The importance of the gut microbiome and its signals for a healthy nervous system and the multifaceted mechanisms of neuropsychiatric disorders.

Increasing evidence links the gut microbiome and the nervous system in health and disease. This narrative review discusses current views on the interaction between the gut microbiota, the intestinal epithelium, and the brain, and provides an overview of the communication routes and signals of the bidirectional interactions between gut microbiota and the brain, including circulatory, immunological, neuroanatomical, and neuroendocrine pathways. Similarities and differences in healthy gut microbiota in humans and mice exist that are relevant for the translational gap between non-human model systems and patients. There is an increasing spectrum of metabolites and neurotransmitters that are released and/or modulated by the gut microbiota in both homeostatic and pathological conditions. Dysbiotic disruptions occur as consequences of critical illnesses such as cancer, cardiovascular and chronic kidney disease but also neurological, mental, and pain disorders, as well as ischemic and traumatic brain injury. Changes in the gut microbiota (dysbiosis) and a concomitant imbalance in the release of mediators may be cause or consequence of diseases of the central nervous system and are increasingly emerging as critical links to the disruption of healthy physiological function, alterations in nutrition intake, exposure to hypoxic conditions and others, observed in brain disorders. Despite the generally accepted importance of the gut microbiome, the bidirectional communication routes between brain and gut are not fully understood. Elucidating these routes and signaling pathways in more detail offers novel mechanistic insight into the pathophysiology and multifaceted aspects of brain disorders.

The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells.

BACKGROUND/AIMS: Glucose-stimulated insulin secretion (GSIS) of pancreatic ß-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem), activation of voltage-activated Ca2+ currents (ICav) and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi) or cell volume, which also affect ß-cell viability, can elicit or modify insulin release. In ß-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. METHODS: In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi) recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. RESULTS: We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICav blocker nifedipine caused a full inhibition of GSIS at 10 and 20 µM. At 20 µM, SCH-28080 inhibited ICav comparable to 20 µM nifedipine and in addition augmented IKATP recorded at -60 mV and hyperpolarized Vmem by ∼15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC50 values of 22.9 µM and 15.3 µM, respectively. At 20 µM the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40-50 µM, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of ΔΨm within 4 h under 50 and 100 µM while 10 and 20 µM had no effect on ΔΨm within 24 h. CONCLUSION: We demonstrate expression of HKα2 in rat INS-1E cells. However, the pump is apparently non-functional under the given conditions. Nonetheless the H+/K+ ATPase blocker SCH-28080 inhibits insulin secretion and induces cell death. Importantly, we show that SCH-28080 inhibits ICav - and activates KATP channels identifying them as novel "off-targets" of the inhibitor, causing hyperpolarization of Vmem and inhibition of insulin secretion.

Quercetin Stimulates Insulin Secretion and Reduces the Viability of Rat INS-1 Beta-Cells.

BACKGROUND/AIMS: Previously we described insulinotropic effects of Leonurus sibiricus L. plant extracts used for diabetes mellitus treatment in Traditional Mongolian Medicine. The flavonoid quercetin and its glycoside rutin, which exert anti-diabetic properties in vivo by interfering with insulin signaling in peripheral target tissues, are constituents of these extracts. This study was performed to better understand short- and long-term effects of quercetin and rutin on beta-cells. METHODS: Cell viability, apoptosis, phospho-protein abundance and insulin release were determined using resazurin, annexin-V binding assays, Western blot and ELISA, respectively. Membrane potentials (Vmem), whole-cell Ca2+ (ICa)- and ATP-sensitive K+ (IKATP) currents were measured by patch clamp. Intracellular Ca2+ (Cai) levels were measured by time-lapse imaging using the ratiometric Ca2+ indicator Fura-2. RESULTS: Rutin, quercetin and the phosphoinositide-3-kinase (PI3K) inhibitor LY294002 caused a dose-dependent reduction in cell viability with IC50 values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively. Quercetin (50 µM) significantly increased the percentage of Annexin-V+ cells within 48 hrs. The mean cell volume (MCV) of quercetin-treated cells was significantly lower. Within 2 hrs, quercetin significantly decreased basal- and insulin-stimulated Akt(T308) phosphorylation and increased Erk1/2 phosphorylation, without affecting P-Akt(S473) abundance. Basal- and glucose-stimulated insulin release were significantly stimulated by quercetin. Quercetin significantly depolarized Vmem by ∼25 mV which was prevented by the KATP-channel opener diazoxide, but not by the L-type ICa inhibitor nifedipine. Quercetin significantly stimulated ICa and caused a 50% inhibition of IKATP. The effects on Vmem, ICa and IKATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute. With a similar time-response quercetin induced an elevation in Cai which was completely abolished in the absence of Ca2+ in the bath solution. Rutin (50 µM) did not significantly alter the percentage of Annexin-V+ cells, MCV, Akt or Erk1/2 phosphorylation, insulin secretion, or the electrophysiological behavior of INS-1 cells. CONCLUSION: We conclude that quercetin acutely stimulates insulin release, presumably by transient KATP channel inhibition and ICa stimulation. Long term application of quercetin inhibits cell proliferation and induces apoptosis, most likely by inhibition of PI3K/Akt signaling.

The putative role of the non-gastric H⁺/K⁺-ATPase ATP12A (ATP1AL1) as anti-apoptotic ion transporter: effect of the H⁺/K⁺ ATPase inhibitor SCH28080 on butyrate-stimulated myelomonocytic HL-60 cells.

BACKGROUND/AIMS: The ATP12A gene codes for a non-gastric H(+)/K(+) ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric H(+)/K(+) ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM) human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the H(+)/K(+) ATPase inhibitor SCH28080 in apoptosis. METHODS: Real-time reverse-transcription PCR (qRT-PCR) was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pHi) after acute intracellular acid load (NH4Cl prepulsing). Mean cell volumes (MCV) and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI) were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle), to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining) and differentiation (CD86 expression). RESULTS: We found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM) induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The H(+)/K(+) ATPase inhibitor SCH28080 (100 µM) diminishes K(+)-dependent pHi recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently decrease it over the course of the next 48 h. This effect can be observed in the overall- and non-apoptotic fraction of both untreated and 1 mM butyrate-treated HL-60 cells, but not in 1 mM butyrate-stimulated phosphatidylserine-positive cells. These cells do not shrink from 24 h to 72 h and have finally a higher MCV than untreated cells unless they are exposed to SCH28080. 10 mM butyrate induces apoptosis within 24 h. CONCLUSION: In summary we show that in HL-60 cells ATP12A is a functionally active H(+)/K(+) ATPase that may counteract events during early apoptosis like intracellular acidosis, loss of intracellular K(+) ions and apoptotic volume decrease. Its expression and/or susceptibility to the H(+)/K(+) ATPase inhibitor SCH28080 becomes most evident in cells exposing phosphatidylserine on the outer leaflet of the cell membrane and therefore during early apoptosis.

The C-terminus of ICln is natively disordered but displays local structural preformation.

ICln is a vital, ubiquitously expressed protein with roles in cell volume regulation, angiogenesis, cell morphology, activation of platelets and RNA processing. In previous work we have determined the 3D structure of the N-terminus of ICln (residues 1-159), which folds into a PH-like domain followed by an unstructured region (residues H134 - Q159) containing protein-protein interaction sites. Here we present sequence-specific resonance assignments of the C-terminus (residues Q159 - H235) of ICln by NMR, and show that this region of the protein is intrinsically unstructured. By applying (13)Cα- (13)Cß secondary chemical shifts to detect possible preferences for secondary structure elements we show that the C-terminus of ICln adopts a preferred α-helical organization between residues E170 and E187, and exists preferentially in extended conformations (ß-strands) between residues D161 to Y168 and E217 to T223.

The molecular and functional interaction between ICln and HSPC038 proteins modulates the regulation of cell volume.

Identifying functional partners for protein/protein interactions can be a difficult challenge. We proposed the use of the operon structure of the Caenorhabditis elegans genome as a "new gene-finding tool" (Eichmüller, S., Vezzoli, V., Bazzini, C., Ritter, M., Fürst, J., Jakab, M., Ravasio, A., Chwatal, S., Dossena, S., Bottà, G., Meyer, G., Maier, B., Valenti, G., Lang, F., and Paulmichl, M. (2004) J. Biol. Chem. 279, 7136-7146) that could be functionally translated to the human system. Here we show the validity of this approach by studying the predicted functional interaction between ICln and HSPC038. In C. elegans, the gene encoding for the ICln homolog (icln-1) is embedded in an operon with two other genes, Nx (the human homolog of Nx is HSPC038) and Ny. ICln is a highly conserved, ubiquitously expressed multifunctional protein that plays a critical role in the regulatory volume decrease after cell swelling. Following hypotonic stress, ICln translocates from the cytosol to the plasma membrane, where it has been proposed to participate in the activation of the swelling-induced chloride current (ICl(swell)). Here we show that the interaction between human ICln and HSPC038 plays a role in volume regulation after cell swelling and that HSPC038 acts as an escort, directing ICln to the cell membrane after cell swelling and facilitating the activation of ICl(swell). Assessment of the NMR structure of HSPC038 showed the presence of a zinc finger motif. Moreover, NMR and additional biochemical techniques enabled us to identify the putative ICln/HSPC038 interacting sites, thereby explaining the functional interaction of both proteins on a molecular level.

The phytostilbene resveratrol induces apoptosis in INS-1E rat insulinoma cells.

We investigated the effect of resveratrol on proliferation and induction of apoptosis of INS-1E rat insulinoma cells by cell counting, crystal violet staining, flow cytometry and immunoblotting. Resveratrol treatment of INS-1E cells at concentrations > or =50 microM resulted in a dose-dependent inhibition of cell proliferation, accumulation of the cells in the S and G0/G1 phase and a significant increase of the percentage of apoptotic cells. This was paralleled by an increase of cell granularity, apoptotic volume decrease (AVD), exposure of phosphatidylserine at the outer leaflet of the plasma membrane, an increase of the 7-AAD signal and caspase activation. The AMP-kinase (AMPK) inhibitor compound C (10 microM) significantly inhibited cell proliferation and induced caspase activation within 48 hours but this effect was not modified by resveratrol suggesting that AMPK is not a major target involved in mediating the proapoptotic effect of resveratrol in INS-1E cells. Immunoblotting revealed a significant inhibition of Akt (PKB) phosphorylation by 100 muM resveratrol within 1 hour. Addition of insulin (10 microM) to the culture medium strongly enhanced basal Akt phosphorylation. This enhancement was significantly attenuated by 50 and 100 microM resveratrol. We conclude that the antiproliferative/proapoptotic effect of resveratrol on INS-1E cells is due to negative interference with Akt signaling and most likely disruption of auto/paracrine insulin signaling.

Quaternary structure assessment of ICln by fluorescence resonance energy transfer (FRET) in vivo.

ICln is a ubiquitously expressed multifunctional protein that plays a critical role in regulatory volume decrease after cell swelling. The majority of ICln is localized in the cytosol and a small fraction of ICln associates with the plasma membrane. In artificial lipid bilayers ICln forms ion channels, and a putative channel model predicts the association of at least two ICln molecules to form a functional ion-conducting pore. Oligomers of ICln have been demonstrated in cytosolic fractions of different cells by native PAGE and gel filtration analysis, but these data have not yet been verified in vivo, and the basis of ICln homooligomerization is unknown. In silico prediction of the quaternary structure of ICln from its primary structure predicts that ICln forms a dimer, and that the C-terminus of ICln may be essential for the intermolecular interaction. To explore the quaternary structure of ICln in living NIH3T3 fibroblasts, we performed fluorescence resonance energy transfer (FRET) experiments using eCFP (donor) and eYFP (acceptor) fused to the C- and/or N-termini of both full length wild type ICln and of C-terminal truncation mutants thereof (ICln(159) and ICln(134)). FRET was assessed by the acceptor photobleaching technique. Here we show that ICln forms oligomers in vivo, and demonstrate intermolecular FRET between the C-, but not the N-termini of full length ICln. In the truncation mutant ICln(159) oligomerization occurs and intermolecular FRET between N-termini can be detected, which indicates that the C-terminus of ICln sterically interferes with interactions between N-termini in full length ICln oligomers. In cells expressing the truncation mutant ICln(134) no FRET between C- and/or N-termini could be measured, suggesting the absence of interaction and a role of amino acids P135-Q159 in the oligomerization of ICln.

LSm4 associates with the plasma membrane and acts as a co-factor in cell volume regulation.

ICln is a ubiquitous, multifunctional protein with functions in cell volume regulation and RNA processing, and is thus part of an intricate protein network critically involved in the homoeostasis of cells. To better understand this vital protein network in health and disease it is fundamental to characterize the interactions between the physiological pathways in which ICln is involved, as well as the spatio-temporal regulation of these interactions. In this study, we focused on the interaction between the two best studied pathways in which ICln is involved--regulatory volume decrease and RNA processing--and asked, whether or not the RNA processing factor and ICln interaction partner LSm4 may also have a function in cell volume regulation in NIH3T3 fibroblasts or HEK293 Phoenix cells. To address this question, we studied in isotonic and hypotonic conditions by FRET, biochemistry and electrophysiology, the intracellular distribution of the RNA processing factor LSm4, its interaction with ICln, as well as the involvement of LSm4 in the activation of the swelling dependent anion and osmolyte channel IClswell. In isotonic conditions, LSm4 associates with ICln, and the plasma membrane. Hypotonic cell swelling leads to the dissociation of LSm4 from the plasma membrane, and from ICln. Over-expression of LSm4 affects the translocation of ICln to the cell membrane and markedly inhibits the activation kinetics and current density of IClswell. These findings indicate that LSm4 not only acts in RNA processing, but also as a co-factor in cell volume regulation.

Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching.

Fluorescence resonance energy transfer (FRET) is a technique used for the study of functional interactions between molecules. The intimate vicinity between two fluorescent molecules (FRET-pair; donor and acceptor) allows for an energy transfer, which can be directly calculated as the so called FRET efficiency. This technique is used in fixed as well as living cells. Here we show first, measured by the FRET technique, that the ICln ion channel is transposed from the cytosol towards the cellular membrane in HEK cells after swelling, and second, that the calculation of the FRET efficiency by de-quenching the donor cyan-fluorescent-protein (CFP) emission due to acceptor-photobleaching leads to erroneous estimate of the FRET efficiency in fixed, mounted and sealed specimens. The acceptor photobleaching leads to a modification of the donor cyan-fluorescent-protein, which shows then a strong emission, thus mimicking functional interaction between CFP (donor) and yellow-fluorescent-protein (YFP; acceptor). Moreover, the procedure of acceptor photobleaching masks physiological (non random) interaction between molecules within the fixed, mounted and sealed cell. We show that no artifactual CFP modifications arise when using the acceptor photobleaching technique under in vivo conditions, and we offer strategies to minimize erroneous FRET efficiency calculations if cells need to be fixed.

Glucose induces anion conductance and cytosol-to-membrane transposition of ICln in INS-1E rat insulinoma cells.

The metabolic coupling of insulin secretion by pancreatic beta cells is mediated by membrane depolarization due to increased glucose-driven ATP production and closure of K(ATP) channels. Alternative pathways may involve the activation of anion channels by cell swelling upon glucose uptake. In INS-1E insulinoma cells superfusion with an isotonic solution containing 20 mM glucose or a 30% hypotonic solution leads to the activation of a chloride conductance with biophysical and pharmacological properties of anion currents activated in many other cell types during regulatory volume decrease (RVD), i.e. outward rectification, inactivation at positive membrane potentials and block by anion channel inhibitors like NPPB, DIDS, 4-hydroxytamoxifen and extracellular ATP. The current is not inhibited by tolbutamide and remains activated for at least 10 min when reducing the extracellular glucose concentration from 20 mM to 5 mM, but inactivates back to control levels when cells are exposed to a 20% hypertonic extracellular solution containing 20 mM glucose. This chloride current can likewise be induced by 20 mM 3-Omethylglucose, which is taken up but not metabolized by the cells, suggesting that cellular sugar uptake is involved in current activation. Fluorescence resonance energy transfer (FRET) experiments show that chloride current activation by 20 mM glucose and glucose-induced cell swelling are accompanied by a significant, transient redistribution of the membrane associated fraction of ICln, a multifunctional 'connector hub' protein involved in cell volume regulation and generation of RVD currents.

Fast fluorometric method for measuring pendrin (SLC26A4) Cl-/I- transport activity.

Malfunction of the SLC26A4 protein leads to Pendred syndrome, characterized by sensorineural hearing loss, often associated with mild thyroid dysfunction and goiter. It is generally assumed that SLC26A4 acts as a chloride/anion exchanger, which in the thyroid gland transports iodide, and in the inner ear contributes to the conditioning of the endolymphatic fluid. Here we describe a fast fluorometric method able to be used to functionally scrutinize SLC26A4 and its mutants described in Pendred syndrome. The validation of the method was done by functionally characterizing the chloride/iodide transport of SLC26A4, and a mutant, i.e. SLC26A4(S28R), which we previously described in a patient with sensorineural hearing loss, hypothyroidism and goiter. Using the fluorometric method we describe here we can continuously monitor and quantify the iodide or chloride amounts transported by the cells, and we found that the transport capability of the SLC26A4(S28R) mutant protein is markedly reduced if compared to wild-type SLC26A4.

Functional characterization of wild-type and a mutated form of SLC26A4 identified in a patient with Pendred syndrome.

BACKGROUND: Malfunction of the SLC26A4 protein leads to prelingual deafness often associated with mild thyroid dysfunction and goiter. It is assumed that SLC26A4 acts as a chloride/anion exchanger responsible for the iodide organification in the thyroid gland, and conditioning of the endolymphatic fluid in the inner ear. METHODS: Chloride uptake studies were made using HEK293-Phoenix cells expressing human wild type SLC26A4 (pendrin) and a mutant (SLC26A4(S28R)) we recently described in a patient with hypothyroidism, goiter and sensorineural hearing loss. RESULTS: Experiments are summarized showing the functional characterization of wild type SLC26A4 and a mutant (S28R), which we described recently. This mutant protein is transposed towards the cell membrane, however, its transport capability is markedly reduced if compared to wild-type SLC26A4. Furthermore, we show that the SLC26A4 induced chloride uptake in HEK293-Phoenix cells competes with iodide, and, in addition, that the chloride uptake can be blocked by NPPB and niflumic acid, whereas DIDS is ineffective. CONCLUSIONS: The functional characteristics of SLC26A4(S28R) we describe here, are consistent with the clinical phenotype observed in the patient from which the mutant was derived.

The expression of wild-type pendrin (SLC26A4) in human embryonic kidney (HEK 293 Phoenix) cells leads to the activation of cationic currents.

OBJECTIVE: The SLC26A4 protein (pendrin) seems to be involved in the exchange of chloride with other anions, therefore being responsible for iodide organification in the thyroid gland and the conditioning of the endolymphatic fluid in the inner ear. Malfunction of SLC26A4 leads to Pendred syndrome, characterized by mild thyroid dysfunction often associated with goiter and/or prelingual deafness. The precise function of the SLC26A4 protein, however, is still elusive. An open question is still whether the SLC26A4-induced ion exchange mechanism is electrogenic or electroneutral. Recently, it has been shown that human pendrin expressed in monkey cells leads to chloride currents. METHODS: We overexpressed the human SLC26A4 isoform in HEK293 Phoenix cells and measured cationic and anionic currents by the patch-clamp technique in whole cell configuration. RESULTS: Here we show that human pendrin expressed in human cells does not lead to the activation of chloride currents, but, in contrast, leads to an increase of cationic currents. CONCLUSION: Our experiments suggest that the SLC26A4-induced chloride transport is electroneutral when expressed in human cellular systems.

ICln159 folds into a pleckstrin homology domain-like structure. Interaction with kinases and the splicing factor LSm4.

ICln is a multifunctional protein involved in regulatory mechanisms as different as membrane ion transport and RNA splicing. The protein is water-soluble, and during regulatory volume decrease after cell swelling, it is able to migrate from the cytosol to the cell membrane. Purified, water-soluble ICln is able to insert into lipid bilayers to form ion channels. Here, we show that ICln159, a truncated ICln mutant, which is also able to form ion channels in lipid bilayers, belongs to the pleckstrin homology (PH) domain superfold family of proteins. The ICln PH domain shows unusual properties as it lacks the electrostatic surface polarization seen in classical PH domains. However, similar to many classical PH domain-containing proteins, ICln interacts with protein kinase C, and in addition, interacts with cAMP-dependent protein kinase and cGMP-dependent protein kinase type II but not cGMP-dependent protein kinase type Ibeta. A major phosphorylation site for all three kinases is Ser-45 within the ICln PH domain. Furthermore, ICln159 interacts with LSm4, a protein involved in splicing and mRNA degradation, suggesting that the ICln159 PH domain may serve as a protein-protein interaction platform.

Thiazide-sensitive NaCl-cotransporter in the intestine: possible role of hydrochlorothiazide in the intestinal Ca2+ uptake.

Thiazides, such as hydrochlorothiazide (HCTZ), are used to control blood pressure and to reduce renal calcium excretion. These effects are a result of interactions with the NaCl-cotransporter (NCC). This is demonstrated by the fact that mutations within the NCC protein lead to salt-resistant hypotension and hypocalciuria, paralleled by an increase in bone mineral density. These symptoms are also known as Gitelman syndrome. It has become increasingly evident that the effect of HCTZ on blood pressure and calcium homeostasis cannot be attributed exclusively to kidney functions, where the primary action of HCTZ on NCC is postulated to occur. We demonstrated the presence of the NCC transporter in the rat small intestine (ileum and jejunum) and human HT-29 cells, by using reverse transcription-PCR, Northern blot, Western blot, and immunofluorescence. Furthermore, we show that HCTZ modulates Ca(2+) uptake by intestinal cells, while affecting the electrical parameters of the cellular membrane, thus suggesting a functional interaction between NCC and the epithelial voltage-dependent calcium channel. The experiments presented here support the hypothesis of a direct involvement of the intestinal cells in the interaction between HCTZ and NaCl, as well as calcium homeostasis.

Membrane thickness changes ion-selectivity of channel-proteins.

The plasma membrane is a highly dynamic cell-barrier if the nature and distribution of its constituents are considered. Ion channels are embedded in these double lipid bilayers, which modulate their 3D-structures. The structure modulations by the lipid bilayer can assume such a degree that channel activation depends on them, as was shown for the KcsA potassium channel. Here we show that the cation-over-anion selectivity of reconstituted ICln channels can be varied by the thickness of a bilayer build of phosphatidylcholines. The shorter the acyl-chains and therefore the thinner the bilayers of the membrane are, the more potassium selective the channels are. In contrast, the longer the acyl-chains and therefore the thicker the membranes are, the more chloride selective the channels become.

Three-dimensional structure of I(to); Kv4.2-KChIP2 ion channels by electron microscopy at 21 Angstrom resolution.

Regulatory KChIP2 subunits assemble with pore-forming Kv4.2 subunits in 4:4 complexes to produce native voltage-gated potassium (Kv) channels like cardiac I(to) and neuronal I(A) subtypes. Here, negative stain electron microscopy (EM) and single particle averaging reveal KChIP2 to create a novel approximately 35 x 115 x 115 Angstrom, intracellular fenestrated rotunda: four peripheral columns that extend down from the membrane-embedded portion of the channel to enclose the Kv4.2 "hanging gondola" (a platform held beneath the transmembrane conduction pore by four internal columns). To reach the pore from the cytosol, ions traverse one of four external fenestrae to enter the rotundal vestibule and then cross one of four internal windows in the gondola.

A new gene-finding tool: using the Caenorhabditis elegans operons for identifying functional partner proteins in human cells.

How can a large number of different phenotypes be generated by a limited number of genotypes? Promiscuity between different, structurally related and/or unrelated proteins seems to provide a plausible explanation to this pertinent question. Strategies able to predict such functional interrelations between different proteins are important to restrict the number of putative candidate proteins, which can then be subjected to time-consuming functional tests. Here we describe the use of the operon structure of the nematode genome to identify partner proteins in human cells. In this work we focus on ion channels proteins, which build an interface between the cell and the outside world and are responsible for a growing number of diseases in humans. However, the proposed strategy for the partner protein quest is not restricted to this scientific area but can be adopted in virtually every field of human biology where protein-protein interactions are assumed.

Ito channels are octomeric complexes with four subunits of each Kv4.2 and K+ channel-interacting protein 2.

Mammalian voltage-gated K+ channels are assemblies of pore-forming alpha-subunits and modulating beta-subunits. To operate correctly, Kv4 alpha-subunits in the heart and central nervous system require recently identified beta-subunits of the neuronal calcium sensing protein family called K+ channel-interacting proteins (KChIPs). Here, Kv4.2.KChIP2 channels are purified, integrity of isolated complexes confirmed, molar ratio of the subunits determined, and subunit valence established. A complex has 4 subunits of each type, a stoichiometry expected for other channels employing neuronal calcium sensing beta-subunits.