Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.     

Calcium-activated chloride conductance of lactotrophs: Comparison of activation in normal and tumoral cells during thyrotropin-releasing-hormone stimulation

We studied a chloride (Cl-) conductance activated by calcium (Ca2+) in normal rat lactotrophs and compared its activation during TRH stimulation in normal rat lactotrophs and in GH3 tumoral lactosomatotrophs cells, using the whole-cell configuration of the patch-clamp technique. The Cl- specificity of the conductance was assessed by manipulation of internal and external Cl- concentrations. The reversal potentials were in agreement with those predicted by the Nernst equation. Ca2+ ionophore A23187 and membrane depolarizations activated the Cl- conductance. However, a feedback effect of Cl- gradient modifications on Ca2+ movements was also observed in normal lactotrophs. In the latter, TRH (100 nM) mobilization of intracellular Ca2+ activated this Cl- conductance together with the potassium (K+) conductance when both ions were present in the intracellular medium (IM) or alone when K+ was absent. Chloride conductance was not activated in the GH3 cells, where mobilization of intracellular Ca2+ by TRH (100 nM) activated only Ca2(+)-dependent K+ conductance. It seems likely that the activation of Cl- conductance in these two different cell types involves different mechanisms.     

Potassium channels along the nephron

The K+ channels that are present in three different nephron segments, the Necturus proximal, Amphiuma early distal (diluting segment), and rabbit collecting tubule have been examined. Ca2+-sensitive K+ channels were present in the apical membranes of the cells lining all these segments. The channels were all voltage-sensitive and their open probability increased with membrane depolarization. Because of the ubiquitous distribution, it is suggested that this channel is responsible for K+ secretion by the nephron and that the same intracellular regulators act throughout the various segments. Basolateral K+ channels have been examined only in Necturus proximal tubules. This channel is apparently insensitive to Ca2+; the voltage dependence is exactly opposite to that of the apical K+ channels; that is, hyperpolarizing potentials caused an increase in open probability. These differences in regulatory factors permit the independent regulation of apical and basolateral membrane K+ permeabilities that must occur in renal cells.     

Distribution of ion channels on taste cells and its relationship to chemosensory transduction

Summary The presence and regional localization of voltagegated ion channels on taste cells inNecturus maculosus were studied. Lingual epithelium was dissected from the animal and placed in a modified Ussing chamber such that individual taste cells could be impaled with intracellular microelectrodes and the chemical environment of the apical and basolateral membranes of cells could be strictly controlled. That is, solutions bathing the the mucosal and serosal surfaces of the epithelium could be exchanged independently and the effects of pharmacological agents could be tested selectively on the apical or basolateral membranes of taste cells. In the presence of amphibian physiological saline, action potentials were elicited by passing brief depolarizing current pulses through the recording electrode. Action potentials provided a convenient assay of voltage-gated ion channels. As in other excitable tissues, blocking current through Na⁺, K⁺, or Ca²⁺ channels had predictable and consistent effects on the shape and magnitude of the action potential. A series of experiments was conducted in which the shape and duration of regenerative action potentials were monitored when the ionic composition was altered and/or pharmacological blocking agents were added to the mucosal or to the serosal chamber. We have found the following: (1) voltage-gated K⁺ channels (delayed rectifier) are found predominately, if not exclusively, on the chemoreceptive apical membrane; (ii) voltage-gated Na⁺ and Ca²⁺ channels are found on the apical (chemoreceptive) and basolateral (synaptic) membrane; (iii) there is a K⁺ leak channel on the basolateral membrane which appears to vary seasonally in its sensitivity to TEA. The nonuniform distribution of voltage-gated K⁺ channels and their predominance on the apical membrane may be important in taste transduction: alterations in apical K⁺ conductance may underlie receptor potentials ellicted by rapid stimuli.     

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.     

K+ secretion across frog skin. Induction by removal of basolateral Cl-

We examined the development of K+ secretion after removing Cl- from the basolateral surface of isolated skins of Rana temporaria using noise analysis. K+ secretion was defined by the appearance of a Lorentzian component in the power density spectrum (PDS) when Ba2+ was present in the apical bath (0.5 mM). No Lorentzians were observed when tissues were bathed in control, NaCl Ringer solution. Replacement of basolateral Cl- by gluconate, nitrate, or SO4- (0-Clb) yielded Lorentzians with corner frequencies near 25 Hz, and plateau values (So) that were used to estimate the magnitude of K+ secretion through channels in the apical cell membranes of the principal cells. The response was reversible and reproducible. In contrast, removing apical Cl- did not alter the PDS. Reduction of basolateral Cl- to 11.5 mM induced Lorentzians, but with lower values of So. Inhibition of Na+ transport with amiloride or by omitting apical Na+ depressed K+ secretion but did not prevent its appearance in response to 0-Clb. Using microelectrodes, we observed depolarization of the intracellular voltage concomitant with increased resistance of the basolateral membrane after 0-Clb. Basolateral application of Ba2+ to depolarize cells also induced K+ secretion. Because apical conductance and channel density are unchanged after 0-Clb, we conclude that K+ secretion is "induced" simply by an increase of the electrical driving force for K+ exit across this membrane. Repolarization of the apical membrane after 0-Clb eliminated K+ secretion, while further depolarization increased the magnitude of the secretory current. The cell depolarization after 0-Clb is most likely caused directly by a decrease of the basolateral membrane K+ conductance. Ba2(+)-induced Lorentzians also were elicited by basolateral hypertonic solutions but with lower values of So, indicating that cell shrinkage per se could not entirely account for the response to 0-Clb and that the effects of 0-Clb may be partly related to a fall of intracellular Cl-.     

KCl cotransport: A mechanism for basolateral chloride exit inNecturus gallbladder

K+ and Cl--selective double-barreled microelectrodes were used to study the effect of changes in external K+ concentration on intracellular Cl- activity (aiCl) in epithelial cells of Necturus gallbladder. Decreasing the K+ concentration simultaneously in both bathing solutions produced a decrease in aiCl. Steady-state values of aiCl were related to the values of the chemical potential gradient for K+ (delta microK) across either the apical or the basolateral cell membrane. A similar dependence between aiCl and delta microK appeared when the K+ concentration was changed in the serosal solution only. This indicates that aiCl depends on delta microK across the basolateral membrane. aiCl was virtually independent of the membrane potential. This supports the idea that both the mucosal and the basolateral membranes of Necturus gallbladder cells have very low passive permeabilities to Cl-. These results indicate that the exit of Cl- from Necturus gallbladder cells is driven by delta microK across the basolateral membrane, and suggest that KCl electroneutral coupled mechanism in this membrane plays an important role in transcellular Cl- transport.     

Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line

Intermediate-conductance K(+) (Kcnn4) channels in the apical and basolateral membranes of epithelial cells play important roles in agonist-induced fluid secretion in intestine and colon. Basolateral Kcnn4 channels have been well characterized in situ using patch-clamp methods, but the investigation of Kcnn4 channels in apical membranes in situ has been hampered by a layer of mucus that prevents seal formation. In the present study, we used patch-clamp methods to characterize Kcnn4 channels in the apical membrane of IEC-18 cells, a cell line derived from rat small intestine. A monolayer of IEC-18 cells grown on a permeable support is devoid of mucus, and tight junctions enable selective access to the apical membrane. In inside-out patches, Ca(2+)-dependent K(+) channels observed with iberiotoxin (a Kcnma1/large-conductance, Ca(2+)-activated K(+) channel blocker) and apamin (a Kcnn1-3/small-conductance, Ca(2+)-activated K(+) channel blocker) present in the pipette solution exhibited a single-channel conductance of 31 pS with inward rectification. The currents were reversibly blocked by TRAM-34 (a Kcnn4 blocker) with an IC(50) of 8.7 ± 2.0 μM. The channels were not observed when charybdotoxin, a peptide inhibitor of Kcnn4 channels, was added to the pipette solution. TRAM-34 was less potent in inhibiting Kcnn4 channels in patches from apical membranes than in patches from basolateral membranes, which was consistent with a preferential expression of Kcnn4c and Kcnn4b isoforms in apical and basolateral membranes, respectively. The expression of both isoforms in IEC-18 cells was confirmed by RT-PCR and Western blot analyses. This is the first characterization of Kcnn4 channels in the apical membrane of intestinal epithelial cells.     

Regulation of cAMP-activated apical membrane chloride conductance in gallbladder epithelium

Regulation of the cAMP-activated apical membrane Cl- conductance (GaCl) in Necturus gallbladder (NGB) epithelial cells was investigated with intracellular-microelectrode techniques. GaCl was increased by exposure to 8-Br-cAMP, theophylline or forskolin. Neither 8-Br-cGMP nor elevation of intracellular [Ca2+] using ionomycin had effects on GaCl or interfered with activation of GaCl by forskolin. N-(2- [methylamino]ethyl)-5-isoquinolinesulfonamide (H8), an inhibitor of cAMP-dependent protein kinase (PKA), slowed but did not prevent the GaCl response to 8-Br-cAMP. Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), stimulated GaCl but had no effects on intracellular [cAMP]. GaCl was unaffected by 4 alpha- phorbol, a PMA analog which does not activate PKC. Okadaic acid (OA), an inhibitor of protein phosphatases (PP) types 1 and 2A, slowed the activation of GaCl by 8-Br-cAMP, hastened the return of GaCl to basal values following removal of 8-Br-cAMP, and significantly reduced the elevation in intracellular [cAMP] produced by forskolin. OA had no effects on the GaCl changes elicited by theophylline. We conclude that: (a) NGB GaCl can be activated by PKA-mediated phosphorylation of apical membrane Cl- channels or a regulatory protein, (b) GaCl can also be activated via PKC, by a cAMP-independent mechanism, (c) OA-sensitive PP are not required for inactivation of GaCl; OA appears to stimulate phosphodiesterase, which lowers intracellular [cAMP] and affects GaCl activation, and (d) the apical membrane of NGB epithelium lacks a Ca(2+)-activated Cl- conductance.     

Cellular NH4+/K+ transport pathways in mouse medullary thick limb of Henle. Regulation by intracellular pH

Fluorescence and electrophysiological methods were used to determine the effects of intracellular pH (pHi) on cellular NH4+/K+ transport pathways in the renal medullary thick ascending limb of Henle (MTAL) from CD1 mice. Studies were performed in suspensions of MTAL tubules (S-MTAL) and in isolated, perfused MTAL segments (IP-MTAL). Steady-state pHi measured using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) averaged 7.42 +/- 0.02 (mean +/- SE) in S-MTAL and 7.26 +/- 0.04 in IP-MTAL. The intrinsic cellular buffering power of MTAL cells was 29.7 +/- 2.4 mM/pHi unit at pHi values between 7.0 and 7.6, but below a pHi of 7.0 the intrinsic buffering power increased linearly to approximately 50 mM/pHi unit at pHi 6.5. In IP-MTAL, NH4+ entered cells across apical membranes via both Ba(2+)-sensitive pathway and furosemide-sensitive Na+:K+(NH4+):2Cl- cotransport mechanisms. The K0.5 and maximal rate for combined apical entry were 0.5 mM and 83.3 mM/min, respectively. The apical Ba(2+)-sensitive cell conductance in IP-MTAL (Gc), which reflects the apical K+ conductance, was sensitive to pHi over a pHi range of 6.0-7.4 with an apparent K0.5 at pHi approximately 6.7. The rate of cellular NH4+ influx in IP-MTAL due to the apical Ba(2+)-sensitive NH4+ transport pathway was sensitive to reduction in cytosolic pH whether pHi was changed by acidifying the basolateral medium or by inhibition of the apical Na+:H+ exchanger with amiloride at a constant pHo of 7.4. The pHi sensitivities of Gc and apical, Ba(2+)-sensitive NH4+ influx in IP-MTAL were virtually identical. The pHi sensitivity of the Ba(2+)-sensitive NH4+ influx in S-MTAL when exposed to (apical+basolateral) NH4Cl was greater than that observed in IP-MTAL where NH4Cl was added only to apical membranes, suggesting an additional effect of intracellular NH4+/NH3 on NH4+ influx. NH4+ entry via apical Na+:K+ (NH4+):2Cl- cotransport in IP-MTAL was somewhat more sensitive to reductions in pHi than the Ba(2+)-sensitive NH4+ influx pathway; NH4+ entry decreased by 52.9 +/- 13.4% on reducing pHi from 7.31 +/- 0.17 to 6.82 +/- 0.14. These results suggest that pHi may provide a negative feedback signal for regulating the rate of apical NH4+ entry, and hence transcellular NH4+ transport, in the MTAL. A model incorporating these results is proposed which illustrates the role of both pHi and basolateral/intracellular NH4+/NH3 in regulating the rate of transcellular N H4+ transport in the MTAL.     

Characterization of the basolateral membrane conductance of Necturus urinary bladder

Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that permitted rapid changes in the ion composition of the serosal solution. The transepithelial electrical properties exhibited a marked seasonal variation that could be attributed to variations in the conductance of the shunt pathway, apical membrane selectivity, and basolateral Na+ transport. In contrast, the passive electrical properties of the basolateral membrane remained constant throughout the year. The apparent transference numbers (Ti) of the basolateral membrane for K+ and Cl- were determined from the effect on the basolateral membrane equivalent electromotive force of a sudden increase in the serosal K+ concentration from 2.5 to 50 mM/liter or a decrease in the Cl- concentration from 101 to 10 mM/liter. TK and TCl were 0.71 +/- 0.05 and 0.04 +/- 0.01, respectively. The basolateral K+ conductance could be blocked by Ba2+ (0.5 mM), Cs+ (10 mM), or Rb+ (10 mM), but was unaffected by 3,4-diaminopyridine (100 microM), decamethonium (100 microM), or tetraethylammonium (10 mM). We conclude that a highly selective K+ conductance dominates the electrical properties of the basolateral membrane and that this conductance is different from those found in nerve and muscle membranes.     

Stable knockdown of CFTR establishes a role for the channel in P2Y receptor-stimulated anion secretion

P2Y receptor regulation of anion secretion was investigated in porcine endometrial gland (PEG) epithelial cells. P2Y2, P2Y4, and P2Y6 receptors were detected in monolayers of PEG cells and immunocytochemistry indicated that P2Y4 receptors were located in the apical membrane. Apical membrane current measurements showed that Ca2+-dependent and PKC-dependent Cl- channels were activated following treatment with uridine triphosphate (UTP) (5 microM). Current-voltage relationships comparing calcium-dependent and PKC-dependent UTP responses under biionic conditions showed significant differences in selectivity between Cl-)and I- for the PKC-dependent conductance (P(I)/P(Cl) = 0.76), but not for Ca2+-dependent conductance (PI/P(Cl) = 1.02). The I-/Cl- permeability ratio for the PKC-dependent conductance was identical to that measured for 8-cpt cAMP. Furthermore, PKC stimulation using phorbol 12-myristate 13-acetate (PMA) activated an apical membrane Cl- conductance that was blocked by the CFTR selective inhibitor, CFTRinh-172. CFTR silencing, accomplished by stable expression of small hairpin RNAs (shRNA), blocked the PKC-activated conductance associated with UTP stimulation and provided definitive evidence of a role for CFTR in anion secretion. CFTR activation increased the initial magnitude of Cl- secretion, and provided a more sustained secretory response compared to conditions where only Ca2+-activated Cl- channels were activated by UTP. Measurements of [cAMP]i following UTP and PMA stimulation were not significantly different than untreated controls. Thus, these results demonstrate that UTP and PMA activation of CFTR occurs independently of increases in intracellular cAMP and extend the findings of earlier studies of CFTR regulation by PKC in Xenopus oocytes to a mammalian anion secreting epithelium.     

Odd behaviour of Li+ in frog skin ion transport

1. Frog skin epithelium has basolateral K+ channels that normally define the basolateral membrane potential between 80 and 100 mV. 2. The membrane mentioned also has almost silent chloride channels and a [Na+, K+, 2Cl-] cotransport, the latter probably maintains the high Cl- in the capital (also called syncytium) cells. 3. If the K+ channels are blocked by Ba2+ (or Li+) it is possible to demonstrate potential gating of the chloride channels of the basolateral membrane. 4. When the normal K+ channels are blocked, a potential-dependent K+ conductance slowly emerges. 5. If Li+ is substituted for outside Na+ the skin shows potential oscillations of about 40 mV at a frequency of about six per hour. 6. The anion channel inhibitor Indacrinone stops these oscillations. 7. The role of Cl- and K+ channels in these oscillations is discussed. 8. The transepithelial inward transport of Li+ requires the presence of Na+ and seems to be due to exchange of cellular Li+ against inside Na+ via the basolateral Na+/H+ exchanger.     

Mechanism of the excitatory Cl- response in mouse olfactory receptor neurons

In vertebrate olfactory receptor neurons (ORNs), the odorant-triggered receptor current flows through two distinct ion channels on the sensory cilia: Ca2+ influx through a cyclic nucleotide-gated (CNG) channel followed by Cl- efflux through a Ca2+-activated anion channel. The excitatory Cl- current amplifies the small CNG current and crucially depends on a high intracellular Cl- concentration. We show here that a (Na+)-(K+)-(2Cl-) cotransporter, NKCC1, is required for this Cl- current, in that ORNs deficient in Nkcc1 or incubated with an NKCC blocker (bumetanide) lack the Cl- current. Surprisingly, immunocytochemistry indicates that NKCC1 is located on the somata and dendrites of ORNs rather than the cilia, where transduction occurs. This topography is remarkably similar to the situation in secretory epithelial cells, where basolateral Cl- uptake and apical Cl- efflux facilitate transepithelial fluid movement. Thus, a single functional architecture serves two entirely different purposes, probably underscoring the epithelial origin of the ORNs.     

Ion channels in the thick ascending limb of Henle's loop.

The thick ascending limb of Henle's loop (TAL) is polarized with respect to its conductances. The luminal membrane contains a K+ conductance which is made up by the synchronous operation of 60- to 80-pS K+ channels. The basolateral membrane contains a chloride conductance. This conductance corresponds most likely to a 30- to 60-pS Cl- channel present in this membrane. Our knowledge on the properties of the K+ channels of these cells has been increased rapidly by patch clamp studies: these K+ channels are inwardly rectifying. They are highly selective for K+ over Na+, Li+ and many other cations. They do not conduct Rb+, Cs+, NH+4 or other larger cations. In fact, all these three cations as well as choline, tetraethylammonium, lidocaine, verapamil, diltiazem, quinine, quinidine and Ba2+ inhibit these K+ channels. As apparent from kinetic studies the mechanisms of inhibition are different for the various blockers. The TAL K+ channels are downregulated by increasing cytosolic Ca2+ activity. Cytosolic adenosine trisphosphate (ATP) has a similar effect. This ATP inhibition is Ca2+ dependent. The affinity to ATP is augmented by increasing Ca2+. Cytosolic alkalinity increases the open probability of these channels, and cytosolic acidification has the opposite effect. This pH dependence is very marked. A change by 0.2 pH units leads to a more than twofold change in the open-channel probability. The basolateral chloride conductance reflects the properties of an outwardly rectifying 30- to 60-pS Cl- channel. This channel behaves, in many respects, like the Cl- channels of a multitude of Cl- transporting epithelia. It is characterized by two open and two closed states. It is highly selective for Cl- as compared with larger anions, and it is inhibited reversibly by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate.     

cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block

Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- >> SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10- fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.     

Effects of changes in mucosal solution Cl- or K+ concentration on cell water volume of Necturus gallbladder epithelium

An electrophysiologic technique was used to measure changes in cell water volume in response to isosmotic luminal solution ion replacement. Intracellular Cl- activity (aCl-i) was measured and net flux determined from the changes in volume and activity. Reduction of luminal solution [Cl-] from 98 to 10 mM (Cl- replaced with cyclamate) resulted in a large fall in aCl-i with no significant change in cell water volume. Elevation of luminal solution [K+] from 2.5 to 83.5 mM (K+ replaced Na+) caused a small increase in aCl-i with no change in cell water volume. Exposure of the Necturus gallbladder epithelium to agents that increase intracellular cAMP levels (forskolin and/or theophylline) induces an apical membrane electrodiffusive Cl- permeability accompanied by a fall in aCl-i and cell shrinkage. In stimulated tissues, reduction of luminal solution [Cl-] resulted in a large fall in aCl-i and rapid cell shrinkage, whereas elevation of luminal solution [K+] caused a large, rapid cell swelling with no significant change in aCl-i. The changes in cell water volume of stimulated tissues elicited by lowering luminal solution [Cl-] or by elevating luminal solution [K+] were reduced by 60 and 70%, respectively, by addition of tetraethylammonium (TEA+) to the luminal bathing solution. From these results, we conclude that: (a) In control tissues, the fall in aCl-i upon reducing luminal solution [Cl-], without concomitant cell shrinkage, indicates that the Cl- entry mechanism is electroneutral (Cl-/HCO3-) exchange. (b) Also in control tissues, the small increase in aCl-i upon elevating luminal solution [K+] is consistent with the recent demonstration of a basolateral Cl- conductance. (c) The cell shrinkage elicited by elevation of intracellular cAMP levels results from conductive loss of Cl- (and probably K+). (d) Elevation of cAMP inhibits apical membrane Cl-/HCO-3-exchange activity by 70%. (e) The cell shrinkage in response to the reduction of mucosal solution [Cl-] in stimulated tissues results from net K+ and Cl- efflux via parallel electrodiffusive pathways. (f) A major fraction of the K+ flux is via a TEA(+)-sensitive apical membrane K+ channel.     

Stimulating effects of dopamine on chloride transport across the rat caudal epididymal epithelium in culture

The present study investigated the effects of dopamine on chloride transport across cultured rat caudal epididymal epithelium. The results showed that dopamine induced a biphasic short-circuit current (Isc) in a concentration-dependent manner. The dopamine-induced response consisted of an initial rapid spike followed by a sustained phase. The alpha and beta adrenoreceptor inhibitors, phentolamine and propranolol, inhibited the initial spike and the sustained phase, respectively, suggesting a contribution of adrenergic receptors. The response was almost abolished by removing the extracellular Cl-, suggesting that the dopamine-induced short-circuit current is primarily a Cl- current. The response was inhibited by the apical Cl- channel blocker, diphenylamine-dicarboxylic acid, and the Ca2+-activated Cl- channel blocker, disulfonic acid stilbene, indicating that Cl- may pass through two types of Cl- channels on the apical side. Preloading monolayers with the intracellular Ca2+ chelator BAPTA/AM abolished the initial spike and greatly reduced the second phase in the Isc response to dopamine. Pretreating the monolayers with an adenylate cyclase inhibitor, MDL12330A, inhibited all of the second Isc response and part of the initial spike. Also, characteristics of the Cl- currents induced by dopamine were observed in whole-cell patch-clamp recording. The increases of intracellular cAMP and Ca2+ induced by dopamine were also measured. The results suggest that extracellular dopamine activates Ca2+-dependent and cAMP-dependent regulatory pathways, leading to activation of both Ca2+-dependent and cAMP-dependent Cl- conductances in epididymal epithelial cells.     

Na(+), Cl(-), Ca(2+) and Zn(2+) transport by fish gills: retrospective review and prospective synthesis

The secondary active Cl(-) secretion in seawater (SW) teleost fish gills and elasmobranch rectal gland involves basolateral Na(+),K(+)-ATPase and NKCC, apical membrane CFTR anion channels, and a paracellular Na(+)-selective conductance. In freshwater (FW) teleost gill, the mechanism of NaCl uptake is more controversial and involves apical V-type H(+)-ATPase linked to an apical Na(+) channel, apical Cl(-)-HCO-3 exchange and basolateral Na(+),K(+)-ATPase. Ca(2+) uptake (in FW and SW) is via Ca(2+) channels in the apical membrane and Ca(2+)-ATPase in the basolateral membrane. Mainly this transport occurs in mitochondria rich (MR) chloride cells, but there is a role for the pavement cells also. Future research will likely expand in two major directions, molded by methodology: first in physiological genomics of all the transporters, including their expression, trafficking, operation, and regulation at the molecular level, and second in biotelemetry to examine multivariable components in behavioral physiological ecology, thus widening the integration of physiology from the molecular to the environmental levels while deepening understanding at all levels.