William C. Valinsky

Impaired intestinal afferent nerve satiety signalling and vagal afferent excitability in diet induced obesity in the mouse

Non‐technical summary  Obesity is known to result from energy intake in excess of expenditure. What is not known is how individuals are able to eat in excess of their energy needs. We show that after chronic consumption of a high fat diet (which causes obesity), intestinal sensory nerves are less responsive to chemicals released from the gut during a meal (cholecystokinin and 5‐hydroxytryptamine) as well as to distension of the gut as might occur during a meal. This appears to be due to the fact that the ability of the nerve cells to be excited is impaired. This suggests that consumption of an unhealthy diet that leads to obesity causes decreased signalling from the intestine, which may lead to increased food intake and contribute to further weight gain, or allow the maintenance of excess weight and obesity.

Ubiquitination-dependent quality control of hERG K+ channel with acquired and inherited conformational defect at the plasma membrane

The role of the plasma membrane quality control machinery is demonstrated in the development of the long QT syndrome phenotype, caused by acquired and inherited conformational defects of the hERG potassium channel in multiple expression systems, including cardiac myocytes.

Aldosterone signaling through transient receptor potential melastatin 7 cation channel (TRPM7) and its α-kinase domain

We demonstrated a role for the Mg(2+) transporter TRPM7, a bifunctional protein with channel and α-kinase domains, in aldosterone signaling. Molecular mechanisms underlying this are elusive. Here we investigated the function of TRPM7 and its α-kinase domain on Mg(2+) and pro-inflammatory signaling by aldosterone. Kidney cells (HEK-293) expressing wild-type human TRPM7 (WThTRPM7) or constructs in which the α-kinase domain was deleted (ΔKinase) or rendered inactive with a point mutation in the ATP binding site of the α-kinase domain (K1648R) were studied. Aldosterone rapidly increased [Mg(2+)]i and stimulated NADPH oxidase-derived generation of reactive oxygen species (ROS) in WT hTRPM7 and TRPM7 kinase dead mutant cells. Translocation of annexin-1 and calpain-II and spectrin cleavage (calpain target) were increased by aldosterone in WT hTRPM7 cells but not in α-kinase-deficient cells. Aldosterone stimulated phosphorylation of MAP kinases and increased expression of pro-inflammatory mediators ICAM-1, Cox-2 and PAI-1 in Δkinase and K1648R cells, effects that were inhibited by eplerenone (mineralocorticoid receptor (MR) blocker). 2-APB, a TRPM7 channel inhibitor, abrogated aldosterone-induced Mg(2+) responses in WT hTRPM7 and mutant cells. In 2-APB-treated ΔKinase and K1648R cells, aldosterone-stimulated inflammatory responses were unchanged. These data indicate that aldosterone stimulates Mg(2+) influx and ROS production in a TRPM7-sensitive, kinase-insensitive manner, whereas activation of annexin-1 requires the TRPM7 kinase domain. Moreover TRPM7 α-kinase modulates inflammatory signaling by aldosterone in a TRPM7 channel/Mg(2+)-independent manner. Our findings identify novel mechanisms for non-genomic actions of aldosterone involving differential signaling through MR-activated TRPM7 channel and α-kinase.

Inhibition of PRL-2·CNNM3 Protein Complex Formation Decreases Breast Cancer Proliferation and Tumor Growth

The oncogenic phosphatase of regenerating liver 2 (PRL-2) has been shown to regulate intracellular magnesium levels by forming a complex through an extended amino acid loop present in the Bateman module of the CNNM3 magnesium transporter. Here we identified highly conserved residues located on this amino acid loop critical for the binding with PRL-2. A single point mutation (D426A) of one of those critical amino acids was found to completely disrupt PRL-2·human Cyclin M 3 (CNNM3) complex formation. Whole-cell voltage clamping revealed that expression of CNNM3 influenced the surface current, whereas overexpression of the binding mutant had no effect, indicating that the binding of PRL-2 to CNNM3 is important for the activity of the complex. Interestingly, overexpression of the CNNM3 D426A-binding mutant in cancer cells decreased their ability to proliferate under magnesium-deprived situations and under anchorage-independent growth conditions, demonstrating a PRL-2·CNNM3 complex-dependent oncogenic advantage in a more stringent environment. We further confirmed the importance of this complex in vivo using an orthotopic xenograft breast cancer model. Finally, because molecular modeling showed that the Asp-426 side chain in CNNM3 buries into the catalytic cavity of PRL-2, we showed that a PRL inhibitor could abrogate complex formation, resulting in a decrease in proliferation of human breast cancer cells. In summary, we provide evidence that this fundamental regulatory aspect of PRL-2 in cancer cells could potentially lead to broadly applicable and innovative therapeutic avenues.

Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels

Cardiac long QT syndrome type 2 is caused by mutations in the human ether a go-go-related gene (hERG) potassium channel, many of which cause misfolding and degradation at the endoplasmic reticulum instead of normal trafficking to the cell surface. The Hsc70/Hsp70 chaperones assist the folding of the hERG cytosolic domains. Here, we demonstrate that the Hsp70 nucleotide exchange factor Bag1 promotes hERG degradation by the ubiquitin-proteasome system at the endoplasmic reticulum to regulate hERG levels and channel activity. Dissociation of hERG complexes containing Hsp70 and the E3 ubiquitin ligase CHIP requires the interaction of Bag1 with Hsp70, but this does not involve the Bag1 ubiquitin-like domain. The interaction with Bag1 then shifts hERG degradation to the membrane-anchored E3 ligase TRC8 and its E2-conjugating enzyme Ube2g2, as determined by siRNA screening. TRC8 interacts through the transmembrane region with hERG and decreases hERG functional expression. TRC8 also mediates degradation of the misfolded hERG-G601S disease mutant, but pharmacological stabilization of the mutant structure prevents degradation. Our results identify TRC8 as a previously unknown Hsp70-independent quality control E3 ligase for hERG.

Aldosterone Upregulates Transient Receptor Potential Melastatin 7 (TRPM7).

Transient receptor potential melastatin 7 (TRPM7) is a ubiquitously expressed Mg2+-permeable ion channel fused to a C-terminal α-kinase domain. Recently, aldosterone was shown to increase intracellular Mg2+ levels and alter inflammatory signaling in TRPM7-expressing HEK293 cells. This study was undertaken to assess whether these effects were related to an aldosterone-mediated increase of TRPM7 current and/or plasma membrane localization. Using HEK293 cells stably expressing WT-TRPM7, we found that 18-h application of aldosterone significantly increased TRPM7 current and TRPM7 plasma membrane protein expression by 48% and 34%, respectively. The aldosterone-mediated increase of TRPM7 current was inhibited by eplerenone, a mineralocorticoid receptor (MR) blocker, and GSK-650394, an inhibitor of the serum- and glucocorticoid-regulated kinase 1 (SGK1). SGK1 blockade also prevented the aldosterone-induced increase of TRPM7 plasma membrane protein. It was further determined that K1648R-TRPM7, the phosphotransferase-inactive TRPM7 mutant, was unresponsive to aldosterone. Therefore, chronic aldosterone treatment increases the plasma membrane expression of TRPM7, which is associated with an increase of TRPM7 current. This process occurs via an MR-dependent, genomic signaling cascade involving SGK1 and a functioning TRPM7 α-kinase domain. We suggest that this mechanism may be of general relevance when interpreting the effects of aldosterone because the MR receptor is found in multiple tissues, and TRPM7 and SGK1 are ubiquitously expressed.

Aldosterone, SGK1, and ion channels in the kidney

Hyperaldosteronism, a common cause of hypertension, is strongly connected to Na+, K+, and Mg2+ dysregulation. Owing to its steroidal structure, aldosterone is an active transcriptional modifier when bound to the mineralocorticoid receptor (MR) in cells expressing the enzyme 11β-hydroxysteroid dehydrogenase 2, such as those comprising the aldosterone-sensitive distal nephron (ASDN). One such up-regulated protein, the ubiquitous serum and glucocorticoid regulated kinase 1 (SGK1), has the capacity to modulate the surface expression and function of many classes of renal ion channels, including those that transport Na+ (ENaC), K+ (ROMK/BK), Ca2+ (TRPV4/5/6), Mg2+ (TRPM7/6), and Cl− (ClC-K, CFTR). Here, we discuss the mechanisms by which ASDN expressed channels are up-regulated by SGK1, while highlighting newly discovered pathways connecting aldosterone to nonselective cation channels that are permeable to Mg2+ (TRPM7) or Ca2+ (TRPV4).

Characterization of constitutive and acid-induced outwardly rectifying chloride currents in immortalized mouse distal tubular cells

Thiazides block Na+ reabsorption while enhancing Ca2+ reabsorption in the kidney. As previously demonstrated in immortalized mouse distal convoluted tubule (MDCT) cells, chlorothiazide application induced a robust plasma membrane hyperpolarization, which increased Ca2+ uptake. This essential thiazide-induced hyperpolarization was prevented by the Cl− channel inhibitor 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), implicating NPPB-sensitive Cl− channels, however the nature of these Cl− channels has been rarely described in the literature. Here we show that MDCT cells express a dominant, outwardly rectifying Cl− current at extracellular pH 7.4. This constitutive Cl− current was more permeable to larger anions (Eisenman sequence I; I− > Br− ≥ Cl−) and was substantially inhibited by > 100 mM [Ca2+]o, which distinguished it from ClC-K2/barttin. Moreover, the constitutive Cl− current was blocked by NPPB, along with other Cl− channel inhibitors (4,4′-diisothiocyanatostilbene-2,2′-disulfonate, DIDS; flufenamic acid, FFA). Subjecting the MDCT cells to an acidic extracellular solution (pH < 5.5) induced a substantially larger outwardly rectifying NPPB-sensitive Cl− current. This acid-induced Cl− current was also anion permeable (I− > Br− > Cl−), but was distinguished from the constitutive Cl− current by its rectification characteristics, ion sensitivities, and response to FFA. In addition, we have identified similar outwardly rectifying and acid-sensitive currents in immortalized cells from the inner medullary collecting duct (mIMCD-3 cells). Expression of an acid-induced Cl− current would be particularly relevant in the acidic IMCD (pH < 5.5). To our knowledge, the properties of these Cl− currents are unique and provide the mechanisms to account for the Cl− efflux previously speculated to be present in MDCT cells.

Increased TASK channel-mediated currents underlie high-fat diet induced vagal afferent dysfunction

We have previously demonstrated that satiety sensing vagal afferent neurons are less responsive to meal-related stimuli in obesity because of reduced electrical excitability. As leak K+ currents are key determinants of membrane excitability, we hypothesized that leak K+ currents are increased in vagal afferents during obesity. Diet-induced obesity was induced by feeding C57Bl/6J mice a high-fat diet (HFF) (60% energy from fat) for 8-10 wk. In vitro extracellular recordings were performed on jejunal afferent nerves. Whole cell patch-clamp recordings were performed on mouse nodose ganglion neurons. Leak K+ currents were isolated using ion substitution and pharmacological blockers. mRNA for TWIK-related acid-sensitive K+ (TASK) subunits was measured using quantitative real-time PCR. Intestinal afferent responses to nutrient (oleate) and non-nutrient (ATP) stimuli were significantly decreased in HFF mice. Voltage clamp experiments revealed the presence of a voltage-insensitive resting potassium conductance that was increased by external alkaline pH and halothane, known properties of TASK currents. In HFF neurons, leak K+ current was approximately doubled and was reduced by TASK1 and TASK3 inhibitors. The halothane sensitive current was similarly increased. Quantitative PCR revealed the presence of mRNA encoding TASK1 (KCNK3) and TASK3 (KCNK9) channels in nodose neurons. TASK3 transcript was significantly increased in HFF mice. The reduction in vagal afferent excitability in obesity is due in part to an increase of resting (leak) K+ conductance. TASK channels may account for the impairment of satiety signaling in diet-induced obesity and thus is a therapeutic target for obesity treatment. NEW & NOTEWORTHY This study characterized the electrophysiological properties and gene expression of the TWIK-related acid-sensitive K+ (TASK) channel in vagal afferent neurons. TASK conductance was increased and contributed to decreased excitability in diet-induced obesity. TASK channels may account for the impairment of satiety signaling in diet-induced obesity and thus is a promising therapeutic target.