Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies

Summary Cystic fibrosis (CF) is characterized by abnormal epithelial Cl^– conductance (G_Cl). In vitro studies that have shown that cAMP regulation is an intrinsic property of the CF-affected G_Cl(CF-G_Cl) have been carried out previously on cultured secretory cells and on nonepithelial cells. Even though G_Cl in absorption is defective in CF, a clear demonstration of cAMP regulation of CF-G_Cl in a purely absorptive tissue is lacking. We studied the cAMP regulation of CF-G_Cl in the microperfused intact human reabsorptive sweat duct. About 40% of the ducts responded to cAMP (responsive) while the remainder of the ducts did not. In responsive ducts, cAMP-elevating agents: -adrenergic agonist isoproterenol (IPR), CPT-cAMP, forskolin, theophylline or IBMX increased G _tby about 2.3-fold (n = no. of ducts = 8). Removal of media Cl^–, but not amiloride pretreatment (in the lumen), abolished the cAMP response, indicating exclusive activation of G_Cl. cAMP activated both apical and basolateral G_Cl. cAMP hyperpolarized gluconate: Cl^– (lumen: bath) transepithelial bionic potentials (V _t=–20.3±5.2 mV, mean ±se, n=9) and transepithelial 3 1 luminal NaCl dilution diffusion potentials (V _t=–8.8±2.9 mV, n=5). cAMP activated basolateral G_Cl as indicated by increased bi-ionic (gluconate: Cl^–, bath: lumen) diffusion potentials (by about 12 mV). The voltage divider ratio in symmetric NaCl solutions increased by 60%. Compared to responsive ducts, nonresponsive ducts were characterized by smaller spontaneous transepithelial potentials in symmetrical Ringer's solution (V _t=–6.9±0.8 mV, n=24, nonresponsive vs. –19.4±1.8 mV, n=22, responsive ducts) but larger bi-ionic potentials (–94±6 mV, n=35, nonresponsive vs. –65±5 mV, n=17, responsive ducts) and dilution diffusion potentials (–40±5 mV, n=11, nonresponsive vs. –29±3 mV, n=7, responsive ducts). These results are consistent with an inherently (prestimulus) maximal activation of G_Cl in nonresponsive ducts and submaximal activation of G_Cl in responsive ducts. We conclude that cAMP activates CF-G _Cl which is expressed and abnormal in both apical and basal membranes of this absorptive epithelium in CF.Abbreviations CF cystic fibrosis - G _t transepithelial conductance - V _b electrical potential across the basolateral membrane - V _a electrical potential across the apical membrane - V _t transepithelial potential - V _b transepithelial currentinduced voltage deflections across the basolateral membrane - V _a transepithelial current-induced voltage deflections across the apical membrane - V _t transepithelial current-induced voltage deflection across the epithelium - VDR voltage divider ratio - G_Cl transepithelial Cl^– conductance - CF-G_Cl cystic fibrosis-affected Cl^– conductance - EMF electromotive force - IPR isoproterenol - IBMX 3-isobutyl-1-methylxanthine - CPT-cAMP chlorophenylthio-adenosine 3-5 cyclic monophosphate - PGE₂ prostaglandin E₂     

Voltage-dependent,monomeric channel activity of colicin E1 in artificial membrane vesicles

Summary The dependence of colicin channel activity on membrane potential and peptide concentration was studied in large unilamellar vesicles using colicin E1, its COOH-terminal thermolytic peptide and other channel-forming colicins. Channel activity was assayed by release of vesicle-entrapped chloride, and could be detected at a peptide: lipid molar ratio as low as 10^–7. The channel activity was dependent on the magnitude of atrans-negative potassium diffusion potential, with larger potentials yielding faster rates of solute efflux. For membrane potentials greater than –60mV (K _in ⁺ /K _out ⁺ 10), addition of valinomycin resulted in a 10-fold increase in the rate of Cl^– efflux. A delay in Cl^– efflux observed when the peptide was added to vesicles in the presence of a membrane potential implied a potential-independent binding-insertion mechanism. The initial rate of Cl^– efflux was about 1% of the single-channel conductance, implying that only a small fraction of channels were initially open, due to the delay or latency of channel formation known to occur in planar bilayers.The amount of Cl^– released as a function of added peptide increased monotonically to a concentration of 0.7 ng peptide/ml, corresponding to release of 75% of the entrapped chloride. It was estimated from this high activity and consideration of vesicle number that 50–100% of the peptide molecules were active. The dependence of the initial rate of Cl^– efflux on peptide concentration was linear to approximately the same concentration, implying that the active channel consists of a monomeric unit.     

Proton conductance caused by long-chain fatty acids in phospholipid bilayer membranes

Summary Mechanisms of proton conductance (G _H) were investigated in phospholipid bilayer membranes containing long-chain fatty acids (lauric, myristic, palmitic, oleic or phytanic). Membranes were formed from diphytanoyl phosphatidylcholine in decane plus chlorodecane (usually 30% vol/vol). Fatty acids were added either to the aqueous phase or to the membrane-forming solution. Proton conductance was calculated from the steadystate total conductance and the H⁺ diffusion potential produced by a transmembrane pH gradient. Fatty acids causedG _H to increase in proportion to the first power of the fatty acid concentration. TheG _H induced by fatty acids was inhibited by phloretin, low pH and serum albumin.G _H was increased by chlorodecane, and the voltage dependence ofG _H was superlinear. The results suggest that fatty acids act as simple (A^– type) proton carriers. The membrane: water partition coefficient (K _p ) and adsorption coefficient () were estimated by finding the membrane and aqueous fatty acid concentrations which gave identical values ofG _H. For palmitic and oleic acidsK _p was about 10⁵ and was about 10^–2 cm. The A^– translocation or flip-flop rate (k _a ) was estimated from the value ofG _H and the fatty acid concentration in the membrane, assuming that A^– translocation was the rate limiting step in H⁺ transport. Thek _A 's were about 10^–4 sec^–1, slower than classical weak-acid uncouplers by a factor of 10⁵. Although long-chain fatty acids are relatively inefficient H⁺ carriers, they may cause significant biological H⁺ conductance when present in the membrane at high concentrations, e.g., in ischemia, hypoxia, hormonally induced lipolysis, or certain hereditary disorders, e.g., Refsum's (phytanic acid storage) disease.     

Analog circuit of theAcetabularia membrane

Summary The high membrane potential ofAcetabularia (E _m=–170 mV) is due to an electrogenic pump in parallel with the passive diffusion system (E _d=–80 mV) which could be studied separately in the cold, when the pump is blocked. Electrical measurements under normal conditions show that the pump pathway consists of its electromotive forceE _p with two elementsP ₁ andP ₂ in series;P ₂ is shunted by a large capacitance (C _p=3 mF cm^–2). The nonlinear current-voltage relationship ofP ₁ (light- and temperature-sensitive) could be determined separately; it reflects the properties of a carrier-mediated electrogenic pump. The value ofE _p (–190 mV) indicates a stoichiometry of 21 between electrogenically transported charges and ATP. The electrical energy, normally stored inC _p, compares well with the metabolic energy, stored in the ATP pool. The nonlinear current-voltage relationship ofP ₂ (attributed to phosphorylating reactions) is also sensitive to light and temperature and is responsible for the region of negative conductance of the overall current-voltage relationship. The power of the pump (1 W cm^–2) amounts to some percent of the total energy turnover. The high Cl^– fluxes (1 nmol cm^–2 sec^–1) and the electrical properties of the plasmalemma are not as closely related as assumed previously. For kinetic reasons, a direct and specific Cl^– pathway between the vacuole and outside is postulated to exist.     

Influence of extracellular Cl concentration on Cl transport across isolated skin ofRana Pipiens

Summary The effect of changes in Cl concentration in the external and/or serosal bath on Cl transport across short-circuited frog skin was studied by measurements of transepithelial Cl influx (J ₁₃ ^Cl ) and efflux (J ₃₁ ^Cl ), short-circuit current, transepithelial potential, and conductance (G _m).J ₁₃ ^Cl as well asJ ₃₁ ^Cl were found to have a saturating component and a component which is apparently linear with Cl concentration. The linear component ofJ ₃₁ ^Cl appears only upon addition of Cl to external medium, and about 3/4 of this component does not contribute toG _m. The saturating component ofJ ₃₁ ^Cl is only 5% of totalJ ₃₁ ^Cl with 115mm Cl in the serosal medium. Replacement of 115mm Cl^– in external medium by SO ₄ ⁼ , NO ₃ ^– , HCO ₃ ^– or I^– results in 87–97% reduction ofJ ₃₁ ^Cl , whereas replacement with Br^– has no effect. As external Cl concentration is raised in steps from 2 to 115mm,J ₁₃ ^Cl andJ ₃₁ ^Cl increase by the same amount butJ ₁₃ ^Cl is persistently 0.15 eq/cm² hr larger thanJ ₃₁ ^Cl . These results indicate that at least 3/4 of linear components ofJ ₁₃ ^Cl andJ ₃₁ ^Cl proceed via an exchange diffusion mechanism which seems to be located at the outer cell border. The saturating component ofJ ₁₃ ^Cl is involved in active Cl transport in an inward direction, and there is evidence suggesting that Cl uptake across outer cell border, which proceeds against an electrochemical gradient, is electroneutral but not directly linked to Na.Reprinted from The Journal of Membrane Biology, Vol. 54, No. 3, pages 191–202. Our apologies for deleting the author's names on the original version.     

Chloride distribution in the proximal convoluted tubule ofNecturus kidney

Summary To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl^– activity ( _i ^Cl ) was studied in the proximal tubule ofNecturus kidney. Paired measurements of cell membrane PD (V _BL) and Cl-selective electrode PD (V _BL ^Cl ) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steadystate _i ^Cl was estimated at 14.6±0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl^– transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased _i ^Cl , whereas addition of the drug in luminal fluid slightly increased _i ^Cl ; both results are consistent with basolateral membrane uphill Cl^– transport from interstitium to the cell. TMA⁺ for Na⁺ substitutions in either luminal or peritubular perfusate had no effect on _i ^Cl . Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO ₃ ^– outflux), resulted in an increase of _i ^Cl , presumably related to enhanced Cl^– cell influx: we infer that Cl^– is exchanged against HCO ₃ ^– at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl^– concentration above equilibrium values: intracellular CO₂ hydration gives rise to cell HCO ₃ ^– concentrations above equilibrium. The passive exit of HCO ₃ ^– at the basolateral membrane energizes an uphill entry of Cl^– into the cell. The resulting increase of _i ^Cl , above equilibrium, generates downhill Cl^– diffusion from cell to lumen. As a result, luminal Cl^– concentration also increases.C.N.R.S. Greco 24. Part of this work was presented at the 12^th annual meeting of the American Society of Nephrology, Boston, Mass. (Edelman et al., 1979).     

Influence of extracellular Cl concentration on Cl transport across isolated skin ofRana pipiens

Summary The effect of changes in Cl concentration in the external and/or serosal bath on Cl transport across short-circuited frog skin was studied by measurements of transepithelial Cl influx (J ₁₃ ^Cl ) and efflux (J ₃₁ ^Cl ), short-circuit current, transepithelial potential, and conductance (G _m).J ₁₃ ^Cl as well asJ ₃₁ ^Cl were found to have a saturating component and a component which is apparently linear with Cl concentration. The linear component ofJ ₃₁ ^Cl appears only upon addition of Cl to external medium, and about 3/4 of this component does not contribute toG _m. The saturating component ofJ ₃₁ ^Cl is only 5% of totalJ ₃₁ ^Cl with 115mm Cl in the serosal medium. Replacement of 115mm Cl^– in external medium by SO ₄ ⁼ , NO ₃ ^– , HCO ₃ ^– or I^– results in 87–97% reduction ofJ ₃₁ ^Cl , whereas replacement with Br^– has no effect. As external Cl concentration is raised in steps from 2 to 115mm,J ₁₃ ^Cl andJ ₃₁ ^Cl increase by the same amount butJ ₁₃ ^Cl is persistently 0.15 eq/cm² hr larger thanJ ₃₁ ^Cl . These results indicate that at least 3/4 of linear components ofJ ₁₃ ^Cl andJ ₃₁ ^Cl proceed via an exchange diffusion mechanism which seems to be located at the outer cell border. The saturating component ofJ ₁₃ ^Cl is involved in active Cl transport in an inward direction, and there is evidence suggesting that Cl uptake across outer cell border, which proceeds against an electrochemical gradient, is electroneutral but not directly linked to Na.     

Membrane potentials and intracellular Cl− activity of toad skin epithelium in relation to activation and deactivation of the transepithelial Cl− conductance

Summary The potential dependence of unidirectional³⁶Cl fluxes through toad skin revealed activation of a conductive pathway in the physiological region of transepithelial potentials. Activation of the conductance was dependent on the presence of Cl^– or Br^– in the external bathing solution, but was independent of whether the external bath was NaCl-Ringer's, NaCl-Ringer's with amiloride, KCl-Ringer's or choline Cl-Ringer's To partition the routes of the conductive Cl^– ion flow, we measured in the isolated epithelium with double-barrelled microelectrodes apical membrane potentialV _a , and intracellular Cl^– activity,a _Cl ^c , of the principal cells indentified by differential interference contrast microscopy. Under short-circuit conditionsI _sc=27.0±2.0 A/cm², with NaCl-Ringer's bathing both surfaces,V _a was –67.9±3.8mV (mean ±se,n=24, six preparations) anda _Cl ^c was 18.0±0.9mM in skins from animals adapted to distilled water. BothV _a anda _Cl ^a were found to be positively correlated withI _sc (r=0.66 andr=0.70, respectively). In eight epithelia from animals adapted to dry milieu/tap waterV _a anda _Cl ^c were measured with KCl Ringer's on the outside during activation and deactivation of the transepithelial Cl^– conductance (G _Cl) by voltage clamping the transepithelial potential (V) at 40 mV (mucosa positive) and –100 mV. AtV=40 mV; i.e. whenG _Cl was deactivated,V _a was –70.1±5.0 mV (n=15, eight preparations) anda _Cl ^c was 40.0±3.8mm. The fractional apical membrane resistance (fR _a) was 0.69±0.03. Clamping toV=–100 mV led to an instantaneous change ofV _a to 31.3±5.6 mV (cell interior positive with respect to the mucosal bath), whereas neithera _Cl ^c norfR _a changed significantly within a 2 to 5-min period during whichG _Cl increased by 1.19±0.10 mS/cm². WhenV was stepped back to 40 mV,V _a instantaneously shifted to –67.8±3.9 mV whilea _Cl ^c andfR _a remained constant during deactivation ofG _Cl. Similar results were obtained in epithelia impaled from the serosal side. In 12 skins from animals adapted to either tap water or distilled water the density of mitochondria-rich (D _MRC) cells was estimated and correlated with the Cl current (I _Cl though the fully activated (V=–100mV) Cl^– conductance). A highly significant correlation was revealed (r=–0.96) with a slope of –2.6 nA/m.r. (mitochondria-rich cell and an I-axis intercept not significantly different from zero. In summary, the voltage-dependent Cl currents were not reflected infR _a anda _Cl ^a of the principal cells but showed a correlation with the m.r. cell density. We conclude that the pricipal cells do not contribute significantly to the voltage-dependent Cl conductance.     

Electrogenic bicarbonate secretion in the turtle bladder: Apical membrane conductance characteristics

Summary We have recently shown that stimulation of electrogenic HCO ₃ ^– secretion is accompanied by a simultaneous increase in short-circuit current (I _sc, equivalent to HCO ₃ ^– secretion rate under these conditions), apical membrane capacitance (C _a , proportional to membrane area), and apical membrane conductance (G _a , proportional to membrane ionic permeability). The current experiments were undertaken to explore the ionic basis for the increase inG _a and the possibility that the rate of electrogenic HCO ₃ ^– secretion is regulated by changes inG _a . Membrane electrical parameters were measured using impedance-analysis techniques before and after stimulation of electrogenic HCO ₃ ^– secretion with cAMP in three solutions which contained different chloride concentrations. In another series of experiments, the effects of an anion channel blocker, anthracene-9-carboxylic acid (9-AA), were measured after stimulation of electrogenic HCO ₃ ^– secretion with cAMP. The major conclusions are: (i) a measurable apical Cl^– conductance exists in control hemibladders; (ii) the transport-associated increase inG _a includes a Cl^–-conductive component; (iii)G _a also appears to reflect a HCO ₃ ^– conductance; (iv) the relative magnitudes of the apical membrane conductances to Cl^– and HCO ₃ ^– are similar; (v) 9-AA reducesG _a andI _sc appear cAMP-stimulated hemibladders; and (vi) alterations inI _sc appear to be mediated by changes inG _a .     

Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C₁ cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C₁ was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10) (m) increased furosemideinsensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanideinsensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloridedepleted cells. When cells with normal chloride content were depolarized (135 mm medium potassium + 10 m valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl^–Br^–>NO ₃ ^– >I^–. The electrogenic transport pathway was inhibited in chloridedepleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.The technical assistance of Dwight Derr is gratefully acknowledged. We also thank Dr. Chahrzad Montrose-Rafizadeh for help in performance of the chloride efflux experiments. This work was supported by National Institutes of Health grants RO1-DK42457 and PO1-DK44484.     

Membrane potential,anion and cation conductances in Ehrlich ascites tumor cell

Summary The fluorescence intensity of the dye 1,1-dipropyloxadicarbocyanine (DiOC₃-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V _m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na⁺-free K⁺/choline⁺ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin null point procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl^–-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN^– as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV _m reported in this paper. Incubation with DiOC₃-(5) for 5 min is demenstrated to reduce the Cl^– permeability by 26±5% and the NO ₃ ^– permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K⁺ permeability. Values forV _m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at –61±1 mV in isotonic standard NaCl medium, –78±3 mV in isotonic Na⁺-free choline medium and –46±1 mV in isotonic NaNO₃ medium. The cell membrane is depolarized by addition of the K⁺ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na⁺-free choline medium, indicating thatV _m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO ₃ ^– for all cellular and extracellular Cl^– leads to a depolarization of the membrane, demonstrating thatV _m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K⁺ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 S/cm² for K⁺, 3.0 S/cm² for Na⁺, 0.6 S/cm² for Cl^– and 8.7 S/cm² for NO ₃ ^– . Addition of the Ca²⁺-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K⁺ permeability exceeds the Cl^– permeability also after the addition of A23187. The K⁺ and Cl^– conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 S/cm², respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl^– permeability following hypotonic exposure exceeds the concommitant increase in the K⁺ permeability. In control experiments where the membrane potentialV _m =E _K =E _Cl =E _Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl^– conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl^– efflux and the change inV _m . The concommitant increase in the K⁺ conductance, as estimated from the net K⁺ efflux, is only twofold.     

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.     

Proton conductance through phospholipid bilayers: Water wires or weak acids?

The proton/hydroxide (H⁺/OH^–) permeability of phospholipid bilayer membranes at neutral pH is at least five orders of magnitude higher than the alkali or halide ion permeability, but the mechanism(s) of H⁺/OH^– transport are unknown. This review describes the characteristics of H⁺/OH^– permeability and conductance through several types of planar phospholipid bilayer membranes. At pH7, the H⁺/OH^– conductances (G _H/OH) range from 2–6 nS cm^–2, corresponding to net H⁺/OH^– permeabilities of (0.4–1.7)×10^–5 cm sec^–1. Inhibitors ofG _H/OH include serum albumin, phloretin, glycerol, and low pH. Enhancers ofG _H/OH include chlorodecane, fatty acids, gramicidin, and voltages >80 mV. Water permeability andG _H/OH are not correlated. The characteristics ofG _H/OH in fatty acid (weak acid) containing membranes are qualitatively similar to the controls in at least eight different respects. The characteristics ofG _H/OH in gramicidin (water wire) containing membranes are qualitatively different from the controls in at least four different respects. Thus, the simplest explanation for the data is thatG _H/OH in unmodified bilayers is due primarily to weakly acidic contaminants which act as proton carriers at physiological pH. However, at low pH or in the presence of inhibitors, a residualG _H/OH remains which may be due to water wires, hydrated defects, or other mechanisms.     

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.     

Electrical properties of chloride transport across theNecturus proximal tubule

Summary The chloride conductance of the basolateral cell membrane of theNecturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral, cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl^–. When extracellular Cl^– activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl^– conductance of the paracellular shunt but a low Cl^– conductance of the cell membranes. Reduction of Cl^– in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl^– perfusion was abolished by exposure to HCO ₃ ^– -free solutions and SITS treatment. In control conditions, intracellular Cl^– activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl^– in only the basolateral solution caused a decrease in intracellular Cl^–. From an estimate of the net Cl^– flux across the basolateral cell membrane and the electrochemical driving force, a Cl^– conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl^– conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl^– by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl^– transport across the basolateral cell membrane of theNecturus proximal tubule cell.     

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.     

Characterization of chloride channels in membrane vesicles from the kidney outer medulla

Summary The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is highly enriched in Na⁺/K⁺ ATPase and has been shown by electrophysiological methods to be highly conductive to Cl^–. In order to study the Cl^– conductive pathways, membrane vesicles were isolated from the TALH-containing region of the porcine kidney, the red outer medulla, and Cl^– channel activity was determined by a³⁶Cl uptake assay where the uptake of the radioactive tracer is driven by the membrane potential (positive inside) generated by an outward Cl^– gradient. The accumulation of³⁶Cl^– inside the vesicles was found to be dependent on the intravesicular Cl^– concentration and was abolished by clamping the membrane potential with valinomycin. The latter finding indicated the involvement of conductive pathways. Cl^– channel activity was also observed using a fluorescent potential-sensitive carbocyanine dye, which detected a diffusion potential induced by an imposed inward Cl^– gradient. The anion selectivity of the channels was Cl^–>NO ₃ ^– =I^– gluconate. Among the Cl^– transport inhibitors tested, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB), 4,4-diisothiocyano-stilbene-2,2-disulfonate (DIDS), and diphenylamine-2-carboxylate (DPC) showed IC₅₀ of 110, 200 and 550 m, respectively. Inhibition of³⁶Cl uptake by NPPAB and two other structural analogues was fully reversible, whereas that by DIDS was not. The nonreactive analogue of DIDS, 4,4-dinitrostilbene-2,2-disulfonate (DNDS), was considerably less inhibitory than DIDS (25% inhibition at 200 m). The irreversible inhibition by DIDS was prevented by NPPAB, whereas DPC was ineffective, consistent with its low inhibitory potency. It is proposed that NPPAB and DIDS bind to the same or functionally related site on the Cl^– channel protein.     

Nitrate and chloride ions have different permeation pathways in skeletal muscle fibers ofRana pipiens

Summary The effects of pH on the permeability and conductance of the membranes to nitrate and to chloride of semitendinosus and lumbricalis muscle fibers were examined.Membrane potential responses to quick solution changes were recorded in semitendinosus fibers initially equilibrated in isotonic, high K₂SO₄ solutions. External solutions were first changed to ones in which either Rb⁺ or Cs⁺ replaced K⁺ and then to solutions containing either NO ₃ ^– or Cl^– to replace SO ₄ ^2– . The hyperpolarizations produced by Cl^– depend on external pH, being smaller in acid than in alkaline solutions. By contrast, hyperpolarizations produced by NO ₃ ^– were independent of external pH over a pH range from 5.5 to 9.0.In addition, voltage-clamp measurements were made on short lumbricalis muscle fibers. Initially they were equilibrated in isotonic solutions containing mainly K₂SO₄ plus Na₂SO₄. KCl or KNO₃ were added to the sulfate solutions and the fibers were equilibrated in these new solutions. When finally equilibrated the fibers had the same volume they had in the sulfate solutions before the additions. Constant hyperpolarizing voltage pulses of 0.6-sec duration were applied when all external K⁺ was replaced by TEA⁺. For these conditions, inward currents flowing during the voltage pulses were largely carried by Cl^– or NO ₃ ^– depending on the final equilibrating solution. Cl^– currents during voltage pulses were both external pH and time dependent. By contrast, NO ₃ ^– currents were independent of both external pH and time.The voltage dependence of NO ₃ ^– currents could be fit by constant field equations with aP _{NO 3} of 3.7·10^–6 cm/sec. The voltage dependence of the initial or instantaneous Cl^– currents at pH 7.5 and 9.0 could also be fit by constant field equations with P_Cl of 5.8·10^–6 and 7.9·10^–6 cm/sec, respectively. At pH 5.0, no measurable instantaneous Cl^– currents were found.From these results we conclude that NO ₃ ^– does not pass through the pH, time-dependent Cl^– channels but rather passes through a distinct set of channels. Furthermore, Cl^– ions do not appear to pass through the channels which allow NO ₃ ^– through. Consequently, the measured ratio ofP _Cl/P _{NO 3} based on membrane potential changes to ionic changes made on intact skeletal muscle fibers is not a measure of the selectivity of a single anion channel but rather is a measure of the relative amounts of different channel types.     

Mechanisms of Cl− uptake through brush border membranes isolated from the posterior intestine of the freshwater trout,Oncorhynchus mykiss,R.

Summary This paper presents a study of the mechanisms of Cl^– transport through the brush border membranes of the posterior part of the intestine in the freshwater trout, Oncorhynchus mykiss. The mechanisms for Cl^– transport in the posterior intestine are distinct from those in the middle intestine; an inwardly directed pH gradient stimulates Cl^– uptake by bursh border membrane vesicles, indicating a Cl^–/OH^– exchange. A pH-regulated Cl^– conductance is present, which is not activated at normal intracellular pH. Cl^– uptake is stimulated by an outwardly directed HCO ₃ ^– gradient revealing the presence of a Cl^–/HCO ₃ ^– exchange but, conversely, Cl^– is not exchanged against SO ₄ ^2- . In addition, carbonic anhydrase activities have been detected in both the intracellular and extracellular leaflets of the bursh border membranes which favour the establishment of a bicarbonate gradient. A model of Cl^– transport mechanisms through the brush-border membranes of the posterior intestine of the freshwater trout is proposed.Abbreviations BBM brush border membrane - CA carbonic anhydrase - EGTA ethylene-bis(oxyethylenenitrilo)tetra-acetic acid - FW fresh water - Hepes N-2-hydroxy-ethyl-piperazine-N'-2-ethanesulphonic acid - Mes 2-(N-morpholino)ethane sulphonic acid - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid - TEA triethanolamine - TMA tetramethylammonium - TRIS tris(hydroxymethyl)aminomethane