Dirk Heitzmann

Physiology and Pathophysiology of Potassium Channels in Gastrointestinal Epithelia

Epithelial cells of the gastrointestinal tract are an important barrier between the milieu interne and the luminal content of the gut. They perform transport of nutrients, salts, and water, which is essential for the maintenance of body homeostasis. In these epithelia, a variety of K(+) channels are expressed, allowing adaptation to different needs. This review provides an overview of the current literature that has led to a better understanding of the multifaceted function of gastrointestinal K(+) channels, thereby shedding light on pathophysiological implications of impaired channel function. For instance, in gastric mucosa, K(+) channel function is a prerequisite for acid secretion of parietal cells. In epithelial cells of small intestine, K(+) channels provide the driving force for electrogenic transport processes across the plasma membrane, and they are involved in cell volume regulation. Fine tuning of salt and water transport and of K(+) homeostasis occurs in colonic epithelia cells, where K(+) channels are involved in secretory and reabsorptive processes. Furthermore, there is growing evidence for changes in epithelial K(+) channel expression during cell proliferation, differentiation, apoptosis, and, under pathological conditions, carcinogenesis. In the future, integrative approaches using functional and postgenomic/proteomic techniques will help us to gain comprehensive insights into the role of K(+) channels of the gastrointestinal tract.

Deoxycholic acid (DOC) affects the transport properties of distal colon.

Abstract. Secondary bile acids can induce diarrhea. In the present study we examined the effects of deoxycholic acid (DOC) on equivalent short-circuit current (Isc) in rabbit colon and the cellular mechanisms involved in DOC action (rabbit and rat). Luminal DOC inhibited amiloride-sensitive Na+ absorption. In the presence of amiloride luminal DOC had a concentration dependent effect on Isc. Low concentrations (1–10xa0µmol/l) induced a lumen-positive current (51±3xa0µA/cm2, 10xa0µmol/l, n=7) which was inhibited by luminal Ba2+ suggesting the activation of a luminal K+ conductance. Higher luminal concentrations induced a lumen-negative current (–76±9xa0µA/cm2, 100xa0µmol/l, n=11). Basolateral application of DOC, also in the presence of amiloride, only induced lumen-negative Isc (–58±10xa0µA/cm2, 100xa0µmol/l, n=6, EC50=3xa0µmol/l). This current could be abolished completely by the K+ channel blocker 293B, a selective inhibitor of cAMP-dependent Cl– secretion. This action of DOC on Isc was additive to the effect of carbachol (CCH) but not additive to that of cAMP. In intact rat colon mucosa pretreated with DOC a significant increase in cAMP production was observed. Fura-2 measurements of cytosolic Ca2+ activity ([Ca2+]i) in isolated colonic crypts (rabbit and rat) showed that 100xa0µmol/l DOC induced a weak [Ca2+]i increase. Whole-cell measurements of membrane voltage in isolated rat colonic crypts revealed a hyperpolarization by DOC (–4.9±0.8xa0mV, 100xa0µmol/l, n=8) but a depolarization by prostaglandin E2 (PGE2, via cAMP) (24±7xa0mV, n=8). The present data show that DOC acts at more than one target in the colon: in the intact mucosa it activates luminal K+ channels and Cl– secretion and this is paralleled by an increase in cAMP production. In isolated crypts DOC probably activates a Ca2+-regulated K+ conductance but has no effect on cAMP. Hence DOC probably activates ion channels or channel-regulating factors in colonocytes and acts on non-epithelial cells to activate Cl– secretion indirectly.

KCNE Beta Subunits Determine pH Sensitivity of KCNQ1 Potassium Channels

Background/Aims: Heteromeric KCNEx/KCNQ1 (=KvLQT1, Kv7.1) K+ channels are important for repolarization of cardiac myocytes, endolymph secretion in the inner ear, gastric acid secretion, and transport across epithelia. They are modulated by pH in a complex way: homomeric KCNQ1 is inhibited by external acidification (low pHe); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported. Methods: The role of KCNE subunits for the effect of pHe on KCNQ1 was analyzed in transfected COS cells and cardiac myocytes by the patch-clamp technique. Results: In outside-out patches of transfected cells, hKCNE2/hKCNQ1 current was increased by acidification down to pH 4.5. Chimeras with the acid-insensitive hKCNE3 revealed that the extracellular N-terminus and at least part of the transmembrane domain of hKCNE2 are needed for activation by low pHe. hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pHe: The slowly activating hKCNE1/hKCNQ1 channels were converted into constitutively open, non-deactivating channels. Experiments on guinea pig and mouse cardiac myocytes pointed to an important role of KCNQ1 during acidosis implicating a significant contribution to cardiac repolarization under acidic conditions. Conclusion: External pH can modify current amplitude and biophysical properties of KCNQ1. KCNE subunits work as molecular switches by modulating the pH sensitivity of human KCNQ1.

Regulation of the Na+2Cl–K+ cotransporter in isolated rat colon crypts

The Na+2Cl–K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20xa0mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δ fluorescence ratio units/1000xa0s). In pilot experiments it was shown that the pH signal caused by the Na+2Cl–K+ cotransporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1xa0mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl– solution (4xa0mmol/l) for 10xa0min enhanced the uptake rate significantly from 1.70±0.11 to 2.54±0.27xa0U/1000xa0s (n=20). The addition of 100xa0mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93±0.17 to 2.84±0.43xa0U/1000xa0s (n=5). Stimulation of NaCl secretion by a solution containing 100xa0µmol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06±0.34 to 2.40±0.30xa0U/1000xa0s (n=11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100xa0µmol/l and 5xa0µmol/l, respectively) was from 2.39±0.24 to 3.06±0.36xa0U/1000xa0s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500xa0µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na+2Cl–K+ cotransporter: (1) lowering of cytosolic Cl– concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.

The Na+2Cl–K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage

Abstractu2002Effects of cAMP on Cl– secretion, intracellular Cl– activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (Isc) showed that cAMP stimulates Na+Cl– secretion in a biphasic manner. The first and rapid phase corresponds to Cl– exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl– conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1–2 min) activation of the Na+2Cl–K+ cotransporter. During the first phase cytosolic Cl– activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of Isc activation. When cytosolic Cl– activity was increased by exposing the RGT to a high K+ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na+2Cl–K+ cotransport is transient cell shrinkage.

The TFIIH Subunit p89 (XPB) Localizes to the Centrosome during Mitosis

Background: The general transcription factor II H (TFIIH), comprised of a core complex and an associated CAK-complex, functions in transcription, DNA repair and cell cycle control. Mutations of the two largest subunits, p89 (XPB) and p80 (XPD), cause the hereditary cancer-prone syndrome xeroderma pigmentosum. Methods: The TFIIH subunit p89 was monitored during interphase and cell division by immunofluorescence staining, GFP-fusion constructs including deletions, live cell imaging and immuno-precipitations. Results: Here we demonstrate that during cell division, from prophase until telophase, the TFIIH core subunit p89, but not other subunits of TFIIH, associates with the centrosomes and the adjacent parts of the mitotic spindle. With overall constant levels throughout mitosis, p89 re-localizes to the newly formed nuclei by the end of mitosis. Furthermore, p89 interacts with the centrosomal protein γ-tubulin. Truncations of p89 result in an abnormal subcellular distribution during interphase and abolished centrosomal association during mitosis. Conclusions: Our observations suggest a so far unappreciated role for p89 in cell cycle regulation, and may be the structural basis for a long known, but hitherto unexplained interaction between p89 and tubulin.

Sex-dependent differences in the in vivo respiratory phenotype of the TASK-1 potassium channel knockout mouse

TASK-1 potassium channels have been implicated in central and peripheral chemoreception; however, the precise contribution of TASK-1 for the control of respiration is still under debate. Here, we investigated the respiration of unrestrained adult and neonatal TASK-1 knockout mice (TASK-1-/-) using a plethysmographic device. Respiration in adult female TASK-1-/- mice under control (21% O2), hypoxia and hypercapnia was unaffected. Under acute hypoxia male TASK-1-/- mice exhibited a reduced increase of the respiratory frequency (fR) compared to wildtypes. However, the tidal volume (VT) of male TASK-1-/- mice was strongly enhanced. The volatile anesthetic isoflurane induced in male TASK-1-/- and male wild type mice (TASK-1+/+) a similar respiratory depression. Neonatal TASK-1-/- mice demonstrated a 30-40% decrease of the minute volume, caused by a reduction of the fR under control condition (21% O2). Under hypoxia, neonatal TASK-1-/- mice more frequently stopped breathing (apnea>3s) suggesting an increased hypoxia-sensitivity. As reported before, this increased hypoxia sensitivity had no influence on the survival rate of neonatal TASK-1-/- mice. In adult and neonatal mice, TASK-1 gene deletion induced a significant prolongation of the relaxation time (RT), which is a parameter for expiration kinetics. Additionally, screening for mutations in the human TASK-1 gene in 155 cases of sudden infant death syndrome (SIDS) was inconclusive. In conclusion, these data are suggestive for an increased hypoxia-sensitivity of neonatal TASK-1-/- mice, however, without causing an increase in neonatal lethality. In adult female TASK-1-/- mice respiration was unaffected, whereas adult male TASK-1-/- mice showed a modified breathing pattern. These results are suggestive for sex-specific mechanisms for compensating the inactivation of TASK-1 in mice.

Evidence for Na+/Ca2+ exchange in the rectal gland of Squalus acanthias.

Abstract. Previously we have shown that stimulation of in vitro perfused rectal gland tubules (RGT) of the dogfish Squalus acanthias by adenosine 3,5-cyclic monophosphate (cAMP), (as a cocktail comprising 0.1xa0mmol/l dibutyryl-cAMP, 10xa0µmol/l forskolin and 0.1xa0mmol/l adenosine, hereafter termed STIM) leads to an increase in cytosolic Ca2+ ([Ca2+]i) and that this assists Cl– secretion by enhancing basolateral K+ conductance. In the present study we examined the mechanism of the cAMP-induced increase in [Ca2+]i. [Ca2+]i was measured using the fura-2 technique in isolated in vitro perfused RGT. As before, STIM enhanced [Ca2+]i. This elevation of [Ca2+]i was prevented completely when STIM was added in the presence of the Na+2Cl–K+ cotransport inhibitor furosemide (0.5xa0mmol/l). This suggests that the increase in [Ca2+]i induced by STIM is caused by a concomitant increase in cytosolic Na+ ([Na+]i) and not by the activation of second messenger cascades. Furosemide prevents this increase in [Na+]i and hence the elevation of [Ca2+]i. Moreover, the plateau phase of the [Ca2+]i transient produced by carbachol (CCH, 0.1xa0mmol/l) was augmented strongly when bath Na+ was reduced to 5xa0mmol/l. These data suggest that the level of [Ca2+]i is determined by Na+-dependent Ca2+ export, most likely via a Na+/Ca2+ exchanger. The increase in [Na+]i accompanying stimulation of Cl– secretion reduces the rate of Ca2+ export leading to an elevation of [Ca2+]i, as does a reduction in bath Na+ which augments the [Ca2+]i plateau produced by CCH.