Apical Conductance Research Articles

Apical potassium channels in Amphiuma diluting segment: effect of barium.

1. Single potassium channels have been studied in the apical membrane of isolated diluting segment tubules of Amphiuma using the patch clamp technique in the cell-attached mode. A kinetic analysis of the interaction of the channel with barium is presented. 2. The channel shows inward rectification, with an inward conductance of 29.2 pS and an outward conductance of 7.5 pS (cell attached, 100 mM-KCl as pipette solution). 3. The channel is mildly voltage sensitive, the open probability of 0.8 at the resting membrane potential being reduced to 0.38 mV at a hyperpolarizing potential of 120 mV. The mean open time also decreased with hyperpolarization (from 51.3 to 11.6 ms at the above potentials). 4. Inclusion of barium in the pipette solution causes a concentration- and voltage-dependent block of the channel. The open probability was reduced by 40 and 79% of control with 0.1 and 1 mM-barium respectively (0 mV pipette potential). This inhibition increases to 70 and 95% at a pipette potential of 80 mV. 5. From the voltage dependence of the blockade by barium, it is estimated that barium ions block at a site one-third of the way across the membrane. 6. Given the high open probability of the channel at resting membrane potentials and the density of the channels on the apical surface of the cells it is concluded that the channels can account for the apical potassium conductance of the diluting segment of Amphiuma.

Apical K+ channels of frog diluting segment: inhibition by acidification.

The apical potassium conductance of the amphibian diluting segment is regulated by the intracellular pH. Alkalinisation of the cytosol, whether directly by bathing the cell in an alkaline medium, or following activation of an apical Na/H exchanger by aldosterone, results in an increase in the K conductance. Early distal tubules were isolated from slices of Frog kidneys and the apical membranes exposed by everting the tubule with the aid of microperfusion pipettes. Single channels in the apical membrane were studied in the cell-attached configuration while the tubules were bathed in Ringer with a pH of either 7.4 (control) or 6.6 (acid). Under control conditions single channel currents were readily seen at the resting membrane potential. Upon acidification of the superfusion solution the open probability of the channels was decreased from 0.76 to 0.15. Thus the reduction in apical conductance is brought about, at least in part, by a reduction in the open probability of the channels upon cellular acidification.

Coupling of volume and Na+ transport in frog skin epithelium

Whole skins and isolated epithelia were bathed with isotonic media (congruent to 244 mOsm) containing sucrose or glucose. The serosal osmolality was intermittently reduced (congruent to 137 mOsm) by removing the nonelectrolyte. Transepithelial and intracellular electrophysiological parameters were monitored while serosal osmolality was changed. Serosal hypotonicity increased the short-circuit current (ISC) and the basolateral conductance, hyperpolarized the apical membrane (psi mc), and increased the intracellular Na+ concentration. The increases in apical conductance and apical Na+ permeability (measured from Goldman fits of the relationship between amiloride-sensitive current and psi mc) were not statistically significant. To verify that the osmotically induced changes in ISC were mediated primarily at the basolateral membrane, the basolateral membrane potential of the experimental area was clamped close to 0 mV by replacing the serosal Na+ with K+ in Cl--free media. The adjoining control area was exposed to serosal Na+. Serosal hypotonicity produced a sustained stimulation of ISC across the control, but not across the adjoining depolarized tissue area. The current results support the concept that hypotonic cell swelling increases Na+ transport across frog skin epithelium by increasing the basolateral K+ permeability, hyperpolarizing the apical membrane, and increasing the electrical driving force for apical Na+ entry.

Coupling of volume and Na+ transport in frog skin epithelium

Whole skins and isolated epithelia were bathed with isotonic media (- 244 mOsm) containing sucrose or glucose. The serosal osmolality was intermittently reduced (to - 137 mOsm) by removing the nonelectrolyte. Transepithelial and intracellular electrophysiological parameters were monitored while serosal osmolality was changed. Serosal hypotonicity increased the short-circuit current (Isc) and the basolateral conductance, hyperpolarized the apical membrane (Φmc), and increased the intracellular Na+ concentration. The increases in apical conductance and apical Na+ permeability (measured from Goldman fits of the relationship between amiloride-sensitive current and Φmc) were not statistically significant. To verify that the osmotically induced changes in Isc were mediated primarily at the basolateral membrane, the basolateral membrane potential of the experimental area was clamped close to 0 mV by replacing the serosal Na+ with K+ in Cl−-free media. The adjoining control area was exposed to serosal Na+. Serosal hypotonicity produced a sustained stimulation of Isc across the control, but not across the adjoining depolarized tissue area. The current results support the concept that hypotonic cell swelling increases Na+ transport across frog skin epithelium by increasing the basolateral K+ permeability, hyperpolarizing the apical membrane, and increasing the electrical driving force for apical Na+ entry.

Influence of serosal Cl on transport properties and cation activities in frog skin.

The effects of serosal substitution of isosmotic Na2SO4-Ringer solution for NaCl-Ringer solution were studied in the short-circuited frog skin (Rana pipiens, Northern variety). Despite prompt changes of transepithelial measurements, initial cellular effects were slight. After 30 to 45 min, however, the transcellular current had decreased and the cell electrical potential had depolarized, in association with decrease of the apical membrane fractional resistance and basolateral membrane conductance. Apical membrane slope conductance was unaffected. Similar effects were obtained with isolated epithelia. With the use of gluconate or NO3 in place of Cl, the effects on cellular current and conductance were minimal or insignificant, despite changes of the cell potential, fractional resistance, and basolateral conductance similar to those seen with sulfate. Following prolonged exposure to serosal SO4-Ringer, the extent of depolarization induced by raising the serosal K concentration decreased, indicating diminution of basolateral K conductance and the existence of other basolateral conductances. Equilibration in serosal gluconate-Ringer enhanced polarization on serosal restoration of Cl or removal of Na, again indicating a time-dependent change in the basolateral conductance pattern. Depolarization on removal of serosal Cl was not attributable to inhibition of the pump. Nor was it the result of decrease of the K equilibrium potential EK: exposure to serosal SO4-Ringer decreased cell K activity aKc from 104 +/- 6 to 58 +/- 4 mM (n = 5), but EK was reduced only slightly; exposure to serosal gluconate increased aKc and EK. Serosal sulfate lowered the cell Na activity aNac, but the electrochemical potential difference for Na across the apical surface was unaffected. The concurrent decrease of both aKc and aNac following serosal substitution of SO4 for Cl raises questions concerning mechanisms of osmoregulation.

Voltage dependence of cellular current and conductances in frog skin.

Knowledge of the voltage dependencies of apical and basolateral conductances is important in determining the factors that regulate transcellular transport. To gain this knowledge it is necessary to distinguish between cellular and paracellular currents and conductances. This is generally done by sequentially measuring transepithelial current/voltage (It/Vt) and conductance/voltage (gt/Vt) relationships before and after the abolition of cellular sodium transport with amiloride. Often, however, there are variable time-dependent and voltage-dependent responses to voltage perturbation both in the absence and presence of amiloride, pointing to effects on the paracellular pathway. We have here investigated these phenomena systematically and found that the difficulties were significantly lessened by the use of an intermittent technique, measuring It and gt before and after brief (less than 10 sec) exposure to amiloride at each setting of Vt. I/V relationships were characterized by these means in frog skins (Rana pipiens, Northern variety, and Rana temporaria). Cellular current, Ic, decreased with hyperpolarization (larger serosa positive clamps) of Vt. Derived Ic/Vt relationships between Vt = 0 and 175 mV (serosa positive) were slightly concave upwards. Because values of cell conductance, gc, remained finite, it was possible to demonstrate reversal of Ic. Values of the reversal potential Vr averaged 156 +/- 14 (SD, n = 18) mV. Simultaneous microelectrode measurements permitted also the calculation of apical and basolateral conductances, ga and gb. The apical conductance decreased monotonically with increasing positivity of Vt (and Va). In contrast, in the range in which the basolateral conductance could be evaluated adequately (Vt less than 125 mV), gb increased with more positive values of Vt (and Vb). That is, there was an inverse relation between gb and cellular current at the quasi-steady state, 10-30 sec after the transepithelial voltage step.

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.

Effects of oxytocin on cation content and electrophysiology of frog skin epithelium

We measured effects of oxytocin on current-voltage (I-V) relations of frog (Rana catesbeiana) skins impaled with an intracellular microelectrode. In both Cl- and Cl(-)-free (SO4(2-) solutions, oxytocin caused an approximate doubling short-circuit current (Isc) and a depolarization of the cell membrane. Increase in apical membrane slope conductance, chord conductance, and permeability after oxytocin correlated with the increase in amiloride-sensitive Isc. Oxytocin also increased basolateral membrane conductance (gb). In Cl-, the shift in the voltage intercept of the apical membrane I-V relation (Ea) implied increased intracellular Na+ activity (a(Na)c) after oxytocin. In isolated frog skin epithelia, a similar increase in intracellular [Na+] after oxytocin was demonstrated by flame photometry. In SO4(2-), changes caused by oxytocin in both Ea and in flame photometrically determined cell [Na+] were minimal. The voltage intercept of the basolateral membrane I-V relations (Eb) was shifted by oxytocin in both Cl- and SO4(2-) solutions. Assuming that the basolateral membrane is selectively permeable to K+, changes in K+ obtained from Eb were in disagreement with those obtained by flame photometry. These results suggest that 1) the increase in a(Na)c caused by oxytocin is not essential to produce either the increase in gb or Isc and 2) ions other than K+ make an important contribution to basolateral membrane conductance.

Effect of a chloride channel blocker on bullfrog corneal epithelium

The effect of the chloride channel blocker, diphenylamine-2-carboxylate (DPC), was investigated on the bullfrog cornea epithelium using microelectrodes. Perfusion of 2 X 10(-4) M DPC on the tear side resulted in a 44% reduction of the corneal transepithelial potential difference (TEP) and a 9% reduction in corneal conductance. The cornea epithelial cells hyperpolarized 7.2 +/- 1.5 mV, and the apical fractional resistance increased 35% (n = 8 tissues). The basolateral potential difference depolarized 5.5 +/- 1.8 mV. The relative apical conductance decreased 29%, whereas relative changes of basolateral membranes increased 56% with prolonged DPC exposure, indicating that the DPC inhibition was acting at the apical cell membrane. DPC-treated corneas could be stimulated with isoproterenol (10(-6) M), indicating that the DPC inhibition could be reversed with the stimulation of chloride conductance. The DPC inhibitory effect was dependent on basolateral chloride. Intracellular chloride activity increases with DPC, indicating that the integrity of the basolateral chloride entry mechanism was not altered. The DPC inhibitory action was due to a reduction in apical chloride conductance.

Detection of left ventricular regional myocardial ischaemia in dogs by intraventricular conductance catheter.

Nine adult mongrel dogs were instrumented with ultrasonic microcrystals to measure left ventricular basal anteroposterior diameter and midwall myocardial segment length near the cardiac apex. Pneumatic cuff occluders were positioned around the left circumflex coronary artery near its origin and around the left anterior descending coronary artery two thirds of the way along its length. A pressure microtransducer was implanted into the left ventricle. Ten days after instrumentation the animals were anaesthetised with morphine chlorhydrate and pentobarbital sodium. An eight electrode catheter was advanced into the left ventricle to measure ventricular apical and basal regional and total electrical conductance. Minor ischaemia caused by occlusion of the left anterior descending artery was detected only by the electrode pair located near the apex, as decreased local ejection fraction. Major ischaemia caused by left circumflex artery occlusion was detected by both apical and basal electrode pairs and by total conductance, the three conductance signals indicating reduced ejection fractions compared with control values. The basal diameter signal indicated that basal regional motility changed only during major ischaemia, thus confirming the specificity of the changes in the basal conductance signals. The apical segment length signal confirmed the altered motility indicated by the apical conductance signal. These results suggest that regional wall motion abnormalities may be detected by the use of a multielectrode conductance catheter.

Effect of rhein on electrogenic chloride secretion in rabbit distal colon.

The effect of the laxative component rhein on electrical properties and Cl transport was investigated in partially stripped epithelial sheets of rabbit distal colon under short-circuit conditions. Whereas mucosal rhein had no effect, serosally applied rhein stimulated electrogenic Cl secretion in a dose-dependent manner with a half-maximal stimulation at 0.05 mmol/l. The stimulation was partially reversible by serosal bumetanide. Indomethacin preincubation inhibited the effect of rhein, but did not prevent theophylline-induced Cl secretion. The results suggest a prostaglandin-mediated effect of rhein on the apical Cl conductance of the colonic cells.

The collecting tubule of Amphiuma. II. Effects of potassium adaptation

Electrophysiological and transport properties were studied in isolated and perfused Amphiuma collecting tubules from two groups of animals, one exposed to a high sodium (NA), the other to a high potassium (KA) environment (both conditions known to modulate blood aldosterone levels). The transepithelial lumen-negative potential was significantly larger (-38 +/- 5 mV) in tubules from KA animals than from NA animals (-15 +/- 3 mV). In addition, we observed an increase in the apical amiloride-sensitive sodium conductance and stimulation of the transepithelial sodium current. Although no measurable potassium conductance was found in the apical cell membrane in either group, a potassium selectivity of the paracellular transport pathway was observed in the KA animals. Net potassium secretion was demonstrated in KA tubules (helium-glow photometry and [3H]inulin analysis). Potassium secretion was abolished by luminal amiloride but imposition of a bath-to-lumen potassium gradient induced potassium secretion. We conclude that in contrast to the mammalian cortical collecting tubule in which potassium secretion is largely transcellular, potassium secretion in the Amphiuma collecting tubule is by diffusion through the paracellular pathway.

Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder.

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.

Transport-dependent alterations of membrane properties of mammalian colon measured using impedance analysis.

Direct current (DC) measurement methods have been commonly used to characterize the conductance properties of the mammalian colon. However, these methods provide no information concerning the effects of tissue morphology on the electrophysiological properties of this epithelium. For example, distribution of membrane resistances along narrow fluid-filled spaces such as the lateral intercellular spaces (LIS) or colonic crypts can influence DC measurements of apical and basolateral membrane properties. We used impedance analysis to determine the extent of such distributed resistance effects and to assess the conductance and capacitance properties of the colon. Because capacitance is proportional to membrane area, this method provides new information concerning membrane areas and specific ionic conductances for these membranes. We measured transepithelial impedance under three conditions: control conditions in which the epithelium was open-circuited and bathed on both sides with NaCl-HCO3 Ringer's solutions, amiloride conditions which were similar to control except that 100 microM amiloride was present in the mucosal bathing solution, and mucosal NaCl-free conditions in which mucosal Na and Cl were replaced by potassium and sulfate or gluconate ("K+ Ringer's"). Three morphologically-based equivalent circuit models were used to evaluate the data: a lumped model (which ignores LIS resistance), a LIS distributed model (distributed basolateral membrane impedance) and a crypt-distributed model (distributed apical membrane impedance). To estimate membrane impedances, an independent measurement of paracellular conductance (Gs) was incorporated in the analysis. Although distributed models yielded improved fits of the data, the distributed and lumped models produced similar estimates of membrane parameters. The predicted effects of distributed resistances on DC microelectrode measurements were largest for the LIS-distributed model. LIS-distributed effects would cause a 12-15% underestimate of membrane resistance ratio (Ra/Rb) for the control and amiloride conditions and a 34% underestimate for the "K Ringer's" condition. Distributed resistance effects arising from the crypts would produce a 1-2% overestimate of Ra/Rb. Apical and basolateral membrane impedances differed in the three different experimental conditions. For control conditions, apical membrane capacitance averaged 21 microF/cm2 and the mean apical membrane specific conductance (Ga-norm) was 0.17 mS/microF. The average basolateral membrane capacitance was 11 microF/cm2 with a mean specific conductance (Gb-norm) of 1.27 mS/microF.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of taurine-conjugated bile salts on ionic transport of the rabbit esophageal mucosa.

Bile salts have been implicated as a cause of esophageal injury. We examined in vitro the changes in ionic transport of the rabbit esophagus resulting from taurine-conjugated bile salts at neutral pH to define and characterize their actions independent of hydrogen ion. In an Ussing chamber changes in potential difference (PD, mV), short-circuit current (SCC, microA X cm-2) and resistance (R, omega X cm2) resulting from taurocholate (TC), taurodeoxycholate (TDC) and taurochenodeoxycholate (TCDC) were studied. Transport properties were unaffected by TC at 5 and 10 mM. With TDC (5 mM) there was an initial rise in SCC and PD. After 60 min PD and R declined in association with an increase in paracellular permeability as measured by [14C]sucrose flux. TCDC (5.0 and 7.5 mM) caused a sustained rise in PD and SCC with a greater rise seen at 7.5 mM. The rise in SCC after TCDC was secondary to an increase in net sodium absorption as basal net sodium absorption increased threefold from 0.15 +/- 0.03 to 0.44 +/- 0.10 mu eq X cm-2 X h-1. Increased net sodium absorption accounted for 73% of the TCDC-induced rise in SCC. Amiloride (10(-4) M) added to the mucosal solution significantly inhibited this bile salt-induced rise in SCC. We conclude that bile salts alter ionic transport in the esophageal mucosa independent of hydrogen ion. These changes are characterized by an early selective increase in apical membrane cation conductance and with longer exposure, particularly in the presence of more hydrophobic bile salts, there is a marked increase in paracellular conductance.

Phenothiazines increase active sodium transport across the isolated toad skin.

Fluphenazine (FPZ) and trifluoperazine (TFP) are phenothiazine derivatives commonly used as antipsychotic tranquilizers. Their mechanism of action is incompletely understood. Epidermal addition of each drug promoted biphasic short-circuit current (SCC) changes across isolated pelvic skin of Bufo arenarum toads. By means of radiotracers fluxes, SCC was found to be given by the algebraic sum of net sodium and chloride transport. A readily stimulant effect was detected a low concentrations (from 1 X 10(-6) mol/l up to 1 X 10(-4) mol/l for FPZ, from 1 X 10(-5) mol/l up to 3.2 X 10(-4) mol/l for TFP) above which inhibition prevailed. Dermal FPZ also stimulated SCC. A higher concentration and time threshold were required. Epidermal 1 X 10(-5) mol/l FPZ stimulation was partially reversible, with a diminished membrane resistance and enhancement of sodium influx, without alteration of sodium efflux or net chloride transport. It could be prevented by amiloride pretreatment, or diminished by dermal sodium removal. Variation of epidermal bulk pH from 5.8 to 8.7 demonstrated that ionized and nonionized molecules contribute to FPZ's effect. Our results suggest that SCC stimulation elicited by FPZ and TFP may be a consequence of direct or indirect modifications on apical sodium conductance.

Characterization of the apical membrane ionic permeability of the rabbit proximal convoluted tubule

Basolateral membrane potential (psi BL), transepithelial potential (psi T), and the ratio of apical to basolateral membrane resistance (RA/RBL) were measured in rabbit proximal convoluted tubules (PCT) perfused in vitro. Analysis of RA/RBL changes using several luminal perfusates indicates that the cotransport of Na with glucose and alanine would represent 19% of the apical conductance in normal conditions; the cotransport of Na with acetate, citrate, sulfate, and phosphate would represent 7%, whereas Na, K, and Cl diffusion would represent 10, 4, and 0% of this apical conductance, respectively. On the other hand, psi BL values can also be analyzed using the equivalent circuit of the epithelium to obtain the apical membrane equivalent electromotive force (EA) in the presence of each perfusate. These values, as well as the preceding values obtained from RA/RBL measurements, indicate that in the absence of cotransported solutes the transference number for Na diffusion is several times larger than for K diffusion. Among the conductance pathways studied, the transference number sequence would be as follows: Na cotransport with alanine and glucose greater than Na cotransport with anions greater than Na diffusion greater than K diffusion greater than Cl diffusion. This study also suggests the presence of another important but unidentified apical ionic permeation pathway, since the total of the transference numbers obtained from RA/RBL analysis represents only 40% of the total apical membrane conductance and the absolute values of EA are difficult to account for using only the tested apical membrane permeation pathways.

Single apical membrane anion channels in primary cultures of canine tracheal epithelium

To investigate the cellular mechanism responsible for the apical membrane Cl conductance in airway epithelia, we used the patch-clamp technique to study single ion channels in primary cultures of canine tracheal epithelium. The cells contained an anion channel that had a single channel conductance of approximately 29 pS at negative voltages in symmetrical 145 mmol/l Cl solutions. In symmetrical Cl solutions the excised single-channel current-voltage relation was nonlinear, with conductance increasing at depolarizing voltages. The channel was inhibited by diphenylamine-2-carboxylate and anthracene-9-carboxylic acid at concentrations similar to those required to inhibit transepithelial Cl secretion. The channel was found in freshly isolated cells, isolated cells in culture, and in the apical membrane of confluent areas of cells. When attached to the cell the channel was activated by addition of isoproterenol in some, but not all cases. Often it was not observed to open until the patch was excised from the cell. The channel was not strongly voltage-gated, nor was it acutely regulated by internal Ca in the excised, inside-out configuration. These results suggest that this channel may be responsible for the apical Cl conductance in canine tracheal epithelium.

CHARACTERIZATION OF A CATION CHANNEL ON THE APICAL SURFACE OF THE FROG LENS EPITHELIUM

The properties of a single conductance pathway of the apical (fibre-facing) surface of the frog lens epithelium are reported. Using the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981), the most common single-channel currents had an amplitude of 1.9 pA, the mean open time 2.1 ms and a conductance of 25 pS. One open-state time constant (to = 3.3 ms) and two closed-state time constants (tau c1 = 0.9 ms, tau c2 = 23.1 ms) were resolved. The channel current and the mean open time were both increased when Ca2+ was removed from the external solution and the open time distribution was no longer fitted by a single exponential. Multiple-channel events in cell-attached patches containing two or more identical channels were distributed in a binomial fashion and the probability that an individual channel was open, obtained by fitting the binomial distribution, was 0.039. The channel was found to have a Na+:K+ selectivity ratio of 3:1. When Ca2+ was removed from the pipette solution the probability that an individual channel was open increased to 0.137 and the Na+:K+ selectivity ratio increased to 4:1. Channel activity was observed in the presence of tetrodotoxin (10(-6) M) in the bathing medium and the pipette solution but was abolished by internal perfusion of the patch pipette with 0.5 x 10(-4) M amiloride. this apical conductance pathway is identified as an amiloride-sensitive cation channel. These channels are clustered in groups on the apical membrane, spontaneously active at the resting potential and with the possibility of altering their Na+:K+ selectivity. They represent a distinct type of channel, that differ from nerve and muscle Na+ channels in their manner of activation, but do share some common features with both Na+ and Ca2+ channels in excitable cells.

Ca2+-dependent, temperature-sensitive regulation of Na+ channels in tight epithelia. A study using membrane vesicles.

Na+ fluxes were measured in toad bladder microsomes. Under favorable conditions, 60-90% of the tracer uptake was blocked by amiloride (Ki = 2.3 X 10(-8) M), i.e. mediated by the apical Na+-specific channels. Vesicles derived from cells maintained at 0 degrees C exhibited relatively small amiloride-sensitive fluxes. However, incubating the scraped cells at 25 degrees C prior to homogenization induced a nearly 5-fold increase of the amiloride-blockable flux in vesicles. This activation was fairly slow (t 1/2 = 5-10 min), irreversible, and strongly dependent on the incubation temperature. On the other hand, the Na+-specific apical conductance measured in mounted bladders was only slightly affected by the incubation temperature. The above activation process could be observed only in Ca2+-free EGTA-containing solutions. Adding Ca2+ (1 mM) to the cell suspension and subsequently removing it before homogenization blocked almost completely the amiloride-sensitive tracer uptake in the vesicles. The data are compatible with the model that the epithelial Na+ channels are down-regulated by a Ca2+-dependent reaction. The incubation of scraped, somewhat permeabilized cells in a Ca2+-free solution releases channels from this down-regulation and increases the Na+ conductance in a temperature-dependent process. The regulation of channels appears to involve a cytoplasmic factor which induces a stable modification of the apical membrane, preserved by the isolated vesicle.