The role of KCNQ1/KCNE1 K+ channels in intestine and pancreas: lessons from the KCNE1 knockout mouse

KCNE1 (IsK, minK) co-assembles with KCNQ1 (KvLQT1) to form voltage-dependent K(+) channels. Both KCNQ1 and KCNE1 are expressed in epithelial cells of gut and exocrine pancreas. We examined the role of KCNQ1/KCNE1 in Cl(-) secretion in small and large intestine and exocrine pancreas using the KCNE1 knockout mouse. Immunofluorescence revealed a similar basolateral localization of KCNQ1 in jejunum and colon of KCNE1 wild-type and knockout mice. Electrogenic Cl(-) secretion in the colon was not affected by gene disruption of KCNE1; in jejunum forskolin-induced short-circuit current was some 40% smaller but without being significantly different. Inhibition of KCNQ1 channels by 293B (IC(50) 1 micromol l(-1)) and by IKS224 (IC(50) 14 nmol l(-1)) strongly diminished intestinal Cl(-) secretion. In exocrine pancreas of wild-type mice, KCNQ1 was predominantly located at the basolateral membrane. In KCNE1 knockout mice, however, the basolateral staining was less pronounced and the distribution of secretory granules was irregular. A slowly activating and 293B-sensitive K(+) current was activated via cholinergic stimulation in pancreatic acinar cells of wild-type mice. In KCNE1 knockout mice this K(+) current was strongly reduced. In conclusion intestinal Cl(-) secretion is independent from KCNE1 but requires KCNQ1. In mouse pancreatic acini KCNQ1 probably co-assembled with KCNE1 leads to a voltage-dependent K(+) current that might be of importance for electrolyte and enzyme secretion.     

Regulation of wild-type and mutant KCNQ1/KCNE1 channels by tyrosine kinase

The objective of the study was to investigate the role of tyrosine phosphorylation in the regulation of KCNQ1/KCNE1 channels. Large whole-cell time- and voltage-dependent K⁺ currents were present in human embryonic kidney 293 cells cotransfected with human KCNQ1 and KCNE1 but not in control nontransfected cells. The time- and voltage-dependent current had biophysical properties typical of cardiac KCNQ1/KCNE1 current and was almost completely abolished by KCNQ1 blocker chromanol 293B (50 μM). Both KCNQ1/KCNE1 and KCNQ1 current were inhibited in a voltage-independent manner by tyrosine kinase (PTK) inhibitor tyrphostin A25 (100 μM), but not by PTK-inactive tyrphostin A1 (100 μM), suggesting involvement of tyrosine phosphorylation in maintaining channel activity. This view was strengthened by the finding that phosphotyrosyl phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) (200 μM) reversed the inhibition of current by tyrphostin A25. However, the channel-pertinent tyrosine phosphorylation modulated by these compounds does not appear to be on the channel itself because inhibition of current by tyrphostin A25 was unaffected by single and multiple mutations of KCNQ1 cytoplasmically accessible tyrosine residues. Inhibition by tyrphostin A25 was unaffected by intracellularly applied diC8 phosphatidylinositol-4,5-bisphosphate (diC8 PIP₂; 25 μM), and based on the results obtained from cell surface biotinylation experiments, it was not due to loss of channels from the membrane. We conclude that tyrphostin A25 inhibits KCNQ1/KCNE1 current by lowering tyrosine phosphorylation on unidentified nonchannel protein(s) that directly or indirectly regulate the open probability of the KCNQ1 pore in a PIP₂-independent manner.     

Block of BK (maxi K) channels of rat pituitary melanotrophs by Na+ and other alkali metal ions

The block of large-conductance calcium-activated potassium (BK) channels by internal and external alkali metal ions was studied in adult rat melanotrophs. Internal but not external 20 mM Na⁺ produced a strongly voltage-dependent, flickery block that was well-fitted to the Woodhull model by using a value of 140 mM for the dissociation rate constant at 0 mV [K _d(0)] and an equivalent valence (zδ) of 0.9. At a concentration of 20 mM external K⁺, Cs⁺ and Rb⁺, but not Li⁺, caused a rightward shift of the voltage dependence of the intracellular Na⁺ (Na⁺ _i ) block. This effect of K⁺, Cs⁺ and Rb⁺ was modelled by an equilibrium knock-out mechanism in which the block-relieving ion binds to a site located within the voltage field and consequently increases the off-rate of Na⁺. Internal Li⁺ caused little or no block whereas internal Cs⁺ caused a voltage-dependent block [K _d(0) ≈150 mM]. Flickery channel block observed in cell-attached patches was consistent with a cytoplasmic Na⁺ activity between 1 and 10 mM. Received: 22 January 1996 /Accepted: 26 March 1996     

Heteromeric KCNE2/KCNQ1 potassium channels in the luminal membrane of gastric parietal cells

Recently, we and others have shown that luminal K⁺ recycling via KCNQ1 K⁺ channels is required for gastric H⁺ secretion. Inhibition of KCNQ1 by the chromanol 293B strongly diminished H⁺ secretion. The present study aims at clarifying KCNQ1 subunit composition, subcellular localization, regulation and pharmacology in parietal cells. Using in situ hybridization and immunofluorescence techniques, we identified KCNE2 as the β subunit of KCNQ1 in the luminal membrane compartment of parietal cells. Expressed in COS cells, hKCNE2/hKCNQ1 channels were activated by acidic pH, PIP₂, cAMP and purinergic receptor stimulation. Qualitatively similar results were obtained in mouse parietal cells. Confocal microscopy revealed stimulation-induced translocation of H⁺,K⁺-ATPase from tubulovesicles towards the luminal pole of parietal cells, whereas distribution of KCNQ1 K⁺ channels did not change to the same extent. In COS cells the 293B-related substance IKs124 blocked hKCNE2/hKCNQ1 with an IC₅₀ of 8 n m . Inhibition of hKCNE1- and hKCNE3-containing channels was weaker with IC₅₀ values of 370 and 440 n m , respectively. In conclusion, KCNQ1 coassembles with KCNE2 to form acid-activated luminal K⁺ channels of parietal cells. KCNQ1/KCNE2 is activated during acid secretion via several pathways but probably not by targeting of the channel to the membrane. IKs124 could serve as a leading compound in the development of subunit-specific KCNE2/KCNQ1 blockers to treat peptic ulcers.     

KCNE1 reverses the response of the human K+ channel KCNQ1 to cytosolic pH changes and alters its pharmacology and sensitivity to temperature

Previous studies have shown that heteromultimeric KCNQ1/KCNE1 (KvLQT1/minK) channels and homomultimeric KCNQ1 (KvLQT1) channels exhibit different current properties, e.g. distinct kinetics and different sensitivities to drugs. In this study we report on the divergent responses to internal pH changes and further characterize some of the current properties of the human isoforms of KCNQ1 and KCNE1 expressed in Chinese hamster ovary (CHO) cells or Xenopus laevis oocytes. Decreasing the bath temperature from 37 degrees C to 20 degrees C increased the half-activation time by a factor of 5 for KCNQ1/KCNE1 currents (IKs) but by only twofold (not significant) for KCNQ1 currents (IK) in CHO cells. Acidification of cytosolic pH (pHi) increased IKs but decreased 1K whereas intracellular alkalinization decreased I(Ks) but increased IK. pHi-induced changes in intracellular Ca2+ activity ([Ca2+]i) did not correlate with the current responses. At 20 degrees C mefenamic acid (0.1 mM) significantly augmented IKs but slightly decreased IK. It changed the slow activation kinetics of I(Ks) to an instantaneous onset. The form of the current/voltage (I/V) curve changed from sigmoidal to almost linear. In contrast, at 37 degrees C, mefenamic acid also increased I(Ks) but slowed the activation kinetics and shifted the voltage activation to more hyperpolarized values without markedly affecting the sigmoidal shape of the I/V curve. The potassium channel blockers clotrimazole and tetrapentylammonium (TPeA) inhibited I(Ks) with a lower potency than I(K). These results show that coexpression of KCNE1 reversed pH regulation of KCNQ1 from inhibition to activation by acidic pHi. In addition, KCNE1 altered the pharmacological properties and sensitivity to temperature of KCNQ1. The pH-dependence of I(Ks) might be of clinical and pathophysiological relevance in the pathogenesis of ischaemic cardiac arrhythmias.     

Interaction of KCNE subunits with the KCNQ1 K+ channel pore

KCNQ1 α subunits form functionally distinct potassium channels by coassembling with KCNE ancillary subunits MinK and MiRP2. MinK-KCNQ1 channels generate the slowly activating, voltage-dependent cardiac I _Ks current. MiRP2-KCNQ1 channels form a constitutively active current in the colon. The structural basis for these contrasting channel properties, and the mechanisms of α subunit modulation by KCNE subunits, are not fully understood. Here, scanning mutagenesis located a tryptophan-tolerant region at positions 338–340 within the KCNQ1 pore-lining S6 domain, suggesting an exposed region possibly amenable to interaction with transmembrane ancillary subunits. This hypothesis was tested using concomitant mutagenesis in KCNQ1 and in the membrane-localized 'activation triplet' regions of MinK and MiRP2 to identify pairs of residues that interact to control KCNQ1 activation. Three pairs of mutations exerted dramatic effects, ablating channel function or either removing or restoring control of KCNQ1 activation. The results place KCNE subunits close to the KCNQ1 pore, indicating interaction of MiRP2-72 with KCNQ1-338; and MinK-59,58 with KCNQ1-339, 340. These data are consistent either with perturbation of the S6 domain by MinK or MiRP2, dissimilar positioning of MinK and MiRP2 within the channel complex, or both. Further, the results suggest specifically that two of the interactions, MiRP2-72/KCNQ1-338 and MinK-58/KCNQ1-340, are required for the contrasting gating effects of MinK and MiRP2.     

Subthreshold inactivation of Na+ and K+ channels supports activity-dependent enhancement of back-propagating action potentials in hippocampal CA1

Back-propagating action potentials in CA1 pyramidal neurons may provide the postsynaptic dendritic depolarization necessary for the induction of long-term synaptic plasticity. The amplitudes of back-propagating action potentials are not all or none but are limited in amplitude by dendritic A-type K+ channels. Previous studies of back-propagating action potentials have suggested that prior depolarization of the dendritic membrane reduces A-type channel availability through inactivation, resulting in an enhanced, or boosted, dendritic action potential. However, inactivation kinetics in the subthreshold potential range have not been directly measured. Furthermore, the corresponding rates of Na+ channel inactivation with depolarization have not been considered. Here we report in cell-attached patches (150-220 microm from the soma, 32 degrees C) that at 20-mV positive to rest, A-type K+ channels inactivated with a single exponential time constant of 6 ms, whereas Na+ channels inactivated with a time constant of 37 ms. The ratio of available Na+ to K+ current increased as the duration of the depolarization increased. Thus the subthreshold properties of Na+ and A-type K+ channels provide a mechanism by which information about the level of synaptic activity may be encoded in the amplitude of back-propagating action potentials.     

ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells

The elementary K⁺ conductance activated by the cAMP or the Ca²⁺ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl^–-free conditions, an inwardly rectifying whole-cell K⁺ current was evoked by either forskolin 10 (mol/l) or acetylcholine 1 (mol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8±2.5 pS (n=26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K⁺ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mol/l free Ca²⁺ at the inner side of the membrane. This channel was blocked by Ba²⁺ (5 mol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca²⁺ with and EC₅₀ of 0.25 mol/l and strongly reduced by intracellular MgATP with an IC₅₀ of 600 mol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K⁺ channel was also recorded, with similar characterictics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K⁺ channel regulated by intracellular Ca²⁺ and phosphorylation supports the main K⁺ current activated by secretory agonists in normal cystic fibrosis cell lines. This conductance possibly represents the major pathway for K⁺ recycling at the basolateral membrane during transepithelial fluid secretion.     

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias

KCNQ1 (KVLQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study the properties and regulation of the cloned sKVLQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (<3 pS) K+ channels, in parallel with other K+ channels. sKCNQ1 generated similar small-conductance K+ channels upon expression in CHO cells and Xenopus oocytes. The results suggest the presence of low-conductance KCNQ1 K+ channels in RGT, which are probably regulated by changes in intracellular cAMP, Ca2+ and pH.     

Maxi K+ channels in leaky epithelia are regulated by intracellular Ca2+, pH and membrane potential

We have studied a Ca²⁺-activated K⁺ channel in the ventricular membrane of the epithelium of choroid plexus by means of the patch-clamp technique, using excised inside-out patches. The channel was highly K⁺ selective. It had a conductance of 200 pS with 112 mM KCl on both sides of the membrane. The probability for the channel being open increased with intracellular Ca²⁺, pH and with membrane potential. The channel shows two gating modes. The primary gating mode has open and closed times which depend strongly on membrane potential, intracellular Ca²⁺ and pH. It accounts for the variation of the channel open probability. Lowering intracellular pH from 7.4 to 6.4 reduced the channel open probability mainly by increasing the channel closed time. It appears, that H⁺ can compete with Ca²⁺ in binding to the same site, thereby preventing channel opening. A second gating mode consisted of short-lived closures, or flickers. The open and closed time for this process were largely independent of membrane potential, intracellular Ca²⁺ and pH. The channel density was 0.4 m^–2 corresponding to a K⁺-permeability of 2.2 10^–5 cm s^–1 if the channels were fully open. In cell-attached patches we measured the open probability of the channel in the intact cell membrane. The channel is almost totally closed under normal cellular conditions. This type of channel is therefore not the membrane component that forms the electrodiffusive pathway for K⁺-ions.     

Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons

C-type dorsal root ganglion (DRG) neurons can generate tetrodotoxin-resistant (TTX-R) sodium-dependent action potentials. However, multiple sodium channels are expressed in these neurons, and the molecular identity of the TTX-R sodium channels that contribute to action potential production in these neurons has not been established. In this study, we used current-clamp recordings to compare action potential electrogenesis in Na(v)1.8 (+/+) and (-/-) small DRG neurons maintained for 2-8 h in vitro to examine the role of sodium channel Na(v)1.8 (alpha-SNS) in action potential electrogenesis. Although there was no significant difference in resting membrane potential, input resistance, current threshold, or voltage threshold in Na(v)1.8 (+/+) and (-/-) DRG neurons, there were significant differences in action potential electrogenesis. Most Na(v)1.8 (+/+) neurons generate all-or-none action potentials, whereas most of Na(v)1.8 (-/-) neurons produce smaller graded responses. The peak of the response was significantly reduced in Na(v)1.8 (-/-) neurons [31.5 +/- 2.2 (SE) mV] compared with Na(v)1.8 (+/+) neurons (55.0 +/- 4.3 mV). The maximum rise slope was 84.7 +/- 11.2 mV/ms in Na(v)1.8 (+/+) neurons, significantly faster than in Na(v)1.8 (-/-) neurons where it was 47.2 +/- 1.3 mV/ms. Calculations based on the action potential overshoot in Na(v)1.8 (+/+) and (-/-) neurons, following blockade of Ca(2+) currents, indicate that Na(v)1.8 contributes a substantial fraction (80-90%) of the inward membrane current that flows during the rising phase of the action potential. We found that fast TTX-sensitive Na(+) channels can produce all-or-none action potentials in some Na(v)1.8 (-/-) neurons but, presumably as a result of steady-state inactivation of these channels, electrogenesis in Na(v)1.8 (-/-) neurons is more sensitive to membrane depolarization than in Na(v)1.8 (+/+) neurons, and, in the absence of Na(v)1.8, is attenuated with even modest depolarization. These observations indicate that Na(v)1.8 contributes substantially to action potential electrogenesis in C-type DRG neurons.     

Distribution of (Na+ + K+)ATPase and sodium channels in skeletal muscle and electroplax

Summary The distributions of (Na⁺ + K⁺)ATPase and sodium channels in skeletal muscle fibres and electrocytes were determined by immunofluorescent and immunoelectron microscopic techniques using antibodies against rat and eel (Na⁺ + K⁺)ATPase and the eel electric organ sodium channel. The extrajunctional sarcolemma of skeletal muscle was uniformly stained by polyclonal antibodies against (Na⁺ + K⁺)ATPase and the sodium channel. The T-tubule system of skeletal muscle was also labelled heavily for both (Na⁺ + K⁺)ATPase and the sodium channel. The terminal cisternae of the sarcoplasmic reticulum was stained for (Na⁺ + K⁺)ATPase but not sodium channels. At the motor endplate, (Na⁺ + K⁺)ATPase-like immunoreactivity was present along the plasmalemma of motor nerve terminals but not along the postsynaptic junctional sarcolemma. Paradoxically, a monoclonal antibody that binds to the form of the catalytic subunit of (Na⁺ + K⁺)ATPase from rat hepatocytes and renal tubule cells did not label the enzyme in rat skeletal muscle. In electrocytes, (Na⁺ + K⁺)ATPase-like irnmunoreactivity was concentrated primarily along the plasmalemma and calveolae of the non-innervated face. In contrast, sodium channel-like immunoreactivity was concentrated along the plasmalemma of the innervated face except in the clefts of the postsynaptic membrane. Thus, we conclude that at endplates both the (Na⁺ + K⁺)ATPase of rat skeletal muscle and sodium channels of eel electrocytes are not concentrated in the juxtaneuronal postsynaptic membrane. We also interpret the failure of the monoclonal anti- (Na⁺ + K⁺)ATPase antibodies to bind to the enzyme in muscle to indicate that the catalytic subunit of skeletal muscle (Na⁺ + K⁺)ATPase displays different epitopes than does the a subunit of kidney and liver.     

Facilitation of Ca2+-activated K+ channels (IKCa1) by mibefradil in B lymphocytes

K⁺ channels play critical roles in the proliferation and activation of lymphocytes. Mouse B cells express large-conductance background K⁺ channel (LK_bg) in addition to the voltage-gated K⁺ channel (Kv) and Ca²⁺-activated K⁺ channel current (IKCa1). Mibefradil, a blocker of T-type Ca²⁺ channels, has been reported to affect the proliferation of immune cells. In this study, we investigated the effects of mibefradil on the membrane potential and ion channels in murine B cell lines, WEHI-231 and Bal-17. In the whole-cell patch clamp experiments, mibefradil blocked Kv and LK_bg current with half inhibitory concentration (IC₅₀), 1.9 and 2.3 μM, respectively. Interestingly, IKCa1 current was increased by mibefradil. In the inside-out patch clamp study with cloned murine IKCa1 (mIKCa1) in HEK-293, mibefradil increased both Ca²⁺ sensitivity and maximum activity of mIKCa1. At high concentrations (>10 μM), mibefradil inhibited mIKCa1 in a voltage-dependent manner. Application of anti-IgM antibody to stimulate B cell receptors (BCR-ligation) induced transient hyperpolarization of Bal-17 and WEHI-231 cells, which became persistent with 1 μM mibefradil. The hyperpolarizing response was abolished by charybdotoxin, a selective blocker for SK4/IKCa1. In summary, our study firstly reports the ion channel-activating effects of mibefradil. The selective potent activation of IKCa1 suggests that mibefradil-derived drugs might be useful in the control of cell responses related with IKCa1. HY Yoo, H Zheng, and JH Nam contributed equally to this study.     

O2 sensing by recombinant TWIK-related halothane-inhibitable K+ channel-1 background K+ channels heterologously expressed in human embryonic kidney cells

Hypoxic inhibition of K+ channels provides a link between low O2 and cell function, and in glossopharyngeal neurons hypoxic inhibition of a TWIK-related halothane-inhibitable K+ channel-1 (THIK-1)-like background K+ channel regulates neuronal function. In the present study, we examined directly the O2 sensitivity of recombinant THIK-1 channels, expressed in human embryonic kidney (HE293) cells. THIK-1 expression conferred a moderately outwardly rectifying halothane-inhibited and arachidonic acid-potentiated K+ current and invoked a strongly hyperpolarized resting membrane potential. Endogenous K+ currents in untransfected cells were unaffected by either agent. Hypoxia (P(O2), 20 mmHg) reversibly inhibited THIK-1 currents and caused membrane depolarization, effects that were occluded by halothane. Neither the mitochondrial complex I inhibitors rotenone, myxothiazol and sodium cyanide, nor the NADPH oxidase inhibitors diphenylene iodonium and phenylarsine oxide, were effective in inhibiting the O2-sensitivity of THIK-1. Thus, hypoxic inhibition of THIK-1 occurs by a mechanism dissimilar to that which regulates the activity of other members of the background K+ channel family. Given the O2 sensitivity of THIK-1 channels and their abundant expression in the CNS, we raise for the first time the possibility of a physiological and/or pathological role for these channels during brain ischemia.     

Decreased expression of apical Na+ channels and basolateral Na+, K+-ATPase in ulcerative colitis

Impaired absorption of sodium (Na+) and water is a major factor in the pathogenesis of diarrhoea in ulcerative colitis (UC). Electrogenic Na+ absorption, present mainly in human distal colon and rectum, is defective in UC, but the molecular basis for this is unclear. The effect of UC on the expression of apical Na+ channels (ENaC) and basolateral Na+, K+-ATPase, the critical determinants of electrogenic Na+ transport, was therefore investigated in this study. Sigmoid colonic and/or proximal rectal mucosal biopsies were obtained from patients with mild to moderate UC, and patients with functional abdominal pain (controls). ENaC subunit expression was studied by immunohistochemistry, western blot analysis, and in situ hybridization, and Na+, K+-ATPase isoform expression was studied by immunohistochemistry, western blotting, and northern blot analysis. UC was associated with substantial decreases in the expression of the ENaC beta- and gamma-subunit proteins and mRNAs, whereas the decrease in ENaC alpha-subunit protein detected by immunolocalization was less marked. The levels of expression of Na+, K+-ATPase alpha1- and beta1-isoform proteins were also lower in UC patients than in controls, although there were no differences in Na+, K+-ATPase alpha1- and beta1-isoform mRNA levels between the two groups. Taken together, these results show that UC results mainly in decreased expression of the apical ENaC beta- and gamma-subunits, as well as the basolateral Na+, K+-ATPase alpha1- and beta1-isoforms. In conclusion, these changes provide a basis for the low/negligible levels of electrogenic Na+ absorption seen in the distal colon and rectum of UC patients, which contribute to the pathogenesis of diarrhoea in this disease.     

The cardioplegic solution HTK: effects on membrane potential,intracellular K+ and Na+ activities in sheep cardiac Purkinje fibres

The effects of the cardioplegic solution HTK on membrane potential (E_M) and intracellular K and Na activities (a _K ⁱ , a _Na ⁱ ) were studied in sheep cardiac Purkinje fibres by means of conventional and ion-selective microelectrodes. HTK contains (mM): Na 15, K 10, Ca 0, Mg 4, histidine 180, (1) In control conditions E_M was –74.3±3.3 mV (n=25), a _K ⁱ was 116.4±4.1 mM (n=7) and a _Na ⁱ was 8.2±1.4 mM (n=15). (2) Exposure to HTK led to a depolarization to –59.7±3.6 mV (n=25) which exceeded by about 5–7 mV that induced in a Tyrode solution of 10 mM K and in a modified HTK solution supplemented by 2 mM Ca (n=6). (3) Addition of 0.5 mM barium eliminated the difference in the steady-state depolarization. (4) HTK superfusion increased a _K ⁱ to 120.1±4.4 mM (n=7) and decreased a _Na ⁱ to 3.9±0.9 mM (n=15). (5) The decrease in a _Na ⁱ was insensitive to amiloride (1 mM) and to external alkalization but was slightly increased by addition of 2 mM calcium. (6) When the calcium in Tyrode solution was lowered from 2.0 mM to 0.05 mM, a _Na ⁱ hardly decreased during subsequent exposure to unmodified HTK and it increased in the presence of 0.1 mM dihydroouabain. We propose the hypothesis (1) that the difference in membrane depolarization between HTK and a 10 mM K-Tyrode is caused by a decrease in K conductance by the HTK solution and (2) that the a _Na ⁱ decline mainly results from a coupled Ca influx via Na-Ca exchange due to a delayed washout of external calcium.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 330 — Organprotektion     

Modulation of recombinant transient-receptor-potential-like (TRPL) channels by cytosolic Ca2+

Whole-cell current recordings were used to examine the involvement of intracellular Ca2+ in the modulation of recombinant transient-receptor-potential like (TRPL) channels of Drosophila photoreceptor cells. TRPL was stably transfected in Chinese hamster ovary (CHO) cells and the expression of a calmodulin-binding protein with a molecular mass that corresponded to TRPL was demonstrated using calmodulin overlays. In cells expressing TRPL, ionic currents that were prominently outwardly rectifying were detected prior to activation of intracellular signalling pathways. The outwardly rectifying currents reversed close to 0 mV and did not occur after removal of permeant cations from the intracellular space. This suggests that TRPL forms non-selective cationic channels that appear to be constitutively active in mammalian cell lines. The TRPL channel currents were enhanced by manoeuvres that activate the phospholipase C (PLC) signalling pathway. These included activation of membrane receptors by thrombin, activation of G proteins by cell dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and release of Ca2+ from intracellular stores by dialysis with inositol 1,4,5-trisphosphate (IP3). After complete depletion of Ca2+ stores, IP3 had no effect on TRPL currents, suggesting that IP3 does not activate recombinant TRPL channels directly. However, thapsigargin, which induces a rise of cytosolic Ca2+, increased TRPL channel currents. Cell dialysis with solutions containing various concentrations of Ca2+ enhanced TRPL currents in a dose-dependent manner (EC50=450 nM Ca2+). Conversely, chelation of cytosolic Ca2+ abolished TRPL channel currents. The present results indicate that the activity of recombinant TRPL channels expressed in mammalian cell lines is up-regulated by a rise of cytosolic Ca2+.     

Regulation of inwardly rectifying K+ channels by intracellular pH in opossum kidney cells

The effects of intracellular pH on an inwardly rectifying K⁺ channel (K_in channel) in opossum kidney (OK) cells were examined using the patch-clamp technique. Experiments with inside-out patches were first carried out in Mg²⁺-and adenosine triphosphate (ATP)-free conditions, where Mg²⁺-induced inactivation and ATP-induced reactivation of K_in channels were suppressed. When the bath (cytoplasmic side) pH was decreased from 7.3 to either 6.8 or 6.3, K_in channels were markedly inhibited. The effect of acid pH was not fully reversible. When the bath pH was increased from 7.3 to 7.8, 8.3 or 8.8, the channels were activated reversibly. The channel activity exhibited a sigmoidal pH dependence with a maximum sensitivity at pH 7.5. Inside-out experiments were also carried out with a solution containing 3 mM Mg-ATP and a similar pH sensitivity was observed. However, in contrast with the results obtained in the absence of Mg²⁺ and ATP, the effect of acid pH was fully reversible. Experiments with cell-attached patches demonstrated that changes in intracellular pH, which were induced by changing extracellular pH in the presence of an H⁺ ionophore, could influence the channel activity reversibly. It is concluded that the activity of K_in channels can be controlled by the intracellular pH under physiological conditions.     

Regulation of alveolar epithelial Na+ channels by ERK1/2 in chlorine-breathing mice

The mechanisms by which the exposure of mice to Cl(2) decreases vectorial Na(+) transport and fluid clearance across their distal lung spaces have not been elucidated. We examined the biophysical, biochemical, and physiological changes of rodent lung epithelial Na(+) channels (ENaCs) after exposure to Cl(2), and identified the mechanisms involved. We measured amiloride-sensitive short-circuit currents (I(amil)) across isolated alveolar Type II (ATII) cell monolayers and ENaC single-channel properties by patching ATII and ATI cells in situ. α-ENaC, γ-ENaC, total and phosphorylated extracellular signal-related kinase (ERK)1/2, and advanced products of lipid peroxidation in ATII cells were measured by Western blot analysis. Concentrations of reactive intermediates were assessed by electron spin resonance (ESR). Amiloride-sensitive Na(+) channels with conductances of 4.5 and 18 pS were evident in ATI and ATII cells in situ of air-breathing mice. At 1 hour and 24 hours after exposure to Cl(2), the open probabilities of these two channels decreased. This effect was prevented by incubating lung slices with inhibitors of ERK1/2 or of proteasomes and lysosomes. The exposure of ATII cell monolayers to Cl(2) increased concentrations of reactive intermediates, leading to ERK1/2 phosphorylation and decreased I(amil) and α-ENaC concentrations at 1 hour and 24 hours after exposure. The administration of antioxidants to ATII cells before and after exposure to Cl(2) decreased concentrations of reactive intermediates and ERK1/2 activation, which mitigated the decrease in I(amil) and ENaC concentrations. The reactive intermediates formed during and after exposure to Cl(2) activated ERK1/2 in ATII cells in vitro and in vivo, leading to decreased ENaC concentrations and activity.