Studies on the perfused plasmalemma ofChara corallina: I. Current-voltage curves: ATP and potassium dependence

Summary The electrical properties of theChara cell membrane have been studied using a perfusion method based on that of Williamson, R.E. 1975.J. Cell Sci. 17655. The vacuole, tonoplast, and inner cytoplasm are removed by a brief rapid perfusion. Electrical properties of the plasmalemma indicate that it remains intact after this perfusion.The membrane potential difference after perfusion and with no ATP was close to the potassium equilibrium potential; the current-voltage characteristic had a slope that was time- and voltage-dependent, indicating that the steady-state potassium conductance increased with depolarization. At –125 mV the membrane conductance of the plasmalemma depended on [K⁺]₀. This dependence was inhibited by perfusing with 2.0mm ATP or by clamping at a more negative membrane potential. The addition of ATP to the perfusion medium of unclamped cells caused a hyperpolarization ofca. 50 mV, presumably by activating the proton pump. In clamped cells, perfusion with ATP caused currents ofca. 20 mA m^–2, whose magnitude depended on pH₀. ATP induced membrane conductance changes which were variable. 2.0mm ADP inhibited the proton pump. The intersection points of current-voltage characteristics can set limits on the stalling potential; the resulting stoichiometry of the proton pump appears to be 1.5–2.0 H⁺ per ATP.     

Chloride conductance of the Amphiuma red cell membrane

Summary Like most other red cells, the giant erythrocytes ofAmphiuma means possess a system for rapid exchange of chloride across the membrane. Also, there are indications that the net transport of chloride in these cells is slow. The size ofAmphiuma erythrocytes allows direct measurements of membrane potential with microelectrodes. The present work exploits the possibility that such measurements can be used to give a quantitative estimate of the chloride conductance (G _Cl) of the Amphiuma red cell membrane. The membrane potential was measured as a function of extracellular chloride concentration (5–120mM), using an impermeant anion (Para-amino-hippurate) as a substitute. Furthermore, the effect of different pH values (6.0–7.2) was studied. For each extracellular chloride concentration the membrane potential was determined at a pH at which hydroxyl, hydrogen, and bicarbonate ions were in electrochemical equilibrium. From these membrane potentials and the corresponding chloride concentrations in the medium (at constant intracellular ion concentrations), theG _Cl of the membrane was calculated to be 3.9×10^–7 {ie27-1} cm^–2. This value is some six orders of magnitude smaller than that calculated from the rate of tracer exchange under equilibrium conditions. The experimental strategy used gives the value for a partial transference number which takes into account only ions which arenot in electrochemical equilibrium. Whereas this approach gives a value forG _Cl, it does not permit calculation of the overall membrane conductance. From the calculated value ofG _Cl it is possible to estimate that the maximal value of the combined conductances of hydroxyl (or proton) and bicarbonate ions is 0.6×10^–7 {ie27-2} cm^–2. The large discrepancy between the rate of exchange of chloride and its conductance is in agreement with measurements on human and sheep red cells employing the ionophore valinomycin to increase the potassium conductance of the membrane. The results in the present study were, however, obtained without valinomycin and an accompanying assumption of a constant field in the membrane. Therefore, the present measurements give independent support to the above mentioned conclusions.     

Chloride transport in apical membrane vesicles from bovine tracheal epithelium: Characterization using a fluorescent indicator

Summary Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J _Cl) was increased significantly from 0.32±0.12 nmol · sec^–1 · mg protein^–1 (mean±sem) to 0.50±0.07 nmol · sec^–1 · mg protein^–1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ _Cl from a control value of 0.37±0.03 nmol · sec^–1 · mg protein^–1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ _Cl significantly (J _Cl values gives as percent change from control): 50mm cis Na (–1±5%), 0.1mm furosemide (–3±4%), 0.1mm furosemide in the presence of 50mm cis Na (–5±2%), 0.1mm H₂DIDS (–18±9%), a 1.5 pH unit inwardly directed H gradient (–7±7%), and 0.1mm H₂DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J _Br andJ ₁, respectively) were not significantly different fromJ _Cl.J _Cl was a saturable function of Cl concentration with apparentK _d of 24mm and apparentV _max of 0.54 nmol · sec^–1 · mg protein^–1. Measurement of the temperature dependence ofJ _Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.     

Apical membrane K conductance in the toad urinary bladder

Summary The conductance of the apical membrane of the toad urinary bladder was studied under voltage-clamp conditions at hyperpolarizing potentials (mucosa negative to serosa). The serosal medium contained high KCl concentrations to reduce the voltage and electrical resistance across the basal-lateral membrane, and the mucosal solution was Na free, or contained amiloride, to eliminate the conductance of the apical Na channels. As the mucosal potential (V _m) was made more negative the slope conductance of the epithelium increased, reaching a maximum at conductance of the epithelium increased, reaching a maximum atV _m=–100 mV. This rectifying conductance activated with a time constant of 2 msec whenV _m was changed abruptly from 0 to –100 mV, and remained elevated for at least 10 min, although some decrease of current was observed. ReturningV _m to+100 mV deactivated the conductance within 1 msec. Ion substitution experiments showed that the rectified current was carried mostly by cations moving from cell to mucosa. Measurement of K flux showed that the current could be accounted for by net movement of K across the apical membrane, implying a voltage-dependent conductance to K (G _K). Mucosal addition of the K channel blockers TEA and Cs had no effect onG _K, while 29mm Ba diminished it slightly. Mucosal Mg (29mm) also reducedG _K, while Ca (29mm) stimulated it.G _K was blocked by lowering the mucosal pH with an apparent pK₁ of 4.5. Quinidine (0.5mm in the serosal bath) reducedG _K by 80%.G _K was stimulated by ADH (20 mU/ml), 8-Br-cAMP (1mm), carbachol (100 m), aldosterone (5×10^–7 m for 18 hr), intracellular Li and extracellular CO₂.     

Microelectrode characterization of the basolateral membrane of rabbit S3 proximal tubule

Summary The purpose of this study was to characterize the basolateral membrane of the S3 segment of the rabbit proximal tubule using conventional and ion-selective microelectrodes. When compared with results from S1 and S2 segments, S3 cells under control conditions have a more negative basolateral membrane potential (V _bl=–69 mV), a higher relative potassium conductance (t _K=0.6), lower intracellular Na⁺ activity (A _Na=18.4mm), and higher intracellular K⁺ activity (A _K=67.8mm). No evidence for a conductive sodium-dependent or sodium-independent HCO ₃ ^– pathway could be demonstrated. The basolateral Na–K pump is inhibited by 10^–4 m ouabain and bath perfusion with a potassium-free (0-K) solution. 0-K perfusion results inA _Na=64.8mm,A _K=18.5mm, andV _bl=–28 mV. Basolateral potassium channels are blocked by barium and by acidification of the bathing medium. The relative K⁺ conductance, as evaluated by increasing bath K⁺ to 17mm, is dependent upon the restingV _bl in both S2 and S3 cells. In summary, the basolateral membrane of S3 cells contains a pump-leak system with similar properties to S1 and S2 proximal tubule cells. The absence of conductive bicarbonate pathways results in a hyperpolarized cell and larger Na⁺ and K⁺ gradients across the cell borders, which will influence the transport properties and intracellular ion activities in this tubule segment.     

Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .     

Electrogenic and diffusive components of the membrane ofHydrodictyon africanum

Summary In cells of the freshwater algaHydrodictyon africanum, in solutions where [K⁺]₀=0.1mm and pH₀>7.0, the membrane in the light is hyperpolarized. The membrane potential difference {ie179-1} has values from –180 to –275 mV, more negative than any ion diffusion potential difference, and is predominantly a function of pH₀, and independent of [K⁺]₀. The hyperpolarization of the membrane appears to arise from an electrogenic efflux of H⁺, estimated from voltage-clamp data to be about 8 nmol m^–2 sec^–1 when pH₀=8.5. In the light the membrane conductanceg _m is about 0.084 S m^–2. At light-off, {ie179-2} becomes less negative, with a halftime for change of 15 to 30 sec andg _m decreases by about 0.052 S m^–2. After dark periods of up to 300 sec, {ie179-3} is largely independent of pH₀ for values greater than 6.0 and usually behaves as a combined K⁺ and Na⁺ diffusion potential with permeability ratioP _Na/P _K=0.05 to 0.2. The membrane potassium conductanceg _K has either a low value of 2–6×10^–2 Sm^–2, or a high value of up to 18×10^–2 S m^–2 depending on [K⁺]₀, the transition from low to high values occurring when {ie179-4} moves over a threshold value that is more negative than {ie179-5}, the electrochemical equilibrium potential for K⁺. The time for half-change of the transition is about 30 sec. The results are consistent with a model of the membrane in which the pump electromotive force and conductance are in parallel with diffusive electromotive forces and conductances. When the pump is operating its properties determine membrane properties, and when it is inoperative, or running at a diminished rate, the membrane properties are determined more by the diffusive pathways. Changes in both pump rate andg _K can account for a variety of characteristic changes in membrane PD and conductance occurring in response to ligh-dark changes, changes in light intensity, pasage of externally applied electric current across the membrane and changes in ionic constituents of the external medium.     

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: I. ADH increases transcellular conductance pathways

Summary This paper reports experiments designed to assess the relations between net salt absorption and transcellular routes for ion conductance in single mouse medullary thick ascending limbs of Henle microperfusedin vitro. The experimental data indicate that ADH significantly increased the transepithelial electrical conductance, and that this conductance increase could be rationalized in terms of transcellular conductance changes. A minimal estimate (G _c ^min ) of the transcellular conductance, estimated from Ba⁺⁺ blockade of apical membrane K⁺ channels, indicated thatG _c ^min was approximately 30–40% of the measured transepithelial conductance. In apical membranes, K⁺ was the major conductive species; and ADH increased the magnitude of a Ba⁺⁺-sensitive K⁺ conductance under conditions where net Cl^– absorption was nearly abolished. In basolateral membranes, ADH increased the magnitude of a Cl^– conductance; this ADH-dependent increase in basal Cl^– conductance depended on a simultaneous hormone-dependent increase in the rate of net Cl^– absorption. Cl^– removal from luminal solutions had no detectable effect onG _e , and net Cl^– absorption was reduced at luminal K⁺ concentrations less than 5mm; thus apical Cl^– entry may have been a Na⁺,K⁺,2Cl^– cotransport process having a negligible conductance. The net rate of K⁺ secretion was approximately 10% of the net rate of Cl^– absorption, while the chemical rate of net Cl^– absorption was virtually equal to the equivalent short-circuit current. Thus net Cl^– absorption was rheogenic; and approximately half of net Na⁺ absorption could be rationalized in terms of dissipative flux through the paracellular pathway. These findings, coupled with the observation that K⁺ was the principal conductive species in apical plasma membranes, support the view that the majority of K⁺ efflux from cell to lumen through the Ba⁺⁺-sensitive apical K⁺ conductance pathway was recycled into cells by Na⁺,K⁺,2Cl^– cotransport.     

Communicating junctions and calmodulin: Inhibition of electrical uncoupling inXenopus embryo by calmidazolium

Summary This paper reports the inhibitory effects of calmidazolium (CDZ), a calmodulin inhibitor, on electrical uncoupling by CO₂. Membrane potential and coupling ratio (V ₂/V₁) are measured in two neighboring cells ofXenopus embryos (16 to 64 cell stage) for periods as long as 5.5 hr. Upon exposure to 100% CO₂, control cells consistently uncouple even if the CO₂ treatments are repeated every 15 min for 2.5 hr. CDZ (5×10^–8–1×10^–7 m) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1×10^–7, 7×10^–8 and 5×10^–8 m CDZ, respectively, is concentration-dependent and partially reversible. In the absence of CO₂, CDZ also improves electrical coupling. CDZ has no significant effect on membrane potential and nonjunctional membrane resistance. These data suggest that calmodulin or a calmodulin-like protein participates in the uncoupling mechanism.     

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance

Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10^–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm² from a mean value of 138±13 · cm² before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl^– in the medium. Subsequent addition of bumetanide (10^–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba²⁺ (1mm), a known inhibitor of basolateral K⁺ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K⁺ conductance.The results suggest that forskolin-induced Cl^– secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl^– conductance of the apical membrane but does not affect K⁺ or Cl^– conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl^– and the basal Ba²⁺-sensitive K⁺ conductance are sufficiently large to maintain the Cl^– efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl^– channel blockers NPPB and DPC on basolateral conductance rather than apical Cl^– conductance.     

Elevation in intracellular calcium activates both chloride and proton currents in human macrophages

The transition of a resting macrophage into the activated state is accompanied by changes in membrane potential, cytoplasmic pH, and intracellular calcium (Ca _i ). Activation of Cl^– as well as H⁺-selective currents may give rise to stimulus-induced changes in membrane potential and counteract changes in intra-cellular pH (pH _i ) which have been observed to be closely associated with respiratory burst activation and superoxide production in macrophages. We carried out whole-cell voltage clamp experiments on human monocyte-derived macrophages (HMDMs) and characterized currents activated following an elevation in Ca _i using isosmotic pipette and bath solutions in which Cl^– was the major permeant species. Ca _i was elevated by exposing cells to the Ca²⁺ ionophore A23187 (1–10 m) in the presence of extracellular Ca²⁺ or by internally exchanging the patch-electrode solution with ones buffered to free Ca²⁺ concentrations between 40 and 2,000 nm. We have identified two Ca²⁺-dependent ion conductances based on differences in their characteristic time-dependent kinetics: a rapidly activating Cl^– conductance that showed variable inactivation at depolarized potentials and a H⁺ conductance with delayed activation kinetics. Both conductances were inhibited by the disulfonic acid stilbene DIDS (100 m). Current activation for both Ca²⁺-dependent conductances was phosphorylation dependent, neither conductance appeared in the presence of the broad spectrum kinase inhibitor H-7 (75 m). Inclusion of the autophosphorylated, Ca²⁺/calmodulin-dependent protein kinase in the pipette in the presence of ATP induced a rapidly activating current similar to that observed following an elevation in Ca _i . Activation of both conductances would contribute to the changes in membrane potential which accompany stimulation-induced activation of macrophages as well as counteract the decrease in pH _i during sustained Superoxide production.The authors wish to thank Dr. H. Schulman for providing us with the purified CaMKII and Jennifer Foss for technical assistance. This work was supported by National Institutes of Health RO1 GM36823.     

Inorganic mercury (Hg2+) transport through lipid bilayer membranes

Summary Diffusion of inorganic mercury (Hg²⁺) through planar lipid bilayer membranes was studied as a function of chloride concentration and pH. Membranes were made from egg lecithin plus cholesterol in tetradecane. Tracer (²⁰³Hg) flux and conductance measurements were used to estimate the permeabilities to ionic and nonionic forms of Hg. At pH 7.0 and [Cl^–] ranging from 10–1000mm, only the dichloride complex of mercury (HgCl₂) crosses the membrane at a significant rate. However, several other Hg complexes (HgOHCl, HgCl ₃ ^– and HgCl ₄ ^2– ) contribute to diffusion through the aqueous unstirred layer adjacent to the membrane. The relation between the total mercury flux (J _Hg), Hg concentrations, and permeabilities is: 1/J _Hg=1/P ^ul[Hg ^t ]+1/P ^m [HgCl₂], where [Hg ^t ] is the total concentration of all forms of Hg,P ^ul is the unstirred layer permeability, andP ^m is the membrane permeability to HgCl₂. By fitting this equation to the data we find thatP ^m =1.3×10^–2 cm sec^–1. At Cl^– concentrations ranging from 1–100mm, diffusion of Hg ^t through the unstirred layer is rate limiting. At Cl^– concentrations ranging from 500–1000mm, the membrane permeability to HgCl₂ becomes rate limiting because HgCl₂ comprises only about 1% of the total Hg. Under all conditions, chemical reactions among Hg²⁺, Cl^– and/or OH^– near the membrane surface play an important role in the transport process. Other important metals, e.g., Zn²⁺, Cd²⁺, Ag⁺ and CH₃Hg⁺, form neutral chloride complexes under physiological conditions. Thus, it is likely that chloride can facilitate the diffusion of a variety of metals through lipid bilayer and biological membranes.     

Precipitation membranes: III. Reversible changes of membrane properties induced by alterations in ionic concentrations

Summary The conditioned state of a precipitation membrane with its particular properties exists within a limited range of membrane potentials and requires certain minimum concentrations,C _lim, of the generating ions in the adjoining solutions. We investigated these quantities for the BaSO₄ cellophane membrane and foundC _lim to be 10×10^–5 n (0.5×10^–4 m), equally for Ba⁺⁺ and SO ₄ ^–– . Beyond these limits, the membrane becomes deconditioned. This transformation is a reversible process provided the limits have not been surpassed too far. The capability for de- and reconditioning is a characteristic and unique property of precipitation membranes, not found in other membrane systems. The phenomenon is explained by the adsorption theory for precipitation membranes. It allows wide modifications and quick variations of the electrical properties and permeability of the membrane in an easy and reversible manner.     

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl− absorption

Summary Cellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage,V _e , which attends net Cl^– absorption,J _Cl ^net , and to assess how ADH might augment bothJ _Cl ^met andV _e in the mouse medullary thick ascending limb of Henle microperfusedin vitro. Substituting luminal 5mm Ba⁺⁺ for 5mm K⁺ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio,R _c /R _bl , and increasing luminal K⁺ from 5 to 50mm in the presence of luminal 10^–4 m furosemide resulted in a 53-mV depolarization of apical membrane voltage,V _a . Thus K⁺ accounted for at least 85% of apical membrane conductance. Either with or without ADH. 10^–4 m luminal furosemide reducedV _e andJ _Cl ^net to near zero values and hyperpolarized bothV _a andV _bl , the voltage across basolateral membranes; however, the depolarization ofV _bl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization ofV _a , Thus ADH-dependent increases inV _b were referable to greater depolarizations ofV _bl in the presence of ADH than in the absence of ADH 68% of the furosemide-induced hyperpolarization ofV _a was referable to a decrease in the K⁺ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization ofV _bl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl^– current. Thus an increase in intracellular Cl^– activity may have contributed to the depolarization ofV _bl during net Cl^– absorption, and the intracellular Cl^– activity was likely greater with ADH than without hormone. Since ADH increases apical K⁺ conductance and since the chemical driving force for electroneutral Na⁺,K⁺,2Cl^– cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba⁺⁺-sensitive conductance K⁺ channels and electroneutral Na⁺,K⁺,2Cl^– cotransport units in apical plasma membranes.     

cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney

Summary The properties of an anion-selective channel observed in basolateral membranes of microdissected, collagenase-treated, cortical thick ascending limbs of Henle's loop from mouse kidney were investigated using patch-clamp single-channel recording techniques. In basal conditions, single Cl^– currents were detected in 8% of cell-attached and excised, inside-out, membrane patches whereas they were observed in 24% of cell-attached and 67% of inside-out membrane patches when tubular fragments were preincubated with Forskolin (10^–5 m) or 8-bromo-cAMP (10^–4 m) and isobutylmethylxanthine (10^–5 m). The channel exhibited a linear current-voltage relationship with conductances of about 40 pS in both cell-attached and cell-free membrane configurations. AP _Na ⁺ P _Cl ^–ratio of 0.05 was estimated in the presence of a 142/42mm NaCl concentration gradient applied to inside-out membrane patches. Anionic selectivity of the channel followed the sequence Cl^–>Br^–>No ₃ ^– F^–; gluconate was not a permeant species. The open-state probability of the channel increased with membrane depolarization in cell-attached, i.e.,in situ membrane patches. In excised, inside-out, membrane patches, the channel was predominantly open with the open-state probability close to 0.8 over the whole range of potentials tested (–60 to +60 mV). The channel activity was not a function of internal calcium concentration between 10^–9 and 10^–3 m. We suggest that this Cl^– channel, whose properties are distinct from those in other epithelia, could account for the well-documented conductance which mediates Cl^– exit in the basolateral step of NaCl absorption in thick ascending limb of Henle's loop.     

Intracellular sodium activity and sodium transport inNecturus gallbladder epithelium

Summary Ion-sensitive glass microelectrodes, conventional microelectrodes and isotope flux measurements were employed inNecturus gallbladder epithelium to study intracellular sodium activity, [Na] _i , electrical parameters of epithelial cells, and properties of active sodium transport. Mean control values were: [Na] _i : 9.2 to 12.1mm; transepithelial potential difference, _ms : –1.5 mV (lumen negative); basolateral cell membrane potential, _es : –62 mV (cell interior negative); sodium conductance of the luminal cell membrane,g _Na: 12 mho cm^–2; active transcellular sodium flux, 88 to 101 pmol cm^–2 sec^–1 (estimated as instantaneous short-circuit current). Replacement of luminal Na by K led to a decrease of the intracellular sodium activity at a rate commensurate to the rate of active sodium extrusion across the basolateral cell membrane. Mucosal application of amphotericin B resulted in an increase of the luminal membrane conductance, a rise of intracellular sodium activity, and an increase of short-circuit current and unidirectional mucosa to serosa sodium flux. Conclusions: (i) sodium transport across the basolateral membrane can proceed against a steeper chemical potential difference at a higher rate than encountered under control conditions; (ii) the luminal Na-conductance is too low to accommodate sodium influx at the rate of active basolateral sodium extrusion, suggesting involvement of an electrically silent luminal transport mechanism; (iii) sodium entry across the luminal membrane is the rate-limiting step of transcellular sodium transport and active sodium extrusion across the basolateral cell membrane is not saturated under control conditions.     

Electrical characteristics of stomatal guard cells: The contribution of ATP-dependent, “Electrogenic” transport revealed by current-voltage and difference-current-voltage analysis

Summary The steady-state, current-voltage (I–V) characteristics of stomatal guard cells fromVicia faba L. were explored by voltage clamp using conventional electrophysiological techniques, but with double-barrelled microelectrodes containing 50mm K⁺-acetate. Attention was focused, primarily, on guard cell response to metabolic blockade. Exposures to 0.3–1.0mm NaCN and 0.4mm salicylhydroxamic acid (SHAM) lead consistently to depolarizing (positive-going) shifts in guard cell potentials (V _m ), as large as +103 mV, which were generally complete within 60–90 sec (mean response half-time, 10.3±1.7 sec); values forV _m in NaCN plus SHAM were close or positive to –100 mV and well removed from the K⁺ equilibrium potential. Guard cell ATP content, which was followed in parallel experiments, showed a mean half-time for decay of 10.8±1.9 ([ATP] _t=0, 1.32±0.28mm; [ATP] _{t=60–180sec}, 0.29±0.40mm). In respiring cells, theI–V relations were commonly sigmoid aboutV _m or gently concave to the voltage axis positive toV _m . Inward- and outward-rectifying currents were also observed, especially near the voltage extremes (nominally –350 and +50 mV). Short-circuit currents (atV=0 mV) were typically about 200–500 mA m^–2. The principal effect of cyanide early on was to linearize theI–V characteristic while shifting it to the right along the voltage axis, to decrease the membrane conductance, and to reduce the short-circuit current by approx. 50–75%. The resulting difference-current-voltage (dI–V) curves (±cyanide) showed a marked sensitivity to voltages negative from –100 mV and, when clamp scans had been extended sufficiently, they revealed a distinct minimum near –300 mV before rising at still more negative potentials. The difference currents, along with changes in guard cell potential, conductance and ATP content are interpreted in context of a primary, ATP-consuming ion pump. FittingdI–V curves to reaction kinetic model for the pump [Hansen, U.-P., et al. (1981)J. Membrane Biol. 63:165; Blatt, M.R. (1986)J. Membrane Biol. 92:91] implicates a stoichiometry of one (+) charge transported outward for each ATP hydrolyzed, with pump currents as high as 200 mA m^–2 at the free-running potential. The analysis indicates that the pump can comprise more than half of the total membrane conductance and argues against modulations of pump activity alone, as an effective means to controlling K⁺ transport for stomatal movements.     

Volume regulation in the early proximal tubule of theNecturus kidney

Summary The ability of early proximal tubule cells of theNecturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes.Necturus proximal tubule cells regulate volume in both hyperand hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO ₃ ^– dependent and is associated with a decrease in the relative K⁺ conductance of the basolateral cell membrane and a decrease in the resistance ratio,R _a /R _bl . Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO ₃ ^– but is also inhibited by 2mm Ba²⁺ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K⁺ conductance of the basolateral cell membrane and an increase inR _a /R _bl . Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO ₃ ^– removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO ₃ ^– -dependent transport systems and the basolateral K⁺ conductance.     

Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.     

Intracellular potassium activity and the role of potassium in transepithelial salt transport in the human reabsorptive sweat duct

Summary We have measured the intracellular potassium activity, [K⁺]_i and the mechanisms of transcellular K⁺ transport in reabsorptive sweat duct (RSD) using intracellular ion-sensitive microelectrodes (ISMEs). The mean value of [K⁺]_i in RSD is 79.8±4.1mm (n=39). Under conditions of microperfusion, the [K⁺]_i is above equilibrium across both the basolateral membrane, BLM (5.5 times) and the apical membrane, APM (7.8 times). The Na⁺/K⁺ pump inhibitor ouabain reduced [K⁺]_i towards passive distribution across the BLM. However, the [K⁺]_i is insensitive to the Na⁺/K⁺/2 Cl^– cotransport inhibitor bumetanide in the bath. Cl^– substitution in the lumen had no effect on [K⁺]_i. In contrast, Cl^– substitution in the bath (basolateral side) depolarized BLM from –26.0±2.6 mV to –4.7^*±2.4 mV (n=3;^* indicates significant difference) and decreased [K⁺]_i from 76.0±15.2mm to 57.7^* ±12.7mm (n=3). Removal of K⁺ in the bath decreased [K⁺]_i from 76.3±15.0mm to 32.3^*±7.6mm (n=4) while depolarizing the BLM from –32.5±4.1 mV to –28.3^*±3.0 mV (n=4). Raising the [K⁺] in the bath by 10-fold increased [K⁺]_i from 81.7±9.0mm to 95.0^*±13.5mm and depolarized the BLM from –25.7±2.4 mV to –21.3^*±2.9 mV (n=4). The K⁺ conductance inhibitor, Ba²⁺, in the bath also increased [K⁺]_i from 85.8±6.7mm to 107.0^*±11.5mm (n=4) and depolarized BLM from –25.8±2.2 mV to –17.0^*±3.1 mV (n=4). Amiloride at 10^–6 m increased [K⁺]_i from 77.5±18.8mm to 98.8^*±21.6mm (n=4) and hyperpolarized both the BLM (from –35.5±2.6 mV to –47.8^*±4.3 mV) and the APM (from –27.5±1.4 mV to –46.0^* ±3.5 mV,n=4). However, amiloride at 10^–4 m decreased [K⁺]_i from 64.5±0.9mm to 36.0^*±9.9mm and hyperpolarized both the BLM (from –24.7±1.4 mV to –43.5^*±4.2 mV) and APM (from –18.3±0.9 mV to –43.5^*±4.2 mV,n=6). In contrast to the observations at the BLM, substitution of K⁺ or application of Ba²⁺ in the lumen had no effect on the [K⁺]_i or the electrical properties of RSD, indicating the absence of a K⁺ conductance in the APM. Our results indicate that (i) [K⁺]_i is above equilibrium due to the Na⁺/K⁺ pump; (ii) only the BLM has a K⁺ conductance; (iii) [K⁺]_i is subject to modulation by transport status; (iv) K⁺ is probably not involved in carrier-mediated ion transport across the cell membranes; and (v) the RSD does not secrete K⁺ into the lumen.