Mechanisms of Anion and Cation Permeations in the Resting Membrane of a Barnacle Muscle Fiber

The resting membrane of a barnacle muscle fiber is mostly permeable to cations in a solution of pH 7.7 whereas it becomes primarily permeable to anions if the pH is below 4.0. Mechanisms of ion permeation for various monovalent cations and anions were investigated at pH 7.7 and 3.9, respectively. Permeability ratios were obtained from the relationship between the membrane potential and the concentration of the test ions, and ionic conductances from current-voltage relations of the membrane. The permeability sequence for anions (SCN > I > NO₃ > Br > ClO₃ > Cl > BrO₃ > IO₃) was different from the conductance sequence for anions (Br, Cl > ClO₃, NO₃ > SCN). In contrast, the permeability and conductance sequences were identical for cations (K > Rb > Cs > Na > Li). The results suggest that anion permeation is governed by membrane charges while cation permeation is via some electrically neutral mechanism.     

Chloride fluxes in isolated dialyzed barnacle muscle fibers

Chloride outflux and influx has been studied in single isolated muscle fibers from the giant barnacle under constant internal composition by means of a dialysis perfusion technique. Membrane potential was continually recorded. The chloride outfluxes and influxes were 143 and 144 pmoles/cm²-sec (mean resting potential: 58 mv, temperature: 22°–24°C) with internal and external chloride concentrations of 30 and 541 mM, respectively. The chloride conductance calculated from tracer measurements using constant field assumptions is about fourfold greater than that calculated from published electrical data. Replacing 97% of the external chloride ions by propionate reduces the chloride efflux by 51%. Nitrate ions applied either to the internal or external surface of the membrane slows the chloride efflux. The external pH dependence of the chloride efflux follows the external pH dependence of the membrane conductance, in the range pH 3.9–4.7, increasing with decreasing pH. In the range pH 5–9, the chloride efflux increased with increasing pH, in a manner similar to that observed in frog muscle fibers. The titration curve for internal pH changes in the range 4.0–7.0 was quantitatively much different from that for external pH change, indicating significant asymmetry in the internal and external pH dependence of the chloride efflux.     

A thermodynamic study of electroneutral K-Cl cotransport in pH- and volume-clamped low K sheep erythrocytes with normal and low internal magnesium

Swelling-induced human erythrocyte K-Cl cotransport is membrane potential independent and capable of uphill transport. However, a complete thermodynamic analysis of basal and stimulated K-Cl cotransport, at constant cell volume, is missing. This study was performed in low K sheep red blood cells before and after reducing cellular free Mg into the nanomolar range with the divalent cation ionophore A23187 and a chelator, an intervention known to stimulate K- Cl cotransport. The anion exchange inhibitor 4,4''diisothiocyanato- 2,2''disulfonic stilbene was used to clamp intracellular pH and Cl or NO3 concentrations. Cell volume was maintained constant as external and internal pH differed by more than two units. K-Cl cotransport was calculated from the K effluxes and Rb (as K congener) influxes measured in Cl and NO3, at constant internal K and external anions, and variable concentrations of extracellular Rb and internal anions, respectively. The external Rb concentration at which net K-Cl cotransport is zero was defined as flux reversal point which changed with internal pH and hence Cl. Plots of the ratio of external Rb concentrations corresponding to the flux reversal points and the internal K concentration versus the ratio of the internal and external Cl concentrations (i.e., the Donnan ratio of the transported ions) yielded slopes near unity for both control and low internal Mg cells. Thus, basal as well as low internal Mg-stimulated net K-Cl cotransport depends on the electrochemical potential gradient of KCl.     

Properties of chloride transport in barnacle muscle fibers.

Unidirectional chloride-36 fluxes were measured in internally dialyzed barnacle giant muscle fibers. About 50--60% of the Cl efflux was irreversibly blocked by the amino-group reactive agent, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS), when it was applied either intra- or extracellularly. Similarly, Cl influx was also blocked by SITS. No significant effect on [Cl]i of SITS was noted in intact muscle fibers. However, the rate of net Cl efflux from muscle fibers which were Cl-loaded by overnight storage at 6 degrees C could be slowed by SITS treatment. Two classes of anions were defined based upon their effects on Cl efflux. Methanesulfonate and nitrate inhibited Cl efflux either when they replaced external chloride or when they were added to a constant external chloride concentration. The other group of anions (propionate, formate) stimulated both Cl efflux and influx and such stimulation could be blocked by SITS. Propionate influx was not nearly as large as the stimulated Cl efflux and was unaffected by SITS. Neither the effects of SITS nor those of the anion substitutes could be simply accounted for by changes in the membrane resting potential or conductance. These results suggest a mediated transport system for chloride across the barnacle sarcolemma.     

Ionic permeability of K, Na, and Cl in potassium-depolarized nerve. Dependency on pH, cooperative effects, and action of tetrodotoxin.

The passive ionic membrane conductances (gj) and permeabilities (Pj) of K, Na, and Cl of crayfish (Procambarus clarkii) medial giant axons were determined in the potassium-depolarized axon and compared with that of the resting axon. Passive ionic conductances and permeabilities were found to be potassium dependent with a major conductance transition occurring around an external K concentration of 12-15 mM (Vm = -60 to -65 mV). The results showed that K, Na, and Cl conductances increased by 6.2, 6.9, and 27-fold, respectively, when external K was elevated from 5.4 to 40 mM. Permeability measurements indicated that K changed minimally with K depolarization while Na and Cl underwent an order increase in permeability. In the resting axon (K0 = 5.4 mM, pH = 7.0) PK = 1.33 X 10(-5), PCl = 1.99 X 10(-6), PNa = 1.92 X 10(-8) while in elevated potassium (K0 = 40 mM, pH 7.0), PK = 1.9 X 10(-5), PCl = 1.2 X 10(-5), and PNa = 2.7 X 10(-7) cm/s. When membrane potential is reduced to 40 mV by changes in internal ions, the conductance changes are initially small. This suggests that resting channel conductances depend also on ion environments seen by each membrane surface in addition to membrane potential. In elevated potassium, K, Na, and Cl conductances and permeabilities were measured from pH 3.8 to 11 in 0.2 pH increments. Here a cooperative transition in membrane conductance or permeability occurs when pH is altered through the imidazole pK (approximately pH 6.3) region. This cooperative conductance transition involves changes in Na and Cl but not K permeabilities. A Hill coefficient n of near 4 was found for the cooperative conductance transition of both the Na and Cl ionic channel which could be interpreted as resulting from 4 protein molecules forming each of the Na and Cl ionic channels. Tetrodotoxin reduces the Hill coefficient n to near 2 for the Na channel but does not affect the Cl channel. In the resting or depolarized axon, crosslinking membrane amino groups with DIDS reduces Cl and Na permeability. Following potassium depolarization, buried amino groups appear to be uncovered. The data here suggest that potassium depolarization produces a membrane conformation change in these ionic permeability regulatory components. A model is proposed where membrane protein, which forms the membrane ionic channels, is oriented with an accessible amino terminal group on the axon exterior. In this model the ionizable groups on protein and phospholipid have varied associations with the different ionic channel access sites for K, Na, and Cl, and these groups exert considerable control over ion permeation through their surface potentials.     

Nonselective ionic channels inAplysia neurones

Summary Single-channel recordings from outside-out patches ofAplysia neurones in K-free solutions revealed the presence in most membrane patches of ionic channels showing surprising selectivity properties, as deduced from reversal potential measurements. After complete substitution of external NaCl by mannitol (in the presence of internal CsCl), these channels are more permeable to Cl than to Cs, but are also slightly permeable to Cs:P _Cl/P _Cs=4. Furthermore, in the presence of external NaCl, their ability to discriminate cations from anions seems lower than in external mannitol. Substitutions of external Cl by various anions showed that the channels are more permeable to NO₃ than to Cl, and that they are appreciably permeable to isethionate, SO₄ and methanesulfonate. Their elementary conductance is about 100 pS in 600mm symmetrical Cl. However, different conductance states (usually 2 or 3) can often be detected in the same membrane patch. By using voltage ramps, we established theI–V curves corresponding to each of these states and found small but significant differences between the reversal potentials of each state.     

Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl⁻ pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl⁻ activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K _m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P_Na/P_Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P_I (1.8) > P_Br (1.3) > P_Cl (1.0) > P_F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F⁻ flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I⁻/Cl⁻ permeability ratio (P_I/P_Cl < 0.4). The switch to low I⁻ permeability was enhanced at potentials that favored Cl⁻ entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl⁻ and I⁻ ions may influence I⁻ permeation and be responsible for the wide range of P_I/P_Cl ratios that have been reported for the CFTR channel. The low P_I/P_Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.     

Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso- thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS- insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.     

Fast Inactivation of Delayed Rectifier K Conductance in Squid Giant Axon and Its Cell Bodies

Inactivation of delayed rectifier K conductance (g_K) was studied in squid giant axons and in the somata of giant fiber lobe (GFL) neurons. Axon measurements were made with an axial wire voltage clamp by pulsing to V_K (∼−10 mV in 50–70 mM external K) for a variable time and then assaying available g_K with a strong, brief test pulse. GFL cells were studied with whole-cell patch clamp using the same prepulse procedure as well as with long depolarizations. Under our experimental conditions (12–18°C, 4 mM internal MgATP) a large fraction of g_K inactivates within 250 ms at −10 mV in both cell bodies and axons, although inactivation tends to be more complete in cell bodies. Inactivation in both preparations shows two kinetic components. The faster component is more temperature-sensitive and becomes very prominent above 12°C. Contribution of the fast component to inactivation shows a similar voltage dependence to that of g_K, suggesting a strong coupling of this inactivation path to the open state. Omission of internal MgATP or application of internal protease reduces the amount of fast inactivation. High external K decreases the amount of rapidly inactivating I_K but does not greatly alter inactivation kinetics. Neither external nor internal tetraethylammonium has a marked effect on inactivation kinetics. Squid delayed rectifier K channels in GFL cell bodies and giant axons thus share complex fast inactivation properties that do not closely resemble those associated with either C-type or N-type inactivation of cloned Kv1 channels studied in heterologous expression systems.     

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.     

Increased chloride conductance as the proximate cause of hydrogen ion concentration effects in Aplysia neurons

A fall in extracellular pH increased membrane conductance of the giant cell in the abdominal ganglion of Aplysia californica. Chloride conductance was trebled whereas potassium conductance was increased by 50%. Half the giant cells were hyperpolarized (2–8 mv) and half were depolarized (3–10 mv) by lowering the pH. The hyperpolarizing response always became a depolarizing response in half-chloride solutions. When internal chloride was increased electrophoretically, the hyperpolarization was either decreased or changed to depolarization. The depolarizing response was reduced or became a hyperpolarizing response after soaking the cell in 10.0 mM chloride, artificial seawater solution for 1 hr. Depolarization was unaffected when either external sodium, calcium, or magnesium was omitted. A glass micropipette having an organic liquid chloride ion exchanger in its tip was used to measure intracellular chloride activity in 14 giant cells; 7 had values of 27.7 ± 1.8 mM (SEM) and 7 others 40.7 ± 1.5 mM. Three of the first group were hyperpolarized when pH was lowered and three of the second group were depolarized. In all six cells, these changes of membrane potential were in the direction of the chloride equilibrium potential. Intracellular potassium activity was measured by means of a potassium ion exchanger microelectrode.     

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(-)).     

Transient removal of alkaline zones after excitation of Chara cells is associated with inactivation of high conductance in the plasmalemma

The action potential (AP) of excitable plant cells is a multifunctional physiological signal. Its generation in characean algae suppresses the pH banding for 15–30 min and enhances the heterogeneity of spatial distribution of photosynthetic activity. This suppression is largely due to the cessation of H⁺ influx (OH⁻ efflux) in the alkaline cell regions. Measurements of local pH and membrane conductance in individual space-clamped alkaline zones (small cell areas bathed in an isolated pool of external medium) showed that the AP generation is followed by the transient disappearance of alkaline zone in parallel with a large decrease in membrane conductance. These changes, specific to alkaline zones, were only observed under continuous illumination following a relaxation period of at least 15 min after previous excitation. The excitation of dark-adapted cells produced no conductance changes in the post-excitation period. The results indicate that the origin of alkaline zones in characean cells is not due to operation of electroneutral H⁺/HCO₃⁻ symport or OH⁻/HCO₃⁻ antiport. It is concluded that the membrane excitation is associated with inactivation of plasmalemma high conductance in the alkaline cell regions.Key words: Chara, membrane conductance, pH banding, action potential, alkaline cell regions, heterogeneity     

Pore dimensions and the role of occupancy in unitary conductance of Shaker K channels

K channels mediate the selective passage of K⁺ across the plasma membrane by means of intimate interactions with ions at the pore selectivity filter located near the external face. Despite high conservation of the selectivity filter, the K⁺ transport properties of different K channels vary widely, with the unitary conductance spanning a range of over two orders of magnitude. Mutation of Pro475, a residue located at the cytoplasmic entrance of the pore of the small-intermediate conductance K channel Shaker (Pro475Asp (P475D) or Pro475Gln (P475Q)), increases Shaker’s reported ∼20-pS conductance by approximately six- and approximately threefold, respectively, without any detectable effect on its selectivity. These findings suggest that the structural determinants underlying the diversity of K channel conductance are distinct from the selectivity filter, making P475D and P475Q excellent probes to identify key determinants of the K channel unitary conductance. By measuring diffusion-limited unitary outward currents after unilateral addition of 2 M sucrose to the internal solution to increase its viscosity, we estimated a pore internal radius of capture of ∼0.82 Å for all three Shaker variants (wild type, P475D, and P475Q). This estimate is consistent with the internal entrance of the Kv1.2/2.1 structure if the effective radius of hydrated K⁺ is set to ∼4 Å. Unilateral exposure to sucrose allowed us to estimate the internal and external access resistances together with that of the inner pore. We determined that Shaker resistance resides mainly in the inner cavity, whereas only ∼8% resides in the selectivity filter. To reduce the inner resistance, we introduced additional aspartate residues into the internal vestibule to favor ion occupancy. No aspartate addition raised the maximum unitary conductance, measured at saturating [K⁺], beyond that of P475D, suggesting an ∼200-pS conductance ceiling for Shaker. This value is approximately one third of the maximum conductance of the large conductance K (BK) channel (the K channel of highest conductance), reducing the energy gap between their K⁺ transport rates to ∼1 kT. Thus, although Shaker’s pore sustains ion translocation as the BK channel’s does, higher energetic costs of ion stabilization or higher friction with the ion’s rigid hydration cage in its narrower aqueous cavity may entail higher resistance.     

Mechanism of Cl- sensitivity in internal ion receptors of the leech; an inward current gated off by Cl- in the nephridial nerve cells

Summary The nephridial nerve cells of the leech, Hirudo medicinalis, 34 sensory cells, each associated with one nephridium, are sensitive to changes in extracellular Cl^- concentration, an important factor in ion homeostasis. Using single-electrode current- and voltage clamp and ion substitution techniques, the specificity and mechanism of Cl^- sensitivity of the nephridial nerve cell was studied in isolated preparations. Increase of the normally low external Cl^- concentration leads to immediate and sustained hyperpolarization, decrease of the frequency of bursts and decrease of membrane conductance. The response is halogen specific: Cl^- can be replaced by Br^–, but not by organic mono- or divalent anions or inorganic divalent anions.At physiological Cl^- concentrations (36mM extra-cellular Cl^-), the nephridial nerve cell has a high resting conductance for Cl^- and the membrane potential is governed by Cl^-. In high extracellular Cl^- concentrations (110–130 mM), membrane conductance is low, most likely due to the gating off of Cl^- channels. Under these conditions, membrane potential is dominated by the K⁺ distribution and the nephridial nerve cell hyperpolarizes towards E_K.Abbreviations NNC nephridial nerve cell - V _m membrane potential - E _Cl(k) equilibrium potential for Cl (K) - IV-curve current-voltage relationship     

Voltage oscillations in the barnacle giant muscle fiber.

Barnacle muscle fibers subjected to constant current stimulation produce a variety of types of oscillatory behavior when the internal medium contains the Ca++ chelator EGTA. Oscillations are abolished if Ca++ is removed from the external medium, or if the K+ conductance is blocked. Available voltage-clamp data indicate that the cell's active conductance systems are exceptionally simple. Given the complexity of barnacle fiber voltage behavior, this seems paradoxical. This paper presents an analysis of the possible modes of behavior available to a system of two noninactivating conductance mechanisms, and indicates a good correspondence to the types of behavior exhibited by barnacle fiber. The differential equations of a simple equivalent circuit for the fiber are dealt with by means of some of the mathematical techniques of nonlinear mechanics. General features of the system are (a) a propensity to produce damped or sustained oscillations over a rather broad parameter range, and (b) considerable latitude in the shape of the oscillatory potentials. It is concluded that for cells subject to changeable parameters (either from cell to cell or with time during cellular activity), a system dominated by two noninactivating conductances can exhibit varied oscillatory and bistable behavior.     

KCl transport across an insect epithelium: II. Electrochemical potentials and electrophysiology

Summary The cellular mechanism of K-stimulated Cl transport in locust hindgut was studied using double-barrelled ionsensitive microelectrodes and electrophysiological techniques. Steady-state net electrochemical potentials for Cl and K and the conductances of apical and basal membranes and paracellular pathway were determined under control conditions, during exposure to 1mm cAMP, and following ion substitutions. Under control open-circuit conditions, intracellular Cl activity (a _Cl ^c ) was 3.5 times that predicted for passive equilibrium across the apical membrane. The net electrochemical potential opposing Cl entry from the mucosal side increased by 50% during cAMP stimulation of transepithelial Cl absorption whereas the net electrochemical potential favoring Cl exit across the basal membrane was unchanged. No correlation was observed between and the net electrochemical potential across the apical membrane for Na. The net electrochemical potential favoring K entry across the apical membrane was negligible underI _sc conditions when Cl transport rate was approximately 10 eq cm^–2 hr^–1. Locust rectal cells showed electrical and dye coupling. The results also indicate that most transepithelial diffusion of ions is transcellular and that epithelial tightness effectively increases during exposure to cAMP becauseR _a andR _b both decrease, by 80% whileR _j is unchanged. The cAMP-induced R _b was abolished in Cl-free saline whereas R _a was insensitive to Cl removal, but was blocked by removing K from the saline. Based on these findings, our model for Cl absorption in locust hindgut features i) an active entry step for Cl at the apical membrane which is stimulated by cAMP and by low levels of K on the mucosal side, but is not energized by or a large cAMP-stimulated Cl conductance in the basal membrane and a similar cAMP-stimulated K conductance in the apical membrane. cAMP dose-response curves are similar for the stimulation of active Cl absorption and Cl-independent (i. e. K) conductance, indicating that cAMP exerts dual control over active Cl transport and counter-ion permeability.     

pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).     

Tension in Skinned Frog Muscle Fibers in Solutions of Varying Ionic Strength and Neutral Salt Composition

The maximal calcium-activated isometric tension produced by a skinned frog single muscle fiber falls off as the ionic strength of the solution bathing this fiber is elevated declining to zero near 0.5 M as the ionic strength is varied using KCl. When other neutral salts are used, the tension always declines at high ionic strength, but there is some difference between the various neutral salts used. The anions and cations can be ordered in terms of their ability to inhibit the maximal calcium-activated tension. The order of increasing inhibition of tension (decreasing tension) at high ionic strength for anions is propionate^- SO₄^-- < Cl^- < Br^-. The order of increasing inhibition of calcium-activated tension for cations is K⁺ Na⁺ TMA⁺ < TEA⁺ < TPrA⁺ < TBuA⁺. The decline of maximal calcium-activated isometric tension with elevated salt concentration (ionic strength) can quantitatively explain the decline of isometric tetanic tension of a frog muscle fiber bathed in a hypertonic solution if one assumes that the internal ionic strength of a muscle fiber in normal Ringer's solution is 0.14–0.17 M. There is an increase in the base-line tension of a skinned muscle fiber bathed in a relaxing solution (no added calcium and 3 mM EGTA) of low ionic strength. This tension, which has no correlate in the intact fiber in hypotonic solutions, appears to be a noncalcium-activated tension and correlates more with a declining ionic strength than with small changes in [MgATP], [Mg], pH buffer, or [EGTA]. It is dependent upon the specific neutral salts used with cations being ordered in increasing inhibition of this noncalcium-activated tension (decreasing tension) as TPrA⁺ < TMA⁺ < K⁺ Na⁺. Measurements of potentials inside these skinned muscle fibers bathed in relaxing solutions produced occasional small positive values (<6 mV) which were not significantly different from zero.     

Roles of external and cellular Cl− ions on the activation of an apical electrodiffusional Cl− pathway in toad skin

Summary This study is concerned with the short-circuit current,I _sc, responses of the Cl^–-transporting cells of toad skin submitted to sudden changes of the external Cl^– concentration. [Cl]₀. Sudden changes of [Cl]₀, carried out under apical membrane depolarization, allowed comparison of the roles of [Cl]₀ and [Cl]_cell on the activation of the apical Cl^– pathways. Equilibration of shortcircuited skins symmetrically in K-Ringer's solutions of different Cl^– concentrations permitted adjustment of [Cl]_cell to different levels. For a given Cl^– concentration (in the range of 11.7 to 117mm) on both sides of a depolarized apical membrane, this structure exhibits a high Cl^– permeability,P _(Cl)apical. On the other hand, for the same range of [Cl]_cell but with [Cl]₀=0,P _(Cl)apical is reduced to negligible values. These observations indicate that when the apical membrane is depolarizedP _(Cl)apical is modulated by [Cl]₀; in the absence of external Cl^– ions, intracellular Cl^– is not sufficient to activateP _(Cl)apical. Computer simulation shows that the fast Cl^– currents induced across the apical membrane by sudden shifts of [Cl]₀ from a control equilibrium value strictly follow the laws of electrodiffusion. For each experimental group, the computer-generatedI _sc versus ([Cl]_cell–[Cl]₀) curve which best fits the experimental data can only be obtained by a unique pair ofP _(Cl)apical andR _b (resistance of the basolateral membrane), thus allowing the calculation of these parameters. The electrodiffusional behavior of the net Cl^– flux across the apical membrane supports the channel nature of the apical Cl^– pathways in the Cl^–-transporting cell. Cl^– ions contribute significantly to the overall conductance of the basolateral membrane even in the presence of a high K concentration in the internal solution.