Siegfried Waldegger

Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family.

Magnesium is an essential ion involved in many biochemical and physiological processes. Homeostasis of magnesium levels is tightly regulated and depends on the balance between intestinal absorption and renal excretion. However, little is known about specific proteins mediating transepithelial magnesium transport. Using a positional candidate gene approach, we identified mutations in TRPM6 (also known as CHAK2), encoding TRPM6, in autosomal-recessive hypomagnesemia with secondary hypocalcemia (HSH, OMIM 602014), previously mapped to chromosome 9q22 (ref. 3). The TRPM6 protein is a new member of the long transient receptor potential channel (TRPM) family and is highly similar to TRPM7 (also known as TRP-PLIK), a bifunctional protein that combines calcium- and magnesium-permeable cation channel properties with protein kinase activity. TRPM6 is expressed in intestinal epithelia and kidney tubules. These findings indicate that TRPM6 is crucial for magnesium homeostasis and implicate a TRPM family member in human disease.

A constitutively open potassium channel formed by KCNQ1 and KCNE3

Mutations in all four known KCNQ potassium channel α-subunit genes lead to human diseases. KCNQ1 (KvLQT1) interacts with the β-subunit KCNE1 (IsK, minK) to form the slow, depolarization-activated potassium current IKs that is affected in some forms of cardiac arrhythmia. Here we show that the novel β-subunit KCNE3 markedly changes KCNQ1 properties to yield currents that are nearly instantaneous and depend linearly on voltage. It also suppresses the currents of KCNQ4 and HERG potassium channels. In the intestine, KCNQ1 and KCNE3 messenger RNAs colocalized in crypt cells. This localization and the pharmacology, voltage-dependence and stimulation by cyclic AMP of KCNQ1/KCNE3 currents indicate that these proteins may assemble to form the potassium channel that is important for cyclic AMP-stimulated intestinal chloride secretion and that is involved in secretory diarrhoea and cystic fibrosis.

Blockade of HERG channels expressed in Xenopus oocytes by the histamine receptor antagonists terfenadine and astemizole.

The widely used histamine receptor antagonists terfenadine and astemizole were shown to prolong the QT interval in electrocardiographic recordings in cases of overdose or inappropriate co‐medications, indicating a possible interaction with cardiac K+ channels. Here, terfenadine and astemizole both inhibited the human ether‐a‐go‐go related gene (HERG) encoded channels expressed in Xenopus oocytes at nanomolar concentrations in a use‐ and voltage‐dependent fashion. In contrast, inhibition of other delayed rectifier (Kvl.1 and IsK) or inward rectifier K+ channels (IRK1) was much weaker and occurred only at high micromolar concentrations. These results suggest that blockade of HERG channels by terfenadine and astemizole might contribute to the cardiac side effects of these compounds.

Claudin-16 and claudin-19 interact and form a cation-selective tight junction complex

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an inherited disorder caused by mutations in the genes encoding the TJ proteins claudin-16 (CLDN16) and CLDN19; however, the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney are not understood. Here we showed that in pig kidney epithelial cells, CLDN19 functioned as a Cl(-) blocker, whereas CLDN16 functioned as a Na(+) channel. Mutant forms of CLDN19 that are associated with FHHNC were unable to block Cl(-) permeation. Coexpression of CLDN16 and CLDN19 generated cation selectivity of the TJ in a synergistic manner, and CLDN16 and CLDN19 were observed to interact using several criteria. In addition, disruption of this interaction by introduction of FHHNC-causing mutant forms of either CLDN16 or CLDN19 abolished their synergistic effect. Our data show that CLDN16 interacts with CLDN19 and that their association confers a TJ with cation selectivity, suggesting a mechanism for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC.

Electrogenic Properties and Substrate Specificity of the Polyspecific Rat Cation Transporter rOCT1

The previously cloned rat cation transporter rOCT1 detected in renal proximal tubules and hepatocytes (Gründemann, D., Gorboulev, V., Gambaryan, S., Veyhl, M., and Koepsell, H. (1994) Nature 372, 549-552) was expressed in Xenopus oocytes, and transport properties were analyzed using tracer uptake studies and electrophysiological measurements. rOCT1 induced highly active transport of a variety of cations, including the classical substrates for cation transport, such as N-1-methylnicotinamide, 1-methyl-4-phenylpyridinium (MPP), and tetraethylammonium (TEA), but also the physiologically important choline. In oocytes rOCT1 also mediated efflux of MPP, which could be trans-stimulated by MPP and TEA. Cation transport via rOCT1 was electrogenic. In voltage-clamped oocytes, transport of TEA and choline via rOCT1 produced inwardly directed currents, which were independent of extracellular ion composition or pH. The choline- and TEA-induced currents were voltage-dependent at nonsaturating concentrations, and the apparent affinity of these cations was decreased at depolarized voltages. Other substrates transported by rOCT1 were the polyamines spermine and spermidine. Interestingly, the previously described potent inhibitors of rOCT1, cyanine 863, quinine, and D-tubocurarine were substrates themselves. The data indicate that rOCT1 is an effective transport system that is responsible for electrogenic uptake of a wide variety of organic cations into epithelial cells of renal proximal tubules and hepatocytes.

Claudin-16 and claudin-19 interaction is required for their assembly into tight junctions and for renal reabsorption of magnesium

Claudins are tight junction integral membrane proteins that are key regulators of the paracellular pathway. Defects in claudin-16 (CLDN16) and CLDN19 function result in the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Previous studies showed that siRNA knockdown of CLDN16 in mice results in a mouse model for FHHNC. Here, we show that CLDN19-siRNA mice also developed the FHHNC symptoms of chronic renal wasting of magnesium and calcium together with defective renal salt handling. siRNA knockdown of CLDN19 caused a loss of CLDN16 from tight junctions in the thick ascending limb (TAL) without a decrease in CLDN16 expression level, whereas siRNA knockdown of CLDN16 produced a similar effect on CLDN19. In both mouse lines, CLDN10, CLDN18, occludin, and ZO-1, normal constituents of TAL tight junctions, remained correctly localized. CLDN16- and CLDN19-depleted tight junctions had normal barrier function but defective ion selectivity. These data, together with yeast two-hybrid binding studies, indicate that a heteromeric CLDN16 and CLDN19 interaction was required for assembling them into the tight junction structure and generating cation-selective paracellular channels.

Colocalization of KCNQ1/KCNE channel subunits in the mouse gastrointestinal tract

Abstract. The KCNQ1 potassium channel α-subunit can associate with various KCNE β-subunits that drastically influence channel gating. Here we show that in the mouse gastrointestinal tract KCNQ1 is prominently expressed in stomach, small intestine and colon, while KCNE3 is expressed in the colon and to a lesser extent in small intestine. Immunostaining revealed that KCNQ1 colocalizes with KCNE3 in the basolateral membranes of crypt cells of the colon and small intestine. Together with the previously shown electrophysiological properties of KCNQ1/KCNE3 channels, this strongly suggests that they form the basolateral potassium conductance that is required for transepithelial cAMP-stimulated chloride secretion. In the stomach, KCNQ1 is expressed together with the H+/K+-ATPase in the luminal membrane of acid-secreting parietal cells of gastric glands. KCNE2, but neither KCNE1 nor KCNE3 was detected in the stomach by Northern analysis. Similar to KCNQ1, KCNE2 was present in gastric glands in only a subset of cells that probably represent parietal cells. The coexpression of KCNQ1 and KCNE2 in HEK293 cells yielded potassium currents that were open at resting voltages, suggesting that these heteromeric channels may underlie the apical potassium conductance in acid-secreting parietal cells that is necessary for the recycling of potassium ions during acid secretion via the H+/K+-ATPase.

h-sgk serine-threonine protein kinase gene as transcriptional target of transforming growth factor β in human intestine ☆ ☆☆ ★

BACKGROUND & AIMS Recently, the immediate early gene h-sgk was cloned as a hypertonicity-induced gene from human hepatoma cells. The aim of this study was to localize h-sgk messenger RNA (mRNA) expression in normal and inflamed intestinal mucosa and to identify potential transcriptional regulators. METHODS h-sgk mRNA in small intestinal mucosa from healthy persons and patients with Crohns disease was determined by in situ hybridization. Transcriptional regulation was studied by Northern blot analysis of total RNA isolated from cultured human Intestine 407, U937, and HepG2 cells. RESULTS In normal ileum, h-sgk mRNA was selectively localized to the apical villus enterocytes, whereas no staining was detected in crypt cells. In Crohns disease, enterocytes of the crypts expressed h-sgk and abundant h-sgk positive inflammatory cells appeared in the lamina propria. Combined h-sgk in situ hybridization and immunohistochemical analysis of CD68 antigen expression identified a part of these cells as macrophages. In addition to spatial correlation of transforming growth factor (TGF)-beta1 protein and h-sgk mRNA expression, h-sgk transcription in human Intestine 407 and HepG2 cells as well as in U937 monocytes/macrophages was strongly induced by TGF-beta1 in vitro. CONCLUSIONS h-sgk expression in normal and inflamed intestinal mucosa may be regulated by TGF-beta1 and may contribute to the pleiotropic actions of TGF-beta1 in mucosal cell populations.

Barttin increases surface expression and changes current properties of ClC-K channels

Abstract. The term Bartter syndrome encompasses a heterogeneous group of autosomal recessive salt-losing nephropathies that are caused by disturbed transepithelial sodium chloride reabsorption in the distal nephron. Mutations have been identified in the NKCC2 (Na+-K+-2Cl–) cotransporter and ROMK potassium channel, which cooperate in the process of apical sodium chloride uptake, and ClC-Kb chloride channels, which mediate basolateral chloride release. Recently, mutations in barttin, a protein not related to any known ion transporter or channel, were described in BSND, a variant of Bartter syndrome associated with sensorineural deafness. Here we show that barttin functions as an activator of ClC-K chloride channels. Expression of barttin together with ClC-K in Xenopus oocytes increased ClC-K current amplitude, changed ClC-K biophysical properties, and enhanced ClC-K abundance in the cell membrane. Co-immunoprecipitation revealed a direct interaction of barttin with ClC-K. We performed in situ hybridization on rat kidney slices and RT-PCR analysis on microdissected nephron segments to prove co-expression of barttin, ClC-K1 and ClC-K2 along the distal nephron. Functional analysis of BSND-associated point mutations revealed impaired ClC-K activation by barttin. The results demonstrate regulation of a CLC chloride channel by an accessory protein and indicate that ClC-K activation by barttin is required for adequate tubular salt reabsorption.

INHIBITION OF IKS IN GUINEA PIG CARDIAC MYOCYTES AND GUINEA PIG ISK CHANNELS BY THE CHROMANOL 293B

The chromanol derivative 293B was previously shown to inhibit a cAMP regulated K+ conductance in rat colon crypts. Subsequent studies on cloned K+ channels from the rat demonstrated that 293B blocks specifically IsK channels expressed in Xenopus oocytes, but does not affect the delayed and inward rectifier Kv1.1 and Kir2.1, respectively. In the present study, the specificity of 293B for the cardiac K+ conductances IKs and IKr, and for the cloned guinea pig IsK channel and the human HERG channel, which underly IKs and IKr, respectively, was analyzed. 293B inhibited both the slowly activating K+ conductance IKs in cardiac myocytes and guinea pig IsK channels expressed in Xenopus oocytes with a similar IC50 (2-6 μmol/1). In contrast, high concentrations of 293B had only a negligible effect on the more rapid activating IKr. Similarly, 293B exerted no effect on HERG channels expressed in Xenopus oocytes. In summary, 293B appears to be a rather specific inhibitor of IKs and the underlying IsK channels.