Diffusion of Small Solutes in the Lateral Intercellular Spaces of MDCK Cell Epithelium Grown on Permeable Supports

The diffusion coefficients of four solutes ranging in molecular weight from 238 to 10,000 in the lateral intercellular spaces (LIS) of cultured kidney cells (MDCK) grown on permeable supports were determined from the spread of fluorescence produced after the release of caged compounds by a pulse from a UV laser. Two types of experiments were performed: measurement of the rate of change of fluorescence after releasing a caged fluorophore, and measurement of the change in fluorescence of a relatively static fluorescent dye produced by the diffusion of an uncaged ligand for the dye. Fluorescence intensity was determined by photon-counting the outputs of a multichannel photomultiplier tube. Diffusion coefficients were determined in free solution as well as in the LIS of MDCK cells grown on permeable supports and the hindrance factor, θ, determined from the ratio of the free solution diffusivity to that in the LIS. The hindrance factors for 3000-MW dextran, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, MW 524) and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES, MW 238) were not significantly different from 1. The diffusion of 10,000-MW dextran was substantially reduced in the LIS with a θ of 5.6 ± 0.3. Enzymatic digestion by neuraminidase of the sialic acid residues of the glycosylation groups in the LIS increased the diffusivity of the 10,000-MW dextran 1.8-fold indicating hindrance by the glycocalyx. We conclude that small solutes, such as Na⁺ and Cl⁻, would not be significantly restricted in their diffusion in the LIS and that solute concentration gradients could not develop along the LIS under physiologic conditions. Received: 7 October 1999     

Hydraulic Properties of MDCK Cell Epithelium

The water permeability of the apical and basolateral cell membranes and the compliance of the lateral intercellular spaces (LIS) of MDCK monolayers were measured on confluent cultures grown on permeable supports. Cell membrane water permeabilities were determined, using quantitative differential interference light microscopy, from the rate of cell volume decrease after exposure to a hyperosmotic bathing solution. Both membranes exhibited osmotic water permeabilities (P_OSM) of ∼10 μm/sec, comparable to that of unmodified lipid bilayers. The compliance of the cell membranes forming the lateral intercellular space (LIS) between cells was determined from the pressure-volume relation. Confocal microscopy of fluorescent labeling of the basolateral cell membranes was used to delineate the LIS geometry as transepithelial hydrostatic pressure was varied. The LIS were poorly deformable as a function of transepithelial hydrostatic pressure until a pressure of ≥8 cm H₂O (basolateral > apical) was reached where catastrophic failure of intercellular connections occurred. The compliance of the LIS was calculated from the geometry changes at pressures <8 cm H₂O and ranged from 0.05–0.11 cm H₂O⁻¹, comparable to that previously predicted in mathematical models of the rat proximal tubule. Received: 10 January 1996/Revised: 9 May 1996     

Regulation of the MDCK Cell Tight Junction

The sodium flux across individual tight junctions (TJ) of low-resistance MDCK cell monolayers grown on glass coverslips was determined as a measure of paracellular permeability. Increases in perfusate glucose concentration from 5 to 25 mm decreased tight junction Na permeability. This permeability decrease was not specific as nonmetabolizable analogues of glucose caused similar diminutions in TJ Na permeability. Stimulation of protein kinase A increased TJ Na permeability, and inhibition of protein kinase A decreased TJ Na permeability. Transepithelial electrical resistance of monolayers grown on permeable supports did not change as predicted from the observed alterations in TJ Na permeability of monolayers grown on glass coverslips. Fluorescent labeling of cell F-actin showed that increased F-actin in the perijunctional ring correlated with higher TJ Na permeability. Although a low dose of cytochalasin D did not change TJ Na permeability, it disrupted the cytoskeleton and blocked the decrease in TJ Na permeability caused by glucose. Cytochalasin D failed to block the effects of protein kinase A stimulation or inhibition on TJ Na permeability. We conclude that tight junction sodium permeability is regulated both by protein kinase A activity and by other processes involving the actin cytoskeleton. Received: 17 June 1997/Revised: 28 August 1997     

The Apical Membrane Glycocalyx of MDCK Cells

The microenvironment near the apical membrane of MDCK cells was studied by quantitation of the fluorescence of wheat germ agglutin attached to fluorescein (WGA). WGA was shown to bind to sialic acid residues attached to galactose at the α-2,3 position in the glycocalyx on the apical membrane. Young MDCK cells (5–8 days after splitting) showed a patchy distribution of WGA at stable sites that returned to the same locations after removal of sialic acid residues by neuraminidase treatment. Other lectins also showed stable binding to patches on the apical membrane of young cells. The ratio of WGA fluorescence emission at two excitation wavelengths was used to measure near-membrane pH. The near-membrane pH was markedly acidic to the pH 7.4 bathing solution in both young and older cells (13–21 days after splitting). Patches on the apical membrane of young cells exhibited a range of near-membrane pH values with a mean ±sem of 6.86 ± 0.04 (n= 121) while the near-membrane pH of older cells was 6.61 ± 0.04 (n= 120) with a uniform WGA distribution. We conclude that the distribution of lectin binding sites in young cells reflects the underlying nonrandom location of membrane proteins in the apical membrane and that nonuniformities in the pH of patches may indicate regional differences in membrane acid-base transport as well as in the location of charged sugars in the glycocalyx. Received: 15 December 1999/Revised: 16 March 2000     

Bending the MDCK Cell Primary Cilium Increases Intracellular Calcium

We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca²⁺-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by calcium release from IP₃-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium. Received: 4 April 2001/Revised: 28 June 2001     

Cytoplasmic pH Responses to Carbonic Anhydrase Inhibitors in Cultured Rabbit Nonpigmented Ciliary Epithelium

Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pH _i ) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pH _i was measured using the fluorescent dye BCECF and the pH _i responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared. ACTZ markedly inhibited the rapid pH _i changes elicited by bicarbonate/CO₂ removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pH _i . We considered whether CA-IV might facilitate H⁺ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mm) reduced pH _i 0.52 ± 0.10 pH units. In the presence of DBI, the magnitude of pH _i reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 ± 0.09 pH units. ACTZ similarly reduced the magnitude of the pH _i reduction. DBI also reduced by ∼40% the rate of pH _i recovery in cells acidified by an ammonium chloride (20 mm) prepulse; a reduction in pH _i recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pH _i alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H⁺ and HCO⁻ ₃ with CO₂ in the unstirred layer outside the plasma membrane, preventing local accumulation of H⁺ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pH _i changes that might alter the function of other ion transporters and channels in the NPE. Received: 24 April 1997/Revised: 4 November 1997     

The Role of Membrane Lateral Tension in Calcium-Induced Membrane Fusion

Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition: at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM) was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca²⁺, Mg²⁺, Cd²⁺ and La³⁺ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely lipidic and protein-mediated membrane fusion is discussed. Received: 27 September 1999/Revised: 22 March 2000     

Lateral Microheterogeneity of Diphenylhexatriene-Labeled Choline Phospholipids in the Erythrocyte Ghost Membrane as Determined by Time-Resolved Fluorescence Spectroscopy

Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane. Received: 22 July 1999/Revised: 6 January 2000     

Studies on H+-ATPase in Cultured Rabbit Nonpigmented Ciliary Epithelium

Studies were conducted to examine the influence of the H⁺-ATPase inhibitor bafilomycin A₁ on cultured rabbit nonpigmented ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A₁ (100 nm) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A₁-treated cells as judged by a 50% increase of ouabain sensitive potassium (⁸⁶Rb) uptake. In bafilomycin A₁-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium (⁸⁶Rb) uptake in a dose-dependent manner with an IC₅₀ of ∼2 μm. DMA (10 μm) also prevented the increase of cytoplasmic sodium caused by bafilomycin A₁. Added alone, DMA (10 μm) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by ∼0.4 pH units. In cells that first received 10 μm DMA, the subsequent addition of bafilomycin A₁ (100 nm) caused a further cytoplasmic pH reduction of ∼0.3 pH units. Taken together, the results suggest H⁺-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A₁, Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport. Received: 19 March 1999/Revised: 20 September 1999     

Transfection Alters Ion Transport in MDCK Cells

In the course of an investigation into the effect of Tamm-Horsfall protein (THP) on ion transport, we performed stable transfection of THP into MDCK cells using the SV40 or the cytomegalovirus (CMV) promoter. As controls, we transfected MDCK cells with an ``empty' plasmid containing SV40 or CMV promoter but without THP cDNA. In another set of controls, we subjected cells to transfection procedures without DNA (mock transfection). K influx was not altered in cells subjected to mock transfection procedures without DNA, but both ouabain sensitive (OS) and ouabain resistant (OR) components of K influx were diminished in cells transfected with THP cDNA using either SV40 or CMV promoter. However, K influx was also reduced in cells transfected with a control plasmid containing either the SV40 promoter alone, or the CMV promoter alone, without the THP cDNA. Thus, the transport alterations were caused by transfection and not by THP. The reduction in ouabain-sensitive K influx was accompanied by a proportional reduction in the abundance of Na-K pump units as assessed by [³H] ouabain binding. [³H] bumetanide binding, a measure of the number of functioning NaK2Cl cotransporter sites, was reduced pari passu with the reduction in bumetanide-sensitive K influx. These results highlight the possibility that alterations in properties of transfected cells may not be solely due to the presence of transfected protein, but the result of some process associated with transfection itself. Without appropriate controls to evaluate this possibility, results of transfection studies are subject to potentially faulty and misleading interpretation. Received: 25 April 1995/Revised: 25 September 1995     

A Mathematical Model of the Pancreatic Ductal Epithelium

A mathematical model of the HCO⁻ ₃-secreting pancreatic ductal epithelium was developed using network thermodynamics. With a minimal set of assumptions, the model accurately reproduced the experimentally measured membrane potentials, voltage divider ratio, transepithelial resistance and short-circuit current of nonstimulated ducts that were microperfused and bathed with a CO₂/HCO⁻ ₃-free, HEPES-buffered solution, and also the intracellular pH of duct cells bathed in a CO₂/HCO⁻ ₃-buffered solution. The model also accurately simulated: (i) the effect of step changes in basolateral K⁺ concentration, and the effect of K⁺ channel blockers on basolateral membrane potential; (ii) the intracellular acidification caused by a Na⁺-free extracellular solution and the effect of amiloride on this acidification; and (iii) the intracellular alkalinization caused by a Cl⁻-free extracellular solution and the effect of DIDS on this alkalinization. In addition, the model predicted that the luminal Cl⁻ conductance plays a key role in controlling both the HCO⁻ ₃ secretory rate and intracellular pH during HCO⁻ ₃ secretion. We believe that the model will be helpful in the analysis of experimental data and improve our understanding of HCO⁻ ₃-transporting mechanisms in pancreatic duct cells. Received: 18 October 1995/Revised: 5 July 1996     

H+-ATPase-Mediated Cytoplasmic pH-Responses Associated with Elevation of Cytoplasmic Calcium in Cultured Rabbit Nonpigmented Ciliary Epithelium

Studies were conducted to test whether an increase of cytoplasmic calcium concentration influences H⁺-ATPase activity in cultured rabbit nonpigmented ciliary epithelium (NPE). Cytoplasmic calcium concentration or cytoplasmic pH was measured by a fluorescence ratio technique in cells loaded with either Fura-2 or BCECF. Cytoplasmic calcium was increased in three ways; by exposure to BAY K 8644 (1 μm), by exposure to a mixture of epinephrine (1 μm) + acetylcholine (10 μm) or by depolarization with potassium-rich solution. In each case cytoplasmic pH increased significantly. In all three cases 100 nm bafilomycin A₁, a specific H⁺-ATPase inhibitor, significantly inhibited the pH increase. These results suggest an increase of cytoplasmic calcium might initiate events that lead to activation of proton export from the cytoplasm by a mechanism involving H⁺-ATPase. This notion is supported by the observation that the pH increase was suppressed when either verapamil or nifedipine was used to prevent the cytoplasmic calcium increase in cells exposed to potassium-rich solution. Protein kinase C activation might also be involved in the mechanism of H⁺-ATPase stimulation since staurosporine suppressed the pH response to potassium-rich solution. A transient rise of cytoplasmic calcium concentration was observed when cytoplasmic acidification was induced by exposure to high pCO₂. This suggests a rise of cytoplasmic calcium might represent part of a physiological mechanism to stimulate H⁺-ATPase-mediated protein export under acid conditions. Received: 11 August 2000/Revised: 29 March 2001     

A Basolateral Chloride Conductance in Rat Lingual Epithelium

We used Ussing chamber measurements and whole-cell recordings to characterize a chloride conductance in rat lingual epithelium. Niflumic acid (NFA) and flufenamic acid (FFA), nonsteroidal anti-inflammatory aromatic compounds known to inhibit Cl⁻ conductances in other tissues, reduced transepithelial short-circuit current (I _sc ) in the intact dorsal anterior rat tongue epithelium when added from the serosal side, and reduced whole-cell currents in rat fungiform taste cells. In both Ussing chamber and patch-clamp experiments, the effect of NFA was mimicked by replacement of bath Cl⁻ with methanesulfonate or gluconate. In low Cl⁻ bath solution, the effect of NFA on whole-cell current was reduced. Replacement of bath Ca²⁺ with Ba²⁺ reduced the whole-cell Cl⁻ current. We conclude that a Ca²⁺-activated Cl⁻ conductance is likely present in the basolateral membrane of the rat lingual epithelium, and is present in the taste receptor cells from fungiform papillae. Further experiments will be required to identify the role of this conductance in taste transduction. Received: 8 September 1997/Revised: 27 March 1998     

Isoform-Specific Function and Distribution of Na/K Pumps in the Frog Lens Epithelium

Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies of the Na/K ATPase. RNase protection assays showed that all cells express α₁- and α₂-isoforms of the Na/K pump but not the α₃-isoform, however the α₂-isoform dominates in anterior cells whereas the α₁-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the Na/K pump current (I _P ), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I _P blockade data indicate the α₁-isoform has a dissociation constant of 100 μm DHO whereas for the α₂-isoform it is 0.75 μm DHO. Both α₁- and α₂-isoforms are half maximally activated at an intracellular Na⁺-concentration of 9 mm. The α₁-isoform is half maximally activated at an extracellular K⁺-concentration of 3.9 mm whereas for the α₂-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the α₁-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the α₂-isoform. Under normal physiological conditions, I _P in equatorial cells is approximately 0.23 μA/μF, and in anterior cells it is about 0.14 μA/μF. These current densities refer to the area of cell membrane assuming a capacitance of around 1 μF/cm². Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 μA/cm² at the equator vs. 0.5 μA/cm² at the anterior pole. Received: 17 May 2000/Revised: 11 August 2000     

Fusion of Human Sperm to Prostasomes at Acidic pH

Prostasomes are membranous vesicles (150–200 nm diameter) present in human semen. They are secreted by the prostate and contain large amounts of cholesterol, sphingomyelin and Ca²⁺. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. The possibility that they may fuse to sperm has never been proved. In this work, we studied the fusion of sperm to prostasomes by using various methods (relief of octadecyl Rhodamine B fluorescence self-quenching, fluorescence microscopy and flow cytometry) and we found that it occurs at acidic pH (4–5), but not at pH 7.5 pH-dependent fusion relies on the integrity of one or more proteins and is different from the Ca²⁺-stimulated fusion between rat liver liposomes and spermatozoa that does not require any protein and occurs at neutral pH. We think that the H⁺-dependent fusion of prostasomes to sperm may have physiological importance by modifying the lipid and protein pattern of sperm membranes. Received: 19 June 1996/Revised: 4 September 1996     

Ion,Fluid and Charge Transport Across Necturus Intestinal Epithelium in Response to Alanine

Fluxes of Na, Cl and volume were followed across Necturus small intestine under zero voltage clamp. 20 mm l-alanine doubles the net Na and fluid transfer. Although there is a ouabain-sensitive Na pump present in Necturus a major fraction of the net Na flux can be measured for an hour after application of 10⁻³ m ouabain. Collected fluid transferred by the epithelium is quasi-isotonic over a range of luminal osmolarities from 100 to 250 milliosmolar in alanine saline. The net Na fluxes account for the Na found in this transported fluid. Fluid transfer also shows a large ouabain-insensitive fraction after the addition of alanine. Compartmental analysis of ²²Na-loaded epithelium was used to separate cellular and paracellular fluxes. The estimated Na concentration in the cell derived from its Na content is 9–10 mm, in agreement with that determined with microelectrodes. The Na efflux from cell to serosa is stimulated by alanine, but this increase accounts for only a quarter of the simultaneous rises in Na, fluid and current flow across the epithelium. The increase of Na efflux from the cell induced by alanine is apparently insensitive to ouabain although the cell Na content rises to circa 20 mm but no higher even after 20 hr. From the initial rate of rise of Na in the cell on treatment with ouabain the activity of the Na pump can be estimated to be ∼92 pM/cm²· sec, a value much smaller than the transepithelial net flux. The results are not consistent with the standard model in which Na-alanine influx stimulates the Na pump and enhances fluid transport by osmotic coupling in the lateral interspace system. A scheme is proposed based upon that for absorption in Necturus gallbladder by which alanine stimulates an active paracellular fluid transfer driven by motile elements of the junction. Received: 5 August 1996/Revised: 7 February 1997     

Directly Observed Membrane Fusion Between Oppositely Charged Phospholipid Bilayers

A novel method was developed for the direct examination of pairwise encounters between positively and negatively charged phospholipid bilayer vesicles. Giant bilayer vesicles (unilamellar, 4–20 μm in diameter) prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a new cationic phospholipid derivative, were electrophoretically maneuvered into contact with individual anionic phospholipid vesicles. Fluorescence video microscopy revealed that such vesicles commonly underwent fusion within milliseconds (1 video field) after contact, without leakage. Fusion occurred at constant volume and, since flaccid vesicles were rare, the excess membrane was not available after fusion. Hemifusion (the outer monolayers of each vesicle fused while the inner monolayers remained intact) was inferred from membrane-bound dye transfer and a change in the contact area. Hemifusion was observed as a final stable state and as an intermediate to fusion of vesicles composed of charged phospholipids plus zwitterionic phospholipids. Hemifusion occurred in one of three ways following adhesion: either delayed with an abrupt increase in area of contact, immediately with a gradual increase in area of contact, or with retraction during which adherent vesicles dissociated from a flat contact to a point contact. Phosphatidylethanolamine strongly promoted immediate hemifusion; the resultant hemifused state was stable and seldom underwent complete fusion. Although sometimes single contacts between vesicles led to rupture of both, in other cases, a single vesicle underwent multiple fusion events. Direct observation has unequivocally demonstrated the fusion of two, isolated bilayer-bounded bodies to yield a stable, non-leaky product, as occurs in cells, in the absence of proteins. Received: 25 November 1998/Revised: 23 March 1999     

Regulation of Ca2+-activated K+ Channel from Rabbit Distal Colon Epithelium by Phosphorylation and Dephosphorylation

The Ca²⁺-activated maxi K⁺ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na⁺ reabsorption from the intestinal lumen. Previous measurements of these basolateral K⁺ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca²⁺ with a K _0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca²⁺. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K⁺ channels lost their high sensitivity to Ca²⁺, yet they could still be activated by high concentrations of Ca²⁺ (10 μm). Furthermore, the high sensitivity to Ca²⁺ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30–120 sec for dephosphorylation and of 10–280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca²⁺ of the maxi K⁺ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca²⁺ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca²⁺ sensitivities for maxi K⁺ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems. Received: 28 February 1995/Revised: 22 December 1995     

External Protons Enhance the Activity of the Hyperpolarization-activated K Channels in Guard Cell Protoplasts of Vicia faba

Hyperpolarization-activated K channels (K _H channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K _H channels in isolated guard cell protoplasts from Vicia faba leaves. Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K _H channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK _a of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing of these charges was ∼30 ? and their apparent dissociation constant was 10^−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence of all four transition rate constants. Received: 26 April 1996/Revised: 29 June 1996