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PubMed1987

Blocking agents of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells.

Ca2+-activated K+ channels with estimated single channel conductances of 127 +/- 2 pS were identified in the apical cell membrane of clone A3 of cultured medullary thick ascending limb (MTAL) cells. Both Ba2+ and the scorpion toxin, charybdotoxin (CTX), are slow blockers of the channels. An application of 0.1 microM Ba2+ to the intracellular face caused a 50% reduction in fractional open time (fv). Ba2+ block is both concentration and voltage dependent. Concentrations of CTX as low as 2 nM in the extracellular solution caused a significant reduction in fv. Tetraethylammonium (TEA) and quinine are fast blockers of Ca2+-activated K+ channels in MTAL cells. TEA, 400 microM, in the extracellular solution caused a voltage-dependent reduction in channel amplitude, whereas it takes 10 mM in the intracellular solution to reduce channel amplitude by 30%. Micromolar amounts of quinine applied to the intracellular face caused the channels to flicker rapidly between open and blocked states. These results suggest that K+ channels in MTAL cells are homologous to those found in muscle cells, and that these blocking agents may be used to probe the nature of K+ conductances in several nephron segments.

Animals↗
PubMed1993

Artifactual expression of maxi-K+ channels in basolateral membrane of gallbladder epithelial cells.

To patch clamp the basolateral cell membrane, sheets of Necturus gallbladder epithelium were stripped of the subepithelial tissue layers and affixed apical side down on cover slips coated with Cell-Tak [F. Wehner, L. Garretson, K. Dawson, Y. Segal, and L. Reuss. Am. J. Physiol. 258 (Cell Physiol. 27): C1159-C1164, 1990]. In 90% of the patches we observed K+ channels identical to the maxi-K+ channels previously demonstrated in the apical membrane (Y. Segal and L. Reuss. J. Gen. Physiol. 95: 791-818, 1990). To ascertain whether these channels were present in the native tissue, we carried out intracellular-microelectrode studies. We tested for activation of basolateral membrane K+ conductance by depolarization or by elevation of intracellular Ca2+ and for tetraethylammonium sensitivity of the basolateral membrane voltage and fractional resistance. The results were negative, indicating that maxi-K+ channels are not expressed in the basolateral membrane of the "intact" epithelium. Using the same intracellular-microelectrode protocol on the apical membrane, we demonstrated the presence of an apical K+ conductance attributable to maxi-K+ channels. Additional experiments revealed a Ba(2+)-sensitive basolateral K+ conductance in the native epithelium. We conclude that in the stripped preparation there is artifactual expression of maxi-K+ channels. In addition, the native basolateral membrane K+ channels either are not expressed in this preparation or have a low conductance and cannot be discerned from the background noise.

Animals↗
PubMed1993

Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells.

Single-channel recording techniques were used to identify and characterize the K+ channel activated by Ca(2+)-mediated secretory agonists in T84 cells. Carbachol (CCh; 100 microM) and taurodeoxycholate (TDC; 0.75 mM) stimulated oscillatory outward K+ currents. With K gluconate in bath and pipette, cell-attached single-channel K+ currents stimulated by CCh and ionomycin (2 microM) were inwardly rectified and reversed at 0 mV. The single-channel chord conductance was 32 pS at -90 mV and 14 pS at +90 mV. Similar properties were observed in excised inside-out patches in symmetric K+, permitting further characterization of channel properties. Partial substitution of bath or pipette K+ with Na+ gave a K(+)-to-Na+ selectivity ratio of 5.5:1. Channel activity increased with increasing bath Ca2+ concentration in the physiological range of 50-800 nM. Maximal channel activity occurred at intracellular pH 7.2 and decreased at more acidic or alkaline pH values. Extracellular charybdotoxin (CTX; 50 nM) blocked inward but not outward currents. Extracellular tetraethylammonium (TEA; 10 mM) reduced single-channel amplitude at all voltages. No apparent block of the channel was observed with extracellular Ba2+ (1 mM), apamin (1 microM), 4-aminopyridine (4-AP; 4 mM), quinine (500 microM), or glyburide (10 microM). Cytosolic quinine and 4-AP blocked both inward and outward currents, whereas Ba2+ blocked only outward currents. Apamin, CTX, TEA, and glyburide did not affect channel activity. The agonist activation and pharmacological profile of this inwardly rectified K+ channel indicate that it is responsible for the increase in basolateral K+ conductance stimulated by Ca(2+)-mediated agonists in T84 cells.

4-Aminopyridine↗
PubMed1994

Ba(2+)-sensitive K+ channels in basal membrane of confluent Madin-Darby canine kidney cells.

A method is described for gaining access to the basolateral membranes of confluent Madin-Darby canine kidney (MDCK) cells by surgical reflection of the cell layer overlying fluid-filled domes. Single-channel recordings from cell-attached inside-out and outside-out configurations revealed two K+ channels located in the basal membranes of the highly differentiated monolayers. With 140 mmol/l KCl in pipette, the intermediate-conductance K+ channel displayed outward rectification in cell-attached configuration with channel conductances of 65 pS for outward part and 17 pS for inward part. In excised-patch recording, this channel had a conductance of 92 pS with 140 mmol/l KCl on the extracellular side of the patch and 5 mmol/l KCl on the cytosolic side. The maximum conductance obtained in symmetrical KCl (140 mmol/l) solution was 140 pS. Ba2+ (1 mmol/l) and tetraethylammonium (5 mmol/l) blocked this channel reversibly. Channel open probability (Po) was reduced from 0.41 at cytosolic pH 7.4 to 0.14 at pH 6.8 and increased to 0.64 at pH 8.0. The channel activity was significantly inhibited by elevation of intracellular Ca2+. A small-conductance K+ channel was also observed mainly in excised patches with single-channel conductance of 48 pS in symmetrical KCl solutions. However, the activity of this channel was partially obscured by the intermediate-conductance K+ channel and further analysis was not possible. A physiological role of these channels in mediating K+ recycling through the monolayer is suggested.

Adenosine Triphosphate↗
PubMed1989

Identification of two calcium currents in acutely dissociated neurons from the rat lateral geniculate nucleus.

1. Intracellular recording in the in vitro slice preparation and whole-cell, patch-clamp recording of acutely dissociated neurons from the rat lateral geniculate nucleus (LGN) were combined to study the Ca currents underlying their electrical responses. In slices from young animals (postnatal days 13-16), we found that dorsal LGN neurons have responses similar to those of adult preparations, including the presence of a low-threshold Ca spike (LTS). After enzymatic isolation of LGN neurons from the same animals, the firing properties appeared well preserved, as indicated by whole-cell, current-clamp recordings from dissociated multipolar cells (presumably geniculocortical relay neurons). 2. Two types of Ca currents were identified in voltage-clamped, isolated LGN neurons on the basis of their voltage dependency, pharmacology, and selectivity properties. These two currents resemble the low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca channels found in rat sensory neurons (9). 3. The LVA current component required negative potentials (less than -80 mV) to deinactivate completely, started to activate around -60 mV and reached a plateau level around -25 mV. It peaked within 30-6 ms and decayed with a single time constant of approximately 24 ms at -20 mV. Its inactivation curve ranged from -100 to -40 mV, with a half-inactivation near -60 mV. The HVA current component could be isolated by holding the membrane potential positive to -60 mV, activated at potentials positive to -30 mV and peaked around +5 mV. The time-to-peak ranged from 30 to 6 ms in the voltage range from -30 to +35 mV and decayed very slowly with sustained depolarizing pulses (time constant ranged between 1,600 and 40 ms over the same voltage range). 4. The inactivation of LVA Ca current during depolarizing voltage steps was consistent with a voltage-dependent process. The recovery from inactivation after short (100 ms), inactivating prepulses displayed two exponential phases. The slower phase was predominant under conditions that induce large current flow through the membrane, suggesting a Ca-mediated mechanism. 5. The LVA current was preferentially blocked by 50 microM Ni2+, leaving the HVA currents almost unaltered. Fifty micromolars Cd2+, in contrast, seemed more effective in blocking the HVA component of the Ca current.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1989

Characterization of aldosterone-induced potassium secretion in rat distal colon.

The role of apical and basolateral membranes in aldosterone-induced active potassium (K) secretion in rat distal colon was investigated by measuring mucosal-to-serosal (Jms) and serosal-to-mucosal (Jsm) 42K fluxes (mueq.h-1.cm-2) across isolated stripped mucosa under short-circuit conditions in normal and secondary-hyperaldosterone animals. In normal colons mucosal tetraethylammonium (TEA; 30 mM) or barium (Ba; 5 mM), but not cesium (Cs; 15 mM), reduced Jsm without affecting Jms. In aldosterone animals (a) net K secretion (-0.54 +/- 0.11) was converted to net K absorption (0.63 +/- 0.15) by mucosal TEA, which produced a marked reduction in Jsm (0.82 +/- 0.07) and an increase in Jms (0.35 +/- 0.07). In contrast mucosal Ba resulted in a relatively smaller reduction in JK(sm) without altering JK(ms), whereas mucosal Cs was ineffective; (b) serosal bumetanide or the removal of serosal Na or Cl markedly inhibited JK(sm and abolished net K secretion; and (c) serosal ouabain (1 mM) produced qualitatively similar effects to those of serosal bumetanide. These results demonstrate that (a) normal rat distal colon contains apical TEA- and Ba-sensitive K channels; (b) aldosterone induces TEA-sensitive and Ba-sensitive apical K channels; (c) aldosterone-induced K secretion requires both the Na,K-pump and Na-K-2Cl cotransport for K uptake across the basolateral membrane; and (d) alteration of any of these processes results in inhibition of aldosterone-induced active K secretion simultaneously with stimulation of K absorption.

Aldosterone↗
PubMed1997

Regulation of K+ channels by cell contact in a cloned folliculo-stellate cell (TtT/GF).

Membrane currents in a folliculo-stellate cell line (TtT/GF) were examined using the whole-cell clamp technique. The cultured cells were morphologically categorized as isolated spread cells, isolated round cells, contact spread cells, and contact round cells. A distinct outward current was observed in isolated spread cells, contact round cells, and contact spread cells, whereas the outward current detected in isolated round cells was barely perceivable. The reversal potentials of the outward tail current obeyed the Nernst equation, indicating that the outward current was carried by K+ ions. The activation and deactivation processes of the K+ current could be fitted by single exponential curves. 4-aminopyridine, tetraethylammonium, and Ba2+ inhibited the K+ current. The concentrations for half-maximal inhibition of these agents to block the K+ current were 0.2 mM, 0.8 mM, and 8 mM, respectively. The biophysical and pharmacological characteristics of the K+ current in TtT/GF cells were similar to those of the K+ current in glial and Schwann cells. According to the results of the present study, it was concluded that TtT/GF cells possessed outward K+ channels, characteristics of which were similar to those of the K+ channels in glial and Schwann cells, and that the amplitude of the K+ current in TtT/GF cells seemed to be regulated by the condition of the cell contact.

4-Aminopyridine↗
PubMed1977

Pharmacologic studies on W10,294A, a new bronchodilator.

W10,294A (1,2,3,4-tetrahydro-8,9-dimethoxy-3(2-piperidinoethyl)-5H-[1] benzopyrano [3,4-c] pyridin-5-one dihydrochloride) is a potent antagonist of the bronchoconstriction induced by histamine, methacholine or acetylcholine. In vitro evaluation on guinea pig trachea indicated that W10,294A had both direct smooth muscle relaxant activity and modest antihistaminic properties. Resistance and dynamic compliance measurements in anesthetized dogs indicated that W10,294A prevented or reversed bronchoconstriction due to histamine or pilocapine. Bronchodilator activity of long duration was observed in dogs after oral administration. W10,294A was compared with aminophylline in several tests. W10,294A was 6 times more potent than aminophylline in relaxing isolated guinea-pig trachea. In the anesthetized dog W10,294A was more potent and longer acting than aminophylline in reversing pilocarpine-induced bronchospasm. The bronchodilator activity of W10,294A was not blocked by the beta adrenoceptor antagonist bunolol. Comparative studies in dogs and guinea pigs, using identical doses, showed that W10,294A had considerably less effect on the cardiovascular system than did aminophylline.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

Chemical analysis and bonding reaction of RelyX Unicem and Bifix composites--a comparative study.

OBJECTIVES: The chemical and physical properties of the dual curing luting composites RelyX Unicem (3M ESPE) and Bifix (VOCO) were analyzed with regard to their elemental composition, surface morphology and polymerization reaction. The bonding of both materials to hydroxyapatite (HAp) was studied. METHODS: The main components were analyzed by XPS and EDX. The minor components were identified with ICP-OES. Moreover, the morphology was examined by SEM and the polymerization reaction products were investigated using GPC. XPS was also applied to study the bonding mechanisms to HAp. RESULTS: The inorganic product particles consist of an Al-Si-Na-glass network, which incorporates radiopaque strontium and barium for Bifix and strontium and lanthanum for RelyX Unicem. RelyX Unicem contains about 10% fluoride and 2% Ca(OH)(2), whereas Bifix comprises 2% fluoride. After polymerization, reaction products of 10(5)-10(6)g/mol were identified with RelyX Unicem. Both products contain mono- and oligomeric compounds. The reaction with HAp generates calcium atoms with a reduced binding energy. They act as an electron acceptor and show chemical interaction between the composite and HAp. With RelyX Unicem 86% of the calcium atoms reacted, compared to 65% with Bifix. SIGNIFICANCE: The intense chemical interaction of RelyX Unicem with HAp seems to be relevant to clinical aspects and explains the mechanical product properties. After setting, a polymer was found with RelyX Unicem but only monomeric/oligomeric products were identified at the surface of Bifix.

Aluminum Silicates↗
PubMed1992

Regulation by calcitonin of Na(+)-K(+)-Cl- cotransport in a rabbit thick ascending limb cell line.

The hormonal regulation of a Na(+)-K(+)-Cl- cotransport was investigated in a renal tubule cell line (RC.SV2 cells) transformed by the simian virus 40. This cell line has the main characteristics of cells from the thick ascending limb of Henle, including the presence of Tamm-Horsfall protein and stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) production by calcitonin (CT). Kinetic studies with 22Na+, 36Cl-, and 86Rb+ indicated the existence of a Na(+)-K(+)-Cl- cotransport with a stoichiometry of 1Na+:1K+: 2Cl-. All compounds stimulating cAMP production enhanced the ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx mediated by Na(+)-K(+)-Cl- cotransport. CT (100 ng/ml) increased the Or-Bs influx twofold by enhancing maximum velocity without changing the apparent Michaelis constant. The K(+)-channel blocker barium blunted the CT-stimulated Or-Bs influx by 64-74%, whereas the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate reduced the CT-stimulated influx by 28-40%. These results suggest that CT stimulates the Na(+)-K(+)-Cl- cotransport by a cAMP-dependent mechanism and that K+ recycling through K+ membrane channels is an important modulator of cotransporter-mediated ion fluxes.

Animals↗
PubMed1996

A cAMP regulated K+-selective channel from the sea urchin sperm plasma membrane.

Ion channels are deeply involved in sperm physiology. In sea urchin sperm cyclic nucleotide levels increase during quimotaxis and in the acrosome reaction (AR). Although cyclic nucleotides are second messengers known to directly or indirectly modulate ion channels, it is not clear how they modulate sperm responses to the egg outer layer. Here, we describe a cAMP regulated K+-selective channel from sea urchin sperm plasma membranes fused into planar bilayers that may have a role during sea urchin sperm quimotaxis and/or the AR. Its single channel conductance in 100 mM KCl is 103 pS. In bi-ionic experiments, the channel displayed a K+/Na+ permeability ratio (PK+/PNa+) of approximately 5. Thus, in sea water its reversal potential would be approximately -13 mV and channel opening would depolarize spermatozoa. The channel has low open probability (Po = 0.8 +/- 0.2% at 0 mV applied voltage) and weak voltage dependence. Channel activity is reversibly up-regulated by cAMP in the cis bilayer side, but not by cGMP. This modulation followed a single Langmuir isotherm with an apparent kd of 200 microM. At this concentration the channel open probability at 0 mV increased up to 11- fold. TEA+ blocked the channel only from the trans side. Also Ba2+ in trans blocked the channel in a voltage-dependent manner.

Animals↗
PubMed1995

The ACh-induced whole-cell currents in sheep parotid secretory cells. Do BK channels really carry the ACh-evoked whole-cell K+ current?

As in other salivary glands, the secretory cells of the sheep parotid have a resting K+ conductance that is dominated by BK channels, which are activated by acetylcholine (ACh) and are blocked by tetraethylammonium (TEA). Nevertheless, perfusion studies indicate that TEA does not inhibit ACh-evoked fluid secretion or K+ efflux from intact sheep parotid glands. In the present study, we have used whole-cell patch clamp techniques to show that ACh activates K+ and Cl- conductances in sheep parotid secretory cells by increasing intracellular free Ca2+, and we have compared the blocker sensitivity of the ACh-evoked whole-cell K+ current to the previously reported blocker sensitivity of the BK channels seen in these cells. The ACh-induced whole-cell K+ current was not blocked by TEA (10 mmol/l) or verapamil (100 mumol/l), both of which block the resting K+ conductance and inhibit BK channels in these cells. Quinine (1 mmol/l) and quinidine (1 mmol/l), although only weak blockers of the resting K+ conductance, inhibited the ACh-evoked current at 0 mV (K+ current), by 68% and 78%, respectively. 4-Aminopyridine (10 mmol/l) partially inhibited the ACh-induced K+ current and caused it to fluctuate. It also caused the resting membrane currents to fluctuate, possibly by altering cytosolic free Ca2+. Ba2+ (100 mumol/l), a blocker of the inwardly rectifying K+ conductance in sheep parotid cells, had no effect on the ACh-induced K+ current. We conclude that the ACh-induced K+ conductance in sheep parotid cells is pharmacologically distinct from both the outwardly rectifying (BK) K+ conductance and the inwardly rectifying K+ conductance seen in unstimulated cells. Given that in vitro perfusion and K+ efflux studies on other salivary glands in which BK channels dominate the resting conductance (e.g., the rat mandibular, rat parotid and mouse mandibular glands) have revealed an insensitivity to TEA, suggesting that BK channels do not carry the ACh-evoked K+ current, we propose that BK channels do not contribute substantially to the K+ current evoked by ACh in the secretory cells of most salivary glands.

4-Aminopyridine↗
PubMed1984

Electrophysiological properties of in vitro hippocampal pyramidal cells from normal and staggerer mutant mice.

Electrophysiological properties of intracellularly recorded CA1 pyramidal cells from normal and staggerer mice were compared by using hippocampal slices maintained in vitro. In staggerer mice, the passive membrane properties of these neurons as well as their synaptic potentials elicited by stratum radiatum stimulation were very similar to those observed in normal mice. In control and mutant mice and in standard Krebs solution, CA1 pyramidal cells mainly fired tetrodoxin (TTX)-sensitive fast spikes but could also generate slow spikes. In both groups, replacement of calcium (Ca) by barium (Ba) or introduction of TEA in the bathing medium prolonged the repolarization of the fast spikes and suppressed the brief spike afterhyperpolarization which normally followed them, thus suggesting that both events involve fast potassium conductances. Furthermore, in both groups of animals, TEA and Ba enhanced the slow spikes and induced the appearance of prolonged depolarizations. These slow events were TTX-resistant and were abolished by the Ca channel blockers cadmium or cobalt, thus suggesting that they are Ca-dependent. On the whole, the present results indicate that the staggerer mutation which yields marked abnormalities in the bioelectrical properties of cerebellar Purkinje cells has no such effect on CA1 pyramidal cells.

Animals↗
PubMed1998

Calcium triggers calcineurin-dependent synaptic vesicle recycling in mammalian nerve terminals.

BACKGROUND: Following exocytosis at the synapse, synaptic vesicle components are recovered by endocytosis. Morphological analysis has suggested that this occurs by a clathrin-mediated pathway, and the GTPase dynamin is thought to be involved in 'pinching off' endocytosing vesicles. The finding that the calcium-dependent phosphatase calcineurin can dephosphorylate dynamin and two other proteins implicated in endocytosis (amphiphysin and synaptojanin) has suggested a potential role for calcium and dephosphorylation in regulating synaptic vesicle endocytosis. RESULTS: We tested this hypothesis with an endocytosis assay in isolated nerve terminals (synaptosomes) that relies on the use of the fluorescent dye FM2-10. In synaptosomes, vesicle recycling occurs predominantly via a pathway dependent on both dynamin and amphiphysin. We found that endocytosis could be stimulated maximally at calcium concentrations that yielded only low levels of exocytosis, suggesting that the two processes had different calcium sensitivities cyclosporin A and Fk506, we identified calcineurin as a calcium sensor for endocytosis and showed that its activity is essential for synaptic vesicle endocytosis in synaptosomes. CONCLUSIONS: Our results suggest that dynamin-dependent synaptic vesicle endocytosis is triggered by calcium influx occurring upon nerve-terminal depolarisation. An essential mediator of calcium's effect is calcineurin, the activation of which leads to dephosphorylation of at least four proteins implicated in endocytosis-dynamin, amphiphysin 1, amphiphysin 2 and synaptojanin. Our findings also imply that endocytosis and exocytosis may occur in tandem in vivo simply because they share a responsiveness to calcium influx, rather than because they are mechanistically coupled.

Animals↗
PubMed1991

Cloning and expression of a rat cardiac delayed rectifier potassium channel.

We have cloned a cDNA (designated RAK) coding for a delayed-rectifier K current (IRAK) from adult rat heart atrium and expressed it in Xenopus oocytes. RAK differs from the cloned rat brain K current, BK2 [McKinnon, D. (1989) J. Biol. Chem. 264, 8230-8236], by one amino acid at residue 411. RAK expressed in oocytes compares closely to the intrinsic adult rat atrial delayed-rectifier current measured by using whole-cell recording of single isolated cells. Northern blot analysis confirmed the presence of the channel in adult rat atrium, and to a lesser extent, in rat ventricle. IRAK activates with time constants ranging from 58 ms at -20 mV to 6 ms at +60 mV and does not show significant inactivation over 800 ms. It is blocked by 4-aminopyridine greater than barium much greater than tetraethylammonium chloride, which is similar to the relative potencies of these blockers on the native delayed rectifier current. We conclude that the main delayed rectifier K current in adult rat atria is virtually identical to a neuronal delayed rectifier, BK2.

4-Aminopyridine↗
PubMed1994

Properties and regulation of basolateral K+ channels in rat duodenal crypts.

1. Patch clamp recording techniques were used to study the properties of K+ channels in the basolateral membrane of rat duodenal crypts, and their regulation by Cl(-)-secretory agonists. 2. High conductance (84-99 pS) K+ channels were activated by 0.1 mM dibutyryl cAMP in 50% of cell-attached patches, while 0.1 mM carbachol had no effect on channel activity. High conductance K+ channels were voltage independent, Ca2+ insensitive, blocked by 5 mM Ba2+ or 20 mM tetraethylammonium chloride (TEA), and stimulated by 57% when intracellular pH was increased from 7.4 to 7.8. 3. In contrast, low conductance (19-28 pS) K+ channels were activated by 0.1 mM dibutyryl cAMP in 67% of cell-attached patches, while 0.1 mM carbachol activated channels in 100% of cell-attached patches. Low conductance K+ channels were voltage independent, Ca2+ sensitive and pH insensitive. Unlike the high conductance K+ channels, 5 mM Ba2+ had no effect on the activity of low conductance K+ channels, although 20 mM TEA decreased channel activity by 53%. 4. The results show that the basolateral membrane of rat duodenal crypts possesses two discrete populations of K+ channels that may have important roles in sustaining the small intestinal Cl(-)-secretory responses triggered by a variety of cAMP- and Ca(2+)-mediated agonists.

Animals↗
PubMed2002

Characterization of basolateral K+ channels underlying anion secretion in the human airway cell line Calu-3.

Transepithelial anion secretion in many tissues depends upon the activity of basolateral channels. Using monolayers of the Calu-3 cell line, a human submucosal serous cell model mounted in an Ussing chamber apparatus, we investigated the nature of the K+ channels involved in basal, cAMP- and Ca2+-stimulated anion secretion, as reflected by the transepithelial short circuit current (I(sc)). The non-specific K+ channel inhibitor Ba2+ inhibited the basal I(sc) by either 77 or 16 % when applied directly to the basolateral or apical membranes, respectively, indicating that a basolateral K+ conductance is required for maintenance of basal anion secretion. Using the K+ channel blockers clofilium and clotrimazole, we found basal I(sc) to be sensitive to clofilium, with a small clotrimazole-sensitive component. By stimulating the cAMP and Ca2+ pathways, we determined that cAMP-stimulated anion secretion was almost entirely abolished by clofilium, but insensitive to clotrimazole. In contrast, the Ca2+-stimulated response was sensitive to both clofilium and clotrimazole. Thus, pharmacologically distinct basolateral K+ channels are differentially involved in the control of anion secretion under different conditions. Isolation of the basolateral K+ conductance in permeabilized monolayers revealed a small basal and forskolin-stimulated I(sc). Finally, using the reverse transcriptase-polymerase chain reaction, we found that Calu-3 cells express the K+ channel genes KCNN4 and KCNQ1 and the subunits KCNE2 and KCNE3. We conclude that while KCNN4 contributes to Ca2+-activated anion secretion by Calu-3 cells, basal and cAMP-activated secretion are more critically dependent on other K+ channel types, possibly involving one or more class of KCNQ1-containing channel complexes.

Anions↗