27 ± 1.5 mV by reduction of external Na results in an exchangeable internal Cl of 49 ± 8 mM. This yields a Cl equilibrium potential of −24 mV, indicating that the Cl distribution shifts in the predicted manner upon a change in cell membrane potential. Tracer flux data indicate that Cl conductance comprises the bulk of the total membrane conductance with Na and K sharing the remainder in approximately equal amounts.