Kinetic Analysis of the Inhibitory Effect of Glibenclamide on KATP Channels of Mammalian Skeletal Muscle

We investigated the block of K_ATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited K_ATP channels with an apparent K _i of 63 nm and a Hill coefficient of 0.85. The inhibition of K_ATP channels by glibenclamide was unaffected by internal Mg²⁺. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased. (3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and K _D to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 × 10⁷ m ⁻¹ sec⁻¹ and an unbinding rate of 6.26 sec⁻¹. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 × 10⁶ m ⁻¹ sec⁻¹. Received: 7 July 1996/Revised: 4 October 1996     

Role of Cl− in Electrogenic H+ Secretion by Cortical Distal Tubule

The presence of an electrogenic H⁺-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting duct. Transepithelial PD (V _t ) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion. The tubules were perfused with electrolyte solutions to which 2 × 10⁻⁷ m bafilomycin or 4.6 × 10⁻⁸ m concanamycin were added. No significant increase in lumen-negative V _t upon perfusion with these inhibitors as compared to control, was observed as well as when 10⁻³ m amiloride, 10⁻⁵ m benzamil or 3 mm Ba²⁺ were perfused alone or in combination. The effect of an inhibition of electrogenic H⁺ secretion, i.e., increase in lumen-negative V _t by 2–4 mV, was observed only when Cl⁻ channels were blocked by 10⁻⁵ m 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this segment from 1.21 ± 0.14 (n= 8) to 0.62 ± 0.03 (8) nmol.cm⁻².sec⁻¹ as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate that: (i) the participation of the vacuolar H⁺ ATPase in the establishment of cortical late distal tubule V _t is minor in physiological conditions, but can be demonstrated after blocking Cl⁻ channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H⁺ secretion in this segment is reduced by a Cl⁻ channel blocker, supporting coupling of H⁺-ATPase with Cl⁻ transport. Received: 6 July 1996/Revised: 27 December 1996     

Large Conductance Ca2+-activated K+ Channels in the Soma of Rat Motoneurones

Properties of large conductance Ca²⁺-activated K⁺ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K⁺ _o //155 mm K⁺ _i ) and 231 ± 4 pS in external high-K _o solution (155 mm K⁺ _o //155 mm K⁺ _i ). The channels were activated by depolarization and by an increase in internal Ca²⁺ concentration. Potentials of half-maximum channel activation (E₅₀) were −13, −34, −64 and −85 mV in the presence of 10⁻⁶, 10⁻⁵, 10⁻⁴ and 10⁻³ m internal Ca²⁺, respectively. Using an internal solution containing 10⁻⁴ m Ca²⁺, averaged K_Ca currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged K_Ca currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC₅₀) of 0.17 ± 0.02 mm. K_Ca channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that K_Ca channels activated by Ca²⁺ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997     

The CFTR Chloride Channel: Nucleotide Interactions and Temperature-dependent Gating

The gating cycle of CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channels requires ATP hydrolysis and can be interrupted by exposure to the nonhydrolyzable nucleotide AMP-PNP. To further characterize nucleotide interactions and channel gating, we have studied the effects of AMP-PNP, protein kinase C (PKC) phosphorylation, and temperature on gating kinetics. The rate of channel locking increased from 1.05 × 10⁻³ sec⁻¹ to 58.7 × 10⁻³ sec⁻¹ when AMP-PNP concentration was raised from 0.5 to 5 mm in the presence of 1 mm MgATP and 180 nm protein kinase A catalytic subunit (PKA). Although rapid locking precluded estimation of P _o or opening rate immediately after the addition of AMP-PNP to wild-type channels, analysis of locking rates in the presence of high AMP-PNP concentrations revealed two components. The appearance of a distinct, slow component at high [AMP-PNP] is evidence for AMP-PNP interactions at a second site, where competition with ATP would reduce P _o and thereby delay locking. All channels exhibited locking when they were strongly phosphorylated by PKA, but not when exposed to PKC alone. AMP-PNP increased P _o at temperatures above 30°C but did not cause locking, evidence that the stabilizing interactions between domains, which have been proposed to maintain CFTR in the open burst state, are relatively weak. The temperature dependence of normal CFTR gating by ATP was strongly asymmetric, with the opening rate being much more temperature sensitive (Q ₁₀= 9.6) than the closing rate (Q ₁₀= 3.6). These results are consistent with a cyclic model for gating of phosphorylated CFTR. Received: 28 August 1997/Revised: 4 February 1998     

Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin

To study vacuolar chloride (Cl⁻) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl⁻ uptake into isolated tonoplast vesicles was measured using the Cl⁻-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl⁻, with a Stern-Volmer constant of 173 m ⁻¹ in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl⁻-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K _m of 17.2 mm and a V _max of 4.8 mm min⁻¹. Vacuolar Cl⁻ transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl⁻ transport was actually significantly increased by 24%. To determine absolute fluxes of Cl⁻ using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 × 10⁷ m⁻¹. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl⁻ transport of 31 nmol m⁻² sec⁻¹ was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. Received: 18 February 2000/Revised: 30 June 2000     

Evidence for Negative Charge in the Conduction Pathway of the Cardiac Ryanodine Receptor Channel Provided by the Interaction of K+ Channel N-type Inactivation Peptides

We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH₂-terminal domain of the Shaker K⁺ channel with purified, ryanodine-modified, cardiac Ca²⁺-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec⁻¹μm ⁻¹ for ShB and 2000 ± 200 sec⁻¹μm ⁻¹ for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K_off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K⁺ channels, this is mediated by a large increase in K_on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel. Received: 15 December 1997/Revised: 13 March 1998     

Kinetics of K-Cl Cotransport in Frog Erythrocyte Membrane: Effect of External Sodium

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO₃) has been shown to mediate a large fraction of the total K⁺ transport. In the present study, Cl⁻-dependent and Cl⁻-independent K⁺ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K⁺ ([K⁺] _e and [K⁺] _i ) concentration. The dependence of ouabain-resistant Cl⁻-dependent K⁺ (⁸⁶Rb) influx on [K⁺] _e over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K _m ) of 8.2 ± 1.3 mm and maximal velocity (V _max ) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl⁻-dependent K⁺ influx increased both K _m (12.8 ± 1.7 mm, P < 0.05) and V _max (20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K⁺] _e above 20 mm in isotonic media significantly reduced the Cl⁻-dependent K⁺ influx due to a reciprocal decrease of the external Na⁺ ([Na⁺] _e ) concentration below 50 mm. Replacing [Na⁺] _e by NMDG⁺ markedly decreased V _max (3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K _m (15.7 ± 2.1 mm, P < 0.03) of Cl⁻-dependent K⁺ influx. Moreover, NMDG⁺ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb⁺ uptake and K⁺ loss from red cells. Cell swelling did not affect the Na⁺-dependent changes in Rb⁺ uptake and K⁺ loss. In a nominally K⁺(Rb⁺)-free medium, net K⁺ loss was reduced after lowering [Na⁺] _e below 50 mm. These results indicate that over 50 mm [Na⁺] _e is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K⁺, Cl⁻-dependent K⁺ loss in K⁺-free media was a linear function of [K⁺] _i , with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr⁻¹ (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K⁺ ions. The residual, ouabain-resistant K⁺ fluxes in NO₃ were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K⁺ effluxes in isotonic (∼0.014 hr⁻¹) and hypotonic (∼0.022 hr⁻¹) media, but cell swelling resulted in a significant increase in the rate constants. Received: 19 November 1998/Revised: 23 August 1999     

The Role of MIP in Lens Fiber Cell Membrane Transport

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in Cat ^Fr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (p _H2O ) on the order of 1 μm/sec whereas normal fiber cell membrane p _H2O was 17 μm/sec frog, 32 μm/sec rabbit and 43 μm/sec mouse. Cat ^Fr mouse lens fiber cell p _H2O was reduced by 13 μm/sec for heterozygous and 30 μm/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the p _H2O conferred by MIP is not sensitive to Hg²⁺ whereas that of CHIP28 (AQP1) is blocked by Hg²⁺. The fiber cell membrane p _H2O was also not sensitive to Hg²⁺ whereas lens epithelial cell p _H2O (136 μm/sec in rabbit) was blocked by Hg²⁺. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous Cat ^Fr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses. Received: 17 February 1999/Revised: 16 April 1999     

An L-type Calcium Channel in Renal Epithelial Cells

A voltage-activated Ca⁺⁺ channel has been identified in the apical membranes of cultured rabbit proximal tubule cells using the patch-clamp technique. With 105 mm CaCl₂ solution in the pipette and 180 NaAsp in the bath, the channel had a conductance of 10.4 ± 1.0 pS (n= 8) in on-cell patches, and 9.8 ± 1.1 pS (n= 8) in inside-out patches. In both on-cell and inside-out patches, the channel is active by membrane depolarization. For this channel, the permeation to Ba⁺⁺ and Ca⁺⁺ is highly selective over Na⁺ and K⁺ (P_Ca(Ba):P_Na(K) >200:1). The sensitivity to dihydropyridines is similar to that for L-type channels where the channel was blocked by nifedipine (10 μm), and activated by Bay K 8644 (5 μm). When activated by Bay K 8644, the channel showed subconductance levels. Treatment with forskolin (12.5 μm), phorbol ester (1 μm), or stretching (40 cm water) did not activate this channel. These results indicate that this Ca⁺⁺ channel is mostly regulated by membrane voltage, and appears to be an epithelial class of L-type Ca⁺⁺ channel. As such, it may participate in calcium reabsorption during periods of enhanced sodium reabsorption, or calcium signaling in volume regulation, where membrane depolarization occurs for prolonged periods. Received: 1 April 1996/Revised: 5 August 1996     

In Vitro Culture of Rudimentary Embryos of Ilex paraguariensis: Responses to Exogenous Cytokinins

The effects of benzyladenine (BAP), kinetin (KIN), zeatin (ZEA), isopentenyladenine (2iP), and thidiazuron (TDZ) were studied on in vitro growth of rudimentary embryos of Ilex paraguariensis St. Hil. Heart stage zygotic embryos were removed from seeds of immature, light green fruits and cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar, and supplemented with or without three concentrations of BAP, KIN, ZEA, 2iP, or TDZ. Cultures were incubated in darkness at 27 ± 2°C. Media containing 4.4 × 10⁻⁶ m BAP, 4.6 × 10⁻⁶ m KIN, or 4.9 × 10⁻⁶ m 2iP were totally ineffective in inducing embryo growth after culture for 28 days. However, lower concentrations of these compounds (4.4 × 10⁻⁸ m BAP, 4.6 × 10⁻⁸ m KIN, 4.5 × 10⁻⁸ m ZEA, or 4.9 × 10⁻⁸ m 2iP) promoted embryo growth. TDZ at 9.9 × 10⁻⁹ m, 9.9 × 10⁻⁸ m, or 9.9 × 10⁻⁷ m induced embryo growth at similar rates. The maximum percentage of embryos converted to seedlings was achieved when the medium was supplemented with 4.5 × 10⁻⁷ m ZEA. Received August 1, 1997; accepted February 19, 1998     

H+ ATPase and Cl− Interaction in Regulation of MDCK Cell pH

MDCK cells display several acid-base transport systems found in intercalated cells, such as Na⁺-H⁺ exchange, H⁺–K⁺ ATPase and Cl⁻/HCO⁻ ₃ exchange. In this work we studied the functional activity of a vacuolar H⁺-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H⁺ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH⁺ ₄ pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na⁺ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10⁻⁸ m (a specific inhibitor of the vacuolar H⁺-ATPase) was added to the zero Na⁺ solution. When using a solution with zero Na⁺, low K⁺ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H⁺–K⁺-ATPase. This result was confirmed by the use of 5 × 10⁻⁵ m Schering 28080. The Na⁺ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10⁻⁵ m (a specific blocker of Cl⁻ channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl⁻/normal Na⁺ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na⁺ and Cl⁻ free solution. From these results we conclude that: (i) MDCK cells have two Na⁺-independent mechanisms of pHi recovery, a concanamycin sensitive H⁺-ATPase and a K⁺ dependent, Schering 28080 sensitive H⁺–K⁺ ATPase; and, (ii) pHi recovery in Na⁺-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl⁻–free medium. This finding supports a role for chloride in the function of the H⁺ ATPase, which might be electrical shunting or a biochemical interaction. Received: 24 October 1997/Revised: 19 February 1998     

Transcellular Chloride Pathways in Ambystoma Proximal Tubule

The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl⁻ absorption linked to Na⁺ were investigated in isolated perfused tubules using Cl⁻-selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl⁻ (a _i ^Cl ) is 11.3 ± 0.5 mm, the basolateral (V ₁ ), apical (V ₂ ), and transepithelial (V ₃ ) potential differences are −68 ± 1.2 mV, +62 ± 1.2 mV and −6.4 ± 0.3 mV, respectively. When Na⁺ absorption is decreased by removal of organic substrates from the lumen, a _i ^Cl falls by 1.3 ± 0.3 mm and V ₂ hyperpolarizes by +11.4 ± 1.7 mV. Subsequent removal of Na⁺ from the lumen causes a _i ^Cl to fall further by 2.3 ± 0.4 mm and V ₂ to hyperpolarize further by +15.3 ± 2.4 mV. The contribution of transporters and channels to the observed changes of a _i ^Cl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on a _i ^Cl . The effects of luminal HCO⁻ ₃ removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO₃ or Cl-OH. The effects of basolateral HCO⁻ ₃ removal, of basolateral Na⁺ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO₃ exchange. Several lines of evidence favor conductive Cl⁻ transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the a _i ^Cl by 2.4 ± 0.6 mm or 2.9 ± 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO⁻ ₃-free Ringer, the equilibrium potential for Cl⁻ does not differ from the membrane potential V ₂ . Finally, the logarithmic changes in a _i ^Cl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl⁻ channels at the apical and basolateral cell membranes of the proximal tubule. Received: 14 November 1997/Revised: 6 July 1998     

Cyanide Inhibition of Chloride Conductance Across Toad Skin

The effect of cyanide (CN⁻) on voltage-activated or cAMP-induced passive chloride conductance (G ^Cl ) was analyzed in isolated toad skin. Comparatively low concentrations of CN⁻ inhibited G ^Cl almost completely and fully reversibly, regardless of whether it was applied from the mucosal or serosal side. The IC₅₀ was 180 ± 12 μm for voltage-activated G ^Cl and 305 ± 30 μm for the cAMP-inducted conductance. At [CN] <100 μm, the initial inhibition frequently declined partly in the continuous presence of CN⁻. Inhibition was independent of the presence of Ca²⁺. Inhibition was stronger at more alkaline pH, which suggests that dissociated CN⁻ is the effective inhibitor. The onset of the inhibition of voltage-activated or cAMP-induced G ^Cl by CN⁻ occurred with half-times of 34 ± 10 sec, whereas reversibility upon washout was twice as fast (18 ± 7 sec). If [CN⁻] <200 μm was applied under inactivating conditions (serosa −30 mV), the reduction of G ^Cl was stronger upon subsequent voltage-activation than under steady-state activated conditions. This effect was essentially complete less than 30 sec after apical addition of CN⁻, but G _t recovered thereafter partially in the continuous presence of CN⁻. Dinitrophenol inhibited G ^Cl similarly, while omission of oxygen did not affect it. These observations, as well as the time course of inhibition and the full reversibility, suggest that interference of CN⁻ with oxidative phosphorylation and subsequent metabolic depletion is not the reason for the inhibition of G ^Cl . We propose that the inhibition is directly on G ^Cl , presumably by competition with Cl⁻ at a rate-limiting site in the pathway. Location and molecular nature of this site remain to be identified. Received: 8 February 1999/Revised: 22 September 1999     

Inhibition of K/HCO(3) cotransport in squid axons by quaternary ammonium ions

Previous squid-axon studies identified a novel K/HCO₃ cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K⁺ and HCO⁻ ₃ out of the cell, tending to lower intracellular pH (pH_i). With an inwardly directed K/HCO₃ gradient, the cotransporter mediates a net uptake of alkali (i.e., K⁺ and HCO⁻ ₃ influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA⁺) inhibit the inwardly directed cotransporter by interacting at the intracellular K⁺ site. We computed the equivalent HCO⁻ ₃ influx (J _HCO3) mediated by the cotransporter from the rate of pH_i increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pH_i 8.0, using a dialysis fluid (DF) free of K⁺, Na⁺ and Cl⁻. Our standard artificial seawater (ASW) also lacked Na⁺, K⁺ and Cl⁻. After halting dialysis, we introduced an ASW containing 437 mm K⁺ and 0.5% CO₂/12 mm HCO⁻ ₃, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pH_i decrease, due to CO₂ influx, followed by a slower plateau-phase pH_i increase, due to inward cotransport of K⁺ and HCO⁻ ₃. With no QA⁺ in the DF, J _HCO3 was ∼58 pmole cm⁻² sec⁻¹. With 400 mm tetraethylammonium (TEA⁺) in the DF, J _HCO3 was virtually zero. The apparent K _i for intracellular TEA⁺ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K⁺ channels. Introducing 100 mm inhibitor into the DF reduced J _HCO3 to ∼20 pmole cm⁻² sec⁻¹ for tetramethylammonium (TMA⁺), ∼24 for TEA⁺, ∼10 for tetrapropylammonium (TPA⁺), and virtually zero for tetrabutylammonium (TBA⁺). The apparent K _i value for TBA⁺ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA⁺), with an apparent K _i of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO₃ influx in the potency sequence PPTEA⁺ > TBA⁺ > TPA⁺ > TEA⁺≅ TMA⁺. The identification of inhibitors of the K/HCO₃ cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter. Received: 21 November 2000/Revised: 14 May 2001     

The Essential Role of Cytosolic Cl− in Ca2+ Regulation of an Amiloride-Sensitive Channel in Fetal Rat Pneumocyte

An amiloride-sensitive, Ca²⁺-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na⁺ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca²⁺ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P _o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca²⁺. The P _o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca²⁺ to 0.26 ± 0.05 (n= 8). The cytosolic Ca²⁺ concentration ([Ca²⁺] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl⁻ concentration ([Cl⁻] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca²⁺] _c from 100 to 20 nm itself had no significant effects on the P _o if the [Cl⁻] _c was reduced to 20 mm; the P _o was 0.58 ± 0.10 at 100 nm [Ca²⁺] _c and 0.55 ± 0.09 at 20 nm [Ca²⁺] _c (n= 8) with 20 mm [Cl⁻] _c in inside-out patches. On the other hand, the P _o (0.28 ± 0.10) at 20 nm [Ca²⁺] _c with 40 mm [Cl⁻] _c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca²⁺] _c with 20 mm [Cl⁻] _c , suggesting that reduction of [Cl⁻] _c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca²⁺ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl⁻] _c . Received: 11 August 2000/Revised: 4 December 2000     

Flow-Dependent Activation of Maxi K+ Channels in Apical Membrane of Rabbit Connecting Tubule

The Ca²⁺-activated maxi K⁺ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean number of open channels (NP _o ) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the stretch-activation of Ca²⁺-permeable cation channels, because the maxi K⁺ channel was not stretch-activated in both the cell-attached configuration using Ca²⁺-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K⁺ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K⁺ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K⁺ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV _t ) when luminal K⁺ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV _t was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced ΔV _t to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV _t to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV _t was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV _t =−7.2 ± 0.5 mV in control vs.ΔV _t =−6.7 ± 0.6 mV in AA). These results suggest that the Ca²⁺-activated maxi K⁺ channel is responsible for flow-dependent K⁺ secretion by coupling with the stretch-activated Ca²⁺-permeable cation channel in the rabbit CNT. Received: 21 August 1997/Revised: 20 March 1998     

Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule

Chloride (Cl⁻) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl₂, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl⁻ current activated by 10⁻⁵ m forskolin, 10⁻³ m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10⁻⁶ m phorbol 12-myristate 13-acetate (PMA), 10⁻³ m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10⁻⁷ m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br⁻ > Cl⁻≫ I⁻ > glutamate. This current was inhibited by 10⁻³ m diphenylamine-2-carboxylate (DPC) and 10⁻⁴ m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10⁻³ m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl⁻ current blocked by DIDS. To determine the exact location of the Cl⁻ conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl⁻ removal from the apical solution induced a Cl⁻ efflux which was stimulated by 10⁻⁵ m forskolin, 10⁻⁷ calcitonin and inhibited by 10⁻⁵ m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br⁻ influx through the Cl⁻ pathway. Forskolin and calcitonin had no effect on the basolateral Cl⁻ permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl⁻ conductance in the apical membrane through a cAMP-dependent mechanism. Received: 5 July 1995/Revised: 21 December 1995     

Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes

When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1 is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino and carboxylic groups are both deprotonated. The pH value mainly influences the V _max in Na⁺ activation curves and l-leucine kinetics. The kinetic parameters are K _mNa = 4.6 ± 2 mm, V _maxNa = 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K _mNa = 2.8 ± 0.7 mm, V _maxNa = 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K _m = 120.4 ± 24.2 μm, V _max = 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K _m = 81.3 ± 24.2 μm, V _max = 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0. On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group, (ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of length and ramification. Received: 27 April 1999/Revised: 10 January 2000     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997