Asipu Sivaprasadarao

TRPC channel activation by extracellular thioredoxin

Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these channels. Here we show the activation of TRPC5 (canonical TRP 5) homomultimeric and TRPC5–TRPC1 heteromultimeric channels by extracellular reduced thioredoxin, which acts by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis, an inflammatory joint disease that disables millions of people worldwide. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, that endogenous TRPC5–TRPC1 channels of the cells are activated by reduced thioredoxin, and that blockade of the channels enhances secretory activity and prevents the suppression of secretion by thioredoxin. The data indicate the presence of a previously unrecognized ion-channel activation mechanism that couples extracellular thioredoxin to cell function.

MEASUREMENT OF THE MOVEMENT OF THE S4 SEGMENT DURING THE ACTIVATION OF A VOLTAGE-GATED POTASSIUM CHANNEL

Abstract Voltage-gated ion channels contain a positively charged transmembrane segment termed S4. Recent evidence suggests that depolarisation of the membrane potential causes this segment to undergo conformational changes that, in turn, lead to the opening of the channel pore. In order to define these conformational changes in structural terms, we have introduced single cysteine substitutions into the S4 segment of the prototypical Shaker K+ channel at various positions and expressed the mutants in Xenopus oocytes. The cells were depolarised to induce K+ currents and the effect of application of 100 μM parachloromercuribenzenesulphonate (PCMBS) on these currents was examined by the two-electrode voltage-clamp technique. PCMBS inhibited K+ currents elicited by mutants L358C, L361C, V363C and L366C, but not those by V367C and S376C. Since PCMBS is a membrane-impermeable cysteine-modifying reagent, the data suggest that depolarisation must have caused the S4 segment to move out of the lipid bilayer into the extracellular phase rendering the residues at positions 358, 361, 363 and 366 susceptible to PCMBS attack. The lack of effect of PCMBS on V367C suggests that the exposure of S4 terminates at L366. Detailed analysis of L361C mutant revealed that the S4 movement can occur even below the resting potential of the cell, at which potential voltage-gated K+ channels are normally in a non-conducting closed state.

Generation of functional ion-channel tools by E3 targeting.

Here we describe a strategy for generating ion-channel inhibitors. It takes advantage of antibody specificity combined with a pattern recognition approach that targets the third extracellular region (E3) of a channel. To test the concept, we first focused on TRPC5, a member of the transient receptor potential (TRP) calcium channel family, the study of which has been hindered by poor pharmacological tools. Extracellular application of E3-targeted anti-TRPC5 antibody led to a specific TRPC5 inhibitor, enabling TRPC5 to be distinguished from its closest family members, and TRPC5 function to be explored in a relatively intractable physiological system. E3 targeting was further applied to voltage-gated sodium channels, leading to discovery of a subtype-specific inhibitor of NaV1.5. These examples illustrate the potential power of E3 targeting as a systematic method for producing gene-type specific ion-channel inhibitors for use in routine assays on cells or tissues from a range of species and having therapeutic potential.

Identification and Pharmacological Correction of a Membrane Trafficking Defect Associated with a Mutation in the Sulfonylurea Receptor Causing Familial Hyperinsulinism

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a genetic disorder characterized by excess secretion of insulin and hypoglycemia. In most patients, the disease is caused by mutations in sulfonylurea receptor-1 (SUR1), which, in association with Kir6.2, constitutes the functional ATP-sensitive potassium (KATP) channel of the pancreatic β-cell. Previous studies reported that coexpression of the PHHI mutant R1394H-SUR1 with Kir6.2 in COS cells produces no functional channels. To investigate if the loss of function could be due to impaired trafficking of mutant channels to the cell membrane, we have cotransfected wild-type and mutant SUR1 subunits with Kir6.2 into HEK293 cells and examined their cellular localization by immunofluorescent staining. Our results show that unlike the wild-type subunits, which showed fluorescence at the cell surface, the mutant subunits displayed fluorescence in punctate structures. Co-immunostaining with antibodies against organelle-specific marker proteins identified these structures as thetrans-Golgi network. Limited localization in clathrin-positive, but transferrin receptor-negative vesicles was also observed. The post-endoplasmic reticulum localization suggests that the mutation does not impair the folding and assembly of the channels so as to cause its retention by the endoplasmic reticulum. Diazoxide, a KATP channel opener drug that is used in the treatment of PHHI, restored the surface expression in a manner that could be prevented by the channel blocker glibenclamide. When expressed in Xenopus oocytes, R1394H-SUR1 formed functional channels with Kir6.2, indicating that the primary consequence of the mutation is impairment of trafficking rather than function. Thus, our data uncover a novel mechanism underlying the therapeutic action of diazoxide in the treatment of PHHI, i.e. its ability to recruit channels to the membrane. Furthermore, this is the first report to describe a trafficking disorder effecting retention of mutant proteins in thetrans-Golgi network.

Activation of the Cl- channel ANO1 by localized calcium signals in nociceptive sensory neurons requires coupling with the IP3 receptor.

Positioning of an excitatory channel where the endoplasmic reticulum and plasma membrane meet enables pain-sensing neurons to distinguish among calcium signals. Insulating Pain Signals Activation of damage-sensing neurons evokes pain. Jin et al. describe a mechanism by which the depolarizing chloride channel anoctamin 1 (ANO1) is coupled only to those calcium signals that arise from activation of proinflammatory G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs), rather than by global calcium signals mediated by voltage-gated calcium channels (VGCCs). ANO1, which is present in the plasma membrane, was found in a lipid raft–based complex that contained the activating GPCRs and the endoplasmic reticulum (ER)–resident calcium channel, IP3R1 (inositol 1,4,5-trisphosphate receptor 1). The interaction of ANO1 with IP3R1 and the ability of the local calcium signals to stimulate ANO1 activity were lost when lipid microdomains were chemically disrupted. Instead, ANO1 became receptive to VGCC activation in the absence of lipid rafts. Thus, a plasma membrane–ER microdomain complex limits activation of nociceptive neurons to only the appropriate signals. We report that anoctamin 1 (ANO1; also known as TMEM16A) Ca2+-activated Cl− channels in small neurons from dorsal root ganglia are preferentially activated by particular pools of intracellular Ca2+. These ANO1 channels can be selectively activated by the G protein–coupled receptor (GPCR)–induced release of Ca2+ from intracellular stores but not by Ca2+ influx through voltage-gated Ca2+ channels. This ability to discriminate between Ca2+ pools was achieved by the tethering of ANO1-containing plasma membrane domains, which also contained GPCRs such as bradykinin receptor 2 and protease-activated receptor 2, to juxtamembrane regions of the endoplasmic reticulum. Interaction of the carboxyl terminus and the first intracellular loop of ANO1 with IP3R1 (inositol 1,4,5-trisphosphate receptor 1) contributed to the tethering. Disruption of membrane microdomains blocked the ANO1 and IP3R1 interaction and resulted in the loss of coupling between GPCR signaling and ANO1. The junctional signaling complex enabled ANO1-mediated excitation in response to specific Ca2+signals rather than to global changes in intracellular Ca2+.

Functional up-regulation of KCNA gene family expression in murine mesenteric resistance artery smooth muscle

This study focused on the hypothesis that KCNA genes (which encode KVα1 voltage‐gated K+ channels) have enhanced functional expression in smooth muscle cells of a primary determinant of peripheral resistance – the small mesenteric artery. Real‐time PCR methodology was developed to measure cell type‐specific in situ gene expression. Profiles were determined for arterial myocyte expression of RNA species encoding KVα1 subunits as well as KVβ1, KVα2.1, KVγ9.3, BKCaα1 and BKCaβ1. The seven major KCNA genes were expressed and more readily detected in endothelium‐denuded mesenteric resistance artery compared with thoracic aorta; quantification revealed dramatic differential expression of one to two orders of magnitude. There was also four times more RNA encoding KVα2.1 but less or similar amounts encoding KVβ1, KVγ9.3, BKCaα1 and BKCaβ1. Patch‐clamp recordings from freshly isolated smooth muscle cells revealed dominant KVα1 K+ current and current density twice as large in mesenteric cells. Therefore, we suggest the increased RNA production of the resistance artery impacts on physiological function, although there is quantitatively less K+ current than might be expected. The mechanism conferring up‐regulated expression of KCNA genes may be common to all the gene family and play a functional role in the physiological control of blood pressure.

Immunolocalisation of adenosine A1 receptors in the rat kidney

The location of adenosine A(1) receptors in the rat kidney was investigated using immunolabelling with antibodies raised to a 15-amino-acid sequence near the C-terminus of the receptor (antibody I) and to a 14-amino-acid sequence in the second extracellular loop (antibody II). In the cortex, antibody I bound to adenosine A(1) receptors in mesangial cells and afferent arterioles, whilst antibody II bound to receptors in proximal convoluted tubules. In the medulla, both antibodies bound to receptors in collecting ducts and the papillary surface epithelium. These observations provide support for the diverse functional roles previously proposed for the adenosine A(1) receptor in the kidney. The labelling of distinct but different structures in the cortex by antibodies raised to different amino acid sequences on the A(1) receptor protein suggests that differing forms of the receptor are present in this region of the kidney.

Inhibition of the K+ channel Kv1.4 by acidosis: protonation of an extracellular histidine slows the recovery from N-type inactivation

1 Acidosis alters the transient outward current, ito, in the heart. We have studied the mechanism underlying the effect of acidosis on one of the K+ channels, Kv1.4 (heterologously expressed in Xenopus laevis oocytes), known to underlie ito. 2 At pH 6.5, wild‐type Kv1.4 current was inhibited during repetitive pulsing, in part as a result of a slowing of recovery from N‐type inactivation. 3 Acidosis still caused slowing of recovery after deletion of just one (either the first or second) of the N‐terminal inactivation ball domains. However, deletion of both the N‐terminal inactivation ball domains greatly reduced the inhibition. 4 As well as the N‐terminus, other parts of the channel are also required for the effect of acidosis, because, whereas the transfer of the N‐terminus of Kv1.4 to Kv1.2 conferred N‐type inactivation, it did not confer acidosis sensitivity. 5 Replacement of an extracellular histidine with a glutamine residue (H508Q) abolished the slowing of recovery by acidosis. Reduction of C‐type inactivation by raising the bathing K+ concentration or by the mutation K532Y also abolished the slowing. 6 It is concluded that binding of protons to H508 enhances C‐type inactivation and this causes a slowing of recovery from N‐type inactivation and, thus, an inhibition of current during repetitive pulsing.

Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP‐sensitive potassium channels

ATP‐sensitive potassium (KATP) channels couple the metabolic status of a cell to its membrane potential—a property that endows pancreatic β‐cells with the ability to regulate insulin secretion in accordance with changes in blood glucose. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1). Here, we report that KATP channels undergo rapid internalisation from the plasma membrane by clathrin‐mediated endocytosis. We present several lines of evidence to demonstrate that endocytosis is mediated by a tyrosine based signal (330YSKF333) located in the carboxy‐terminus of Kir6.2 and that SUR1 has no direct role. We show that genetic mutations, Y330C and F333I, which cause permanent neonatal diabetes mellitus, disrupt this motif and abrogate endocytosis of reconstituted mutant channels. The resultant increase in the surface density of KATP channels would predispose β‐cells to hyperpolarise and may account for reduced insulin secretion in these patients. The data imply that endocytosis of KATP channels plays a crucial role in the (patho)‐physiology of insulin secretion.

Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density

Pancreatic ATP-sensitive potassium (KATP) channels control insulin secretion by coupling the excitability of the pancreatic β-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed KATP channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that KATP channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases KATP channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic β-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native KATP channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic β-cells, we propose that PKC-regulated KATP channel trafficking may play a role in the regulation of insulin secretion.