Nonselective Cation Research Articles

Cytotoxic activity of TRPV4 antagonist RN-1734 in G-361 human melanoma cancer cell line

Purpose: Intracellular calcium (Ca2+) signaling plays a role in many cellular events, such as cell proliferation and differentiation, gene transcription, oxidative stress, the antioxidant system, and apoptosis. Transient receptor potential vanilloid 4 (TRPV4) channels are non-selective cation (Ca2+) channels. The present study aims to investigate the cytotoxic activity of RN-1734, a transient receptor potential vanilloid 4 (TRPV4) antagonist, in the G361 human melanoma cancer cell line. 
 Materials and Methods: The effects of RN-1734 on G361 cell viability at concentrations of 1, 5, 25, 50, and 100 μM were measured using the 3-(4,5-dimethylthiazol-2-il)-2,5-diphenyltetrazolium bromide (MTT) method. Total antioxidant status (TAS) and total oxidant status (TOS) levels were determined using a ready-made commercial kit, after which oxidative stress index (OSI) values were calculated. To determine the apoptotic effects of RN-1734, Bcl-2, Bax, and p53 expression levels, caspase-3 and -8 activities were examined via quantitative real-time PCR analysis. 
 Results: G361 cell viability significantly decreased to 82.72, 72.81, 56.36, 39.16 and 18.96% in RN-1734 groups (1, 5, 25, 50 and 100 μM) compared to the control group (100.00%). At IC50 concentration (39.48 μM), RN-1734 application (3.35 mmol/g prot.-TAS, 45.87 μmol/g prot.-TOS, and 1501.97 AU-OSI) increased the TAS level (2.17 mmol/g prot.) and decreased the TOS level (55.41 μmol/g prot.) and OSI value (3142.76 AU) compared to the control group. 
 Conclusion: Our findings show that RN-1734 may be a novel therapeutic approach to treating melanoma by decreasing the cell viability of G361 human melanoma cancer cells.

The Chloride Conductance Inhibitor NS3623 Enhances the Activity of a Non-selective Cation Channel in Hyperpolarizing Conditions

Handbooks of physiology state that the strategy adopted by red blood cells (RBCs) to preserve cell volume is to maintain membrane permeability for cations at its minimum. However, enhanced cation permeability can be measured and observed in specific physiological and pathophysiological situations such as in vivo senescence, storage at low temperature, sickle cell anemia and many other genetic defects affecting transporters, membrane or cytoskeletal proteins. Among cation pathways, cation channels are able to dissipate rapidly the gradients that are built and maintained by the sodium and calcium pumps. These situations are very well-documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. In addition, non-selective cation (NSC) channels present in the RBC membrane have proven difficult to molecular identification and functional characterization. For instance, NSC channel activity can be elicited by Low Ionic Strength conditions (LIS): the associated change in membrane potential triggers its opening in a voltage dependent manner. But, whereas this depolarizing media produces a spectacular activation of NSC channel, Gárdos channel-evoked hyperpolarization's have been shown to induce sodium entry through a pathway thought to be conductive and termed Pcat. Using the CCCP method, which allows to follow fast changes in membrane potential, we show here (i) that hyperpolarization elicited by Gárdos channel activation triggers sodium entry through a conductive pathway, (ii) that chloride conductance inhibition unveils such conductive cationic conductance, (iii) that the use of the specific chloride conductance inhibitor NS3623 (a derivative of Neurosearch compound NS1652), at concentrations above what is needed for full anion channel block, potentiates the non-selective cation conductance. These results indicate that a non-selective cation channel is likely activated by the changes in the driving force for cations rather than a voltage dependence mechanism per se.

Abstract MP240: Site-specific O-Glcnacylation Of Transient Receptor Potential Canonical Type 6 (trpc6) Channel Is Required For Normal Structure And Function

O-GlcNAcylation is a dynamic, reversible posttranslational modification (PTM) that regulates a multitude of biological processes. Fluctuations in O-GlcNAC of various calcium handling proteins impact their functionality in cardiomyocytes. Here, we show for the first time that TRPC6, a nonselective receptor-operated cation channel and mediator of hypertrophy and fibrosis, is constitutively O-GlcNAcylated in the ankyrin repeat domain (AR4), at Ser 14, Thr 70, and Thr 221 within the N-terminal cytoplasmic segment. Of these, only substitution of Thr 221 with alanine (T221A) results in a change of function, notably a hyperactive TRPC6 channel with 5X greater increase in consequent NFAT promoter activity, which is a marker of TRPC6 calcium signaling. Patch-clamp analysis of T221A mutant channels found a 75-80% increased conductance compared to WT. Myocardial injection of T221A in homozygous TRPC6 KO mice by AAV-9 mediated gene transfer results in systolic dysfunction, hypertrophy, and cardiac fibrosis, by loss of OGlcNAc modification at site T221. T221 is highly conserved across species and found in the AR4 domain, which forms the core structure of TRPC6 intracellular domain. Mutating the site in its closest homologs, TRPC3 and TRPC7, also activates channel activity. T221 O-GlcNAcylation also protects the nascent protein from premature proteasomal degradation. Molecular modeling from the crystal structure of human TRPC6 predicts that OGlcNACylation stabilizes electrostatic interactions with the 193-203 loop near AR4, and loop connecting AR4 to the linker helix 1 (LH1) at S199, E200, and E246. Mutating these sites to alanine also increases TRPC6-NFAT signaling similar to what was observed in the T221A mutant. In summary, this study highlights that O-GlcNAcylation of TRPC6 is an important PTM needed to stabilize channel function, and its decline results in gain-of-function related diseases.

РАЗДЕЛЕНИЕ ИОНОВ КОБАЛЬТА(II) И ЖЕЛЕЗА(III) С ИСПОЛЬЗОВАНИЕМ НЕСЕЛЕКТИВНОГО СУЛЬФОКАТИОНИТА КУ-2-8

A separation method of ions of cobalt (II) and iron (III) has been proposed and studied for the case of their simultaneous presence in a solution. This method includes preliminary separation of an iron portion by alkaline precipitation with pH = 2.3, obtaining the product of simultaneous precipitation of iron and cobalt and extraction of cobalt from it by the method of cation-exchange leaching with the use of nonselective sulfonic cation resin KU-2-8.

The Neuroprotective Effect of L-Stepholidine on Methamphetamine-Induced Memory Deficits in Mice.

Repeated methamphetamine (METH) exposure can cause severe neurotoxicity to the central nervous system, and lead to memory deficits. L-Stepholidine (L-SPD) is a structurally identified alkaloid extract of the Chinese herb Stephania intermedia, which elicits dopamine (DA) D1-type receptors partial agonistic activity and D2-type receptors antagonistic activity. In this study, we investigated the effect of L-SPD on METH-induced memory deficits in mice and its underlying mechanisms. We found that repeated exposure to METH (10mg/kg, i.p., once per day for 7 consecutive days) impaired memory functions in the novel object recognition experiment. Pretreatment of L-SPD (10mg/kg, i.p.) significantly improved METH-induced memory deficits in mice. Meanwhile, the protein expression of dopaminergic D2 receptors in hippocampus area was significantly increased by repeated METH exposure, while the protein expression of dopamine transporter (DAT) was significantly reduced. Additionally, the protein expression of phospho-protein kinase A (p-PKA) was significantly increased by repeated METH exposure. The hyperpolarization-activated cyclic-nucleotide-gated non-selective cation 1 (HCN1) channel, which was a key regulator of memory functions and could be regulated by p-PKA, was also significantly increased by repeated METH exposure. These changes caused by METH could be prevented by L-SPD pretreatment. Therefore, our data firstly showed that pretreatment of L-SPD exhibited the protective effect against METH-induced memory deficits, possibly through reducing METH-induced upregulation of dopaminergic pathway and HCN1 channels.

STIM1-dependent membrane insertion of heteromeric TRPC1-TRPC4 channels in response to muscarinic receptor stimulation.

Muscarinic receptor stimulation results in activation of nonselective cation (NSC) channels in guinea pig adrenal medullary (AM) cells. The biophysical and pharmacological properties of the NSC channel suggest the involvement of heteromeric channels of TRPC1 with TRPC4 or TRPC5. This possibility was explored in PC12 cells and guinea pig AM cells. Proximity ligation assay (PLA) revealed that when exogenously expressed in PC12 cells, TRPC1 forms a heteromeric channel with TRPC4, but not with TRPC5, in a STIM1-dependent manner. The heteromeric TRPC1-TRPC4 channel was also observed in AM cells and trafficked to the cell periphery in response to muscarine stimulation. To explore whether heteromeric channels are inserted into the cell membrane, tags were attached to the extracellular domains of TRPC1 and TRPC4. PLA products developed between the tags in cells stimulated by muscarine, but not in resting cells, indicating that muscarinic stimulation results in the membrane insertion of channels. This membrane insertion required expression of full-length STIM1. We conclude that muscarinic receptor stimulation results in the insertion of heteromeric TRPC1-TRPC4 channels into the cell membrane in PC12 cells and guinea pig AM cells.

Downregulation of HCN1 channels in hippocampus and prefrontal cortex in methamphetamine re-exposed mice with enhanced working memory.

The hyperpolarization-activated cyclic-nucleotide-gated non-selective cation (HCN) channels play a potential role in the neurological basis underlying drug addiction. However, little is known about the role of HCN channels in methamphetamine (METH) abuse. In the present study, we examined the changes in working memory functions of METH re-exposed mice through Morris water maze test, and investigated the protein expression of HCN1 channels and potential mechanisms underlying the modulation of HCN channels by Western blotting analysis. Mice were injected with METH (1 mg/kg, i.p.) once per day for 6 consecutive days. After 5 days without METH, mice were re-exposed to METH at the same concentration. We found that METH re-exposure caused an enhancement of working memory, and a decrease in the HCN1 channels protein expression in both hippocampus and prefrontal cortex. The phosphorylated extracellular regulated protein kinase 1/2 (p-ERK1/2), an important regulator of HCN channels, was also obviously reduced in hippocampus and prefrontal cortex of mice with METH re-exposure. Meanwhile, acute METH exposure did not affect the working memory function and the protein expressions of HCN1 channels and p-ERK1/2. Overall, our data firstly showed the aberrant protein expression of HCN1 channels in METH re-exposed mice with enhanced working memory, which was probably related to the down-regulation of p-ERK1/2 protein expression.

Involvement of ion channels in allergy.

Allergic asthma is a complex disease, often characterized by an inappropriate Th2 response to normally harmless allergens. Epithelial cells damaged or activated by the allergen produce IL-33, TSLP and IL-25, activating ILC2 and dendritic cells. The latter migrate into lymph nodes where they induce Th2-cell commitment. Th2 and other type 2 innate inflammatory cells trigger inflammation and airway hyper-reactivity. The toolbox consisting of the ion channels varies from one cellular type to another and depends on its activation state, offering the possibility to design novel drugs in the field of allergy. We will discuss about some channels as calcium, nonselective cation, potassium and chloride channels that appear as good candidates in allergy.

Ion Channel Activity in a 3‐hydroxyacyl‐coA dehydratase 1 (HACD1) Deficient Muscle Cell Line

Centronuclear myopathy in Labrador retrievers is the most common inherited neuromuscular disorder in dogs: this is caused by a mutation in a fatty acid processing gene, HACD1. The mutation is carried by 15–20% of the breed worldwide (Pele et al., 2005). These dogs present with generalised progressive weakness. A congenital myopathy due to a HACD1 mutation has also been documented in a human family, where infants have hypotonia and delayed motor milestones (Muhammed et al., 2013). The cellular mechanisms by which HACD1 mutation lead to muscle weakness are unknown, but since fatty acids interact with membrane ion channels we hypothesised that dysregulation of membrane function could be involved. To investigate this we have used a muscle cell line (C2C12) with shRNA knock down (KD) of HACD1 in combination with ion channel gene qPCR and patch‐clamp electrophysiology.To quantify changes in C2C12 myogenic differentiation following HACD1 KD, we used a fusion index calculation on day 8 myotubes stained for the differentiation marker MF20 and counted nuclei that were present in MF20 positive myotubes against those that were in undifferentiated, MF20 negative cells. No significant difference in myotube fusion was observed in HACD1‐KD cells compared to control (n=2).Before beginning patch‐clamp studies on C2C12 cells we used qPCR to identify potential ion channel targets in control C2C12 myoblasts. We found expression of (<30ct) of 21 ion channels, including those of particular interest to calcium homeostasis such as the voltage‐gated Ca2+ channels; Cacna1c, cacna1g, the non‐selective cation (NSC) channels; TRPV4, TRPV2, TRPC1 and a Ca2+‐activated K+ channel; Kcnn3.To functionally identify channel gating we used cell‐attached patch‐clamp electrophysiology of control and HACD1‐KD C2C12 myoblasts. K‐means clustering revealed five statistically significant clusters of ion channel activity in these cells (p<0.05). Assuming an RMP of −15mV (Tanaka et al., 2017) clusters had centeroids of;(i) conductance 21 ± 0.4pS, Vrev 30 ± 6mV (ii) 28 ± 6pS, −38 ± 4mV (iii) 29 ± 0.7pS, −78 ± 4mV (iv) 40 ± 6pS, 5 ± 3mV (v) 94 ± 13, 16 ± 44mV.In conclusion, the phenotype of the smallest ion channel conductance (28pS) would be consistent with the Ca2+‐activated K+ channel KCNN3, and the 94pS consistent with any of the three TRP channels identified by qPCR. These will be further characterised with pharmacological inhibitors and in the future we will examine if the populations change as the control and HACD1‐KD myoblasts differentiate into myotubes.Support or Funding InformationAuthors would like to thank J Blondelle & L Tiret from the Alfort Veterinary School for the cells used in these experiments.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transient potential receptor melastatin-2 (Trpm2) does not influence murine MLL-AF9-driven AML leukemogenesis or invitro response to chemotherapy.

Transient potential receptor melastatin-2 (TRPM2) is a nonselective cationic, Ca(2+)-permeable transmembrane pore that is preferentially expressed in cells of the myeloid lineage and modulates signaling pathways converging into NF-kB. This is of potential interest for acute myeloid leukemia (AML) therapy, as NF-κB signaling is emerging as a key pathway, mediating drug resistance and leukemia-initiating cell survival in AML. Inhibition of NF-κB signaling has been found to be synergistic with chemotherapy. TRPM2 is overexpressed in AML compared with normal bone marrow, with the highest levels in the FAB M3-6 subtypes. To determine the effect of TRPM2 depletions in a defined genetic model, we established MLL-AF9-driven AML on a Trpm2(-/-) genetic background. Trpm2(-/-) MLL-AF9 leukemias displayed reduced NF-κB phosphorylation as well as nuclear translocation. Invivo, primary and secondary recipients of Trpm2(-/-) MLL-AF9 leukemias exhibit increased latency compared with recipients of wild-type leukemia cells. However, the difference in latency was small and was lost in tertiary transplants. The effect of loss of Trpm2 in a BCR-ABL/NUP98-HOXA9 fusion model was even smaller. Given reports that loss or inhibition of TRPM2 enhanced killing by DNA-damaging agents in neuroblastoma, breast cancer, and prostate cancer cell lines, we exposed Trpm2(-/-) and Trpm2(wt) primary MLL-AF9 leukemias to doxorubicin, cytarabine, and etoposide, but found no difference in IC50 values. The invitro response to decitabine was also unaffected. In summary, Trpm2 does not seem toplay a major role in myeloid leukemogenesis. Additionally, loss of Trpm2 does not augment the cytotoxicity of standard AML chemotherapeutic agents.

Imbalance of HCN1 and HCN2 expression in hippocampal CA1 area impairs spatial learning and memory in rats with chronic morphine exposure.

The hyperpolarization-activated cyclic-nucleotide-gated non-selective cation (HCN) channels play a vital role in the neurological basis underlying nervous system diseases. However, the role of HCN channels in drug addiction is not fully understood. In the present study, we investigated the expression of HCN1 and HCN2 subunits in hippocampal CA1 and the potential molecular mechanisms underlying the modulation of HCN channels in rats with chronic morphine exposure with approaches of electrophysiology, water maze, and Western blotting. We found that chronic morphine exposure (5 mg/kg, sc, for 7 days) caused an inhibition of long-term potentiation (LTP) and impairment of spatial learning and memory, which is associated with a decrease in HCN1, and an increase in HCN2 on cell membrane of hippocampal CA1 area. Additional experiments showed that the imbalance of cell membrane HCN1 and HCN2 expression under chronic morphine exposure was related to an increase in expression of TPR containing Rab8b interacting protein (TRIP8b) (1a-4) and TRIP8b (1b-2), and phosphorylation of protein kinase A (PKA) and adaptor protein 2 μ2 (AP2 μ2). Our results demonstrate the novel information that drug addiction-induced impairment of learning and memory is involved in the imbalance of HCN1 and HCN2 subunits, which is mediated by activation of TRIP8b (1a-4), TRIP8b (1b-2), PKA and AP2 μ2.

Sulfonylurea Receptor 1 Contributes to the Astrocyte Swelling and Brain Edema in Acute Liver Failure

Astrocyte swelling (cytotoxic brain edema) is the major neurological complication of acute liver failure (ALF), a condition in which ammonia has been strongly implicated in its etiology. Ion channels and transporters are known to be involved in cell volume regulation, and a disturbance in these systems may result in cell swelling. One ion channel known to contribute to astrocyte swelling/brain edema in other neurological disorders is the ATP-dependent, nonselective cation (NCCa-ATP) channel. We therefore examined its potential role in the astrocyte swelling/brain edema associated with ALF. Cultured astrocytes treated with 5 mM ammonia showed a threefold increase in the sulfonylurea receptor type 1 (SUR1) protein expression, a marker of NCCa-ATP channel activity. Blocking SUR1 with glibenclamide significantly reduced the ammonia-induced cell swelling in cultured astrocytes. Additionally, overexpression of SUR1 in ammonia-treated cultured astrocytes was significantly reduced by cotreatment of cells with BAY 11-7082, an inhibitor of NF-κB, indicating the involvement of an NF-κB-mediated SUR1 upregulation in the mechanism of ammonia-induced astrocyte swelling. Brain SUR1 mRNA level was also found to be increased in the thioacetamide (TAA) rat model of ALF. Additionally, we found a significant increase in SUR1 protein expression in rat brain cortical astrocytes in TAA-treated rats. Treatment with glibenclamide significantly reduced the brain edema in this model of ALF. These findings strongly suggest the involvement of NCCa-ATP channel in the astrocyte swelling/brain edema in ALF and that targeting this channel may represent a useful approach for the treatment of the brain edema associated with ALF.

Application of ion-sensitive field effect transistors for ion channel screening

Cell-based screening assays are now widely used for identifying compounds that serve as ion channel modulators. However, instrumentation for the automated, real-time analysis of ion flux from clonal and primary cells is lacking. This study describes the initial development of an ion-sensitive field effect transistor (ISFET)-based screening assay for the acquisition of K+ efflux data from cells cultured in multi-well plates. Silicon-based K+-sensitive ISFETs were tested for their electrical response to varying concentrations of KCl and were found to display a linear response relationship to KCl in the range of 10µM–1mM. The ISFETs, along with reference electrodes, were inserted into fast-flow chambers containing either human colonic T84 epithelial cells or U251-MG glioma cells. Application of the Ca2+ ionophore A23187 (1µM), to activate Ca2+-activated non-selective cation (NSC) channels (T84 cells) and large conductance Ca2+-activated K+ (BK) channels (U251 cells), resulted in time-dependent increases in the extracellular K+ concentration ([K+]o) as measured with the ISFETs. Treatment of the cells with blockers of either the NSC or BK channels, caused a strong inhibition of the A23187-induced increase in [K+]o. These results were consistent with ion current measurements obtained using the whole-cell arrangement of the patch clamp procedure. In addition, K+ efflux data could be acquired in parallel from multiple cell chambers using the ISFET sensors. Given the non-invasive properties of the probes, the ISFET-based assay should be adaptable for screening ion channels in various cell types.

Identification of a nonselective cation channel in isolated lens fiber cells that is activated by cell shrinkage

The initiation of lens cataract has long been associated with the development of a membrane "leak" in lens fiber cells that depolarizes the lens intracellular potential and elevates intracellular Na(+) and Ca(2+) concentrations. It has been proposed that the leak observed in cataractous lenses is due to the activation of a nonselective cation (NSC) conductance in the normal electrically tight fiber cells. Studies of the membrane properties of isolated fiber cells using the patch-clamp technique have demonstrated a differentiation-dependent shift in membrane permeability from K(+)-dominated in epithelial and short fiber cells toward larger contributions from anion and NSC conductances as fiber cells elongate. In this study, the NSC conductances in elongating lens fiber cells are demonstrated to be due to at least two distinct classes: a Gd(3+)-sensitive, mechanosensitive channel whose blockade is essential for obtaining viable isolated fiber cells, and a second Gd(3+)-insensitive, La(3+)-sensitive conductance that appears to be activated by cell shrinkage. This second conductance was eliminated by the replacement of extracellular Na(+) with the impermeant cation N-methyl-d-glucamine and was potentiated by both hypertonic stress and isosmotic cell shrinkage evoked by the replacement of extracellular Cl(-) with the impermeant anion gluconate. This additional cation conductance may play a role in normal lens physiology by mediating regulatory volume increase under osmotic or other physiological challenges. Since the inappropriate activation of NSC channels is implicated in the initiation of lens cataract, they represent potential targets for the development of novel anticataract therapies.

β-Adrenergic agonists differentially regulate highly selective and nonselective epithelial sodium channels to promote alveolar fluid clearance in vivo

β-Adrenergic receptors (β-AR) increase epithelial sodium channel (ENaC) activity to promote lung fluid clearance. However, the effect of selective β-AR agonist on highly selective cation (HSC) channels or nonselective cation (NSC) channels in alveolar type 1 (T1) and type 2 (T2) cells is unknown. We hypothesized that stimulation with β(1)-AR agonist (denopamine) or β(2)-AR agonist (terbutaline) would increase HSC and/or NSC channel activity in alveolar epithelial cells. We performed single-channel measurements from T1 and T2 cells accessed from rat lung slices. Terbutaline (20 μM) increased HSC ENaC activity (open probability, NP(o)) in T1 (from 0.96 ± 0.61 to 1.25 ± 0.71, n = 5, P <0.05) and T2 cells (from 0.28 ± 0.14 to 1.0 ± 0.30, n = 8, P = 0.02). Denopamine (20 μM) increased NSC NP(o) in T1 cells (from 0.34 ± 0.09 to 0.63 ± 0.14, n = 7, P = 0.02) and in T2 cells (from 0.47 ± 0.09 to 0.68 ± 0.10, P = 0.004). In vivo X-ray imaging of lung fluid clearance and ICI 118,551 selective inhibition of β(2)-ARs confirmed patch-clamp findings. cAMP concentrations increased following treatment with denopamine or terbutaline (n = 3, P < 0.002). The effects of systemic (intraperitoneal, IP) and local (intratracheal, IT) modes of delivery on lung fluid clearance were assessed. IT delivery of denopamine promoted alveolar flooding, whereas IP delivery promoted delayed fluid clearance. In summary, β-AR agonists differentially regulate HSC and NSC in T1 and T2 cells to promote lung fluid clearance in vivo, and the mode of drug delivery is critical for maximizing β-AR agonist efficacy.

Epithelial Sodium Channels Play a Role in Air &amp; Water Borne Diseases

+, Cl and water from the airway lumen into the vascular space. ENaC is considered the rate limiting step in the net movement of fluid out of the alveolar space. Thus, strategies to enhance ENaC activity at the apical membrane are of interest in order to improve outcomes associated with diseases such as pneumonia and acute lung injury. ENaC in the airways is regulated by a variety of agents including G-coupled protein receptors (e.g. adrenergic, purinergic and dopaminergic), chemokines (TGF-β, TNF-α), reactive oxygen/nitrogen species (H 2 O 2 , O 2 ., NO) and hormones (glucocorticoids). The multitude of regulatory pathways highlights the importance of ENaC in the lung [3-6]. Single channel electrophysiology measurements from lung tissue demonstrate that there are two types of ENaC—highly selective cation (HSC) and non-selective cation (NSC) channels. HSC ENaC channels preferentially transport Na + (Na + /K + selectivity >40) while NSC ENaC channels are less discriminant (Na + /K + selectivity 1.4) [7]. In addition to differences in cation selectivity, HSC channels and NSC channels differ in their unitary conductance and other biophysical properties. However, they both play an important role in epithelial Na + reabsorption.

On the Potential Interaction between Non-Selective Cation Channel TRPM4 and the Sulfonylurea Receptor SUR1

The sulfonylurea receptor SUR1, of the ABC protein superfamily, associates with inward-rectifying K channel subunits Kir6.2 or Kir6.1 to form KATP channels, which link metabolism to electrical activity in multiple cell types. The strong coupling of SUR1 with Kir6 appears exclusive, but recent studies argue that SUR1 also associates with and regulates TRPM4, of the transient receptor potential (TRP) channel family of non-selective cation (NSC) channels. It has been reported that, following stroke, brain or spinal cord trauma, SUR1 is expressed de novo in neurovascular cells at the injury site. In these cells, there appears to be dramatic upregulation of a NSC conductance with TRPM4-like properties, which seems to be reduced by sulfonylureas and enhanced in presence of the KATP channel opener diazoxide, leading to the postulation that post-traumatic NSC currents are determined by SUR1-modulated TRPM4 channels (Simard et al., J Neurosurg 113: 622-9; 2010). However, direct evidence for functional association of TRPM4 and SUR1 is lacking. To address this, we performed inside-out membrane patch-clamp of COSm6 cells expressing TRPM4 channels with or without SUR1. TRPM4-mediated, non-selective currents were Ca2+-activated, voltage-dependent, underwent desensitization, and were inhibited by ATP, but were insensitive to diazoxide, glibenclamide and tolbutamide. These properties were not affected by co-transfection with SUR1 albeit functional, sulfonylurea-sensitive KATP channels were formed when the same SUR1 was co-transfected with Kir6.2. In cells co-transfected with Kir6.2, SUR1 and TRPM4, we measured Ca2+-independent, KATP-mediated K+ currents and Ca2+-activated, sulfonylurea-insensitive Na+ currents in the same patch, further showing that SUR1 subunits control KATP channel activity, without affecting TRPM4 channel properties. Co-transfection of TRPM4 and SUR1 does not elicit sulfonylurea-sensitive NSC currents as reported in stroke and brain injury models, suggesting that functional association of TRPM4 and SUR1 is unlikely.

Abstract 16945: Role of Thromboxane A 2 -Activated Nonselective Cation Channels in Hypoxic Pulmonary Vasoconstriction of Rat

Hypoxia-induced pulmonary vasoconstriction (HPV) is a physiological mechanism responsible for controlling the ventilation-perfusion ratio in the lungs. Maintenance of the ventilation-perfusion ratio at 0.8-1 is critical for the optimization and maximization of gas exchange in the lung. Inhibition of K + channels has been shown to be a mechanism for hypoxia-induced membrane depolarization in pulmonary arterial smooth muscle cells (PASMCs). Pharmacological inhibition of K + channels, however, does not induce significant contraction in isolated rat pulmonary arteries, but significantly enhances HPV. These results suggest that, in addition to K + channel inhibition, other mechanisms are involved in hypoxia-induced membrane depolarization. We recently found that a partial contraction by thromboxane A 2 (TXA 2 ) is required to induce HPV. Therefore we hypothesize that TXA 2 receptor (TP) stimulation is involved in eliciting membrane polarization by activating nonselective cation (NSC) channels. Consistently, we found that 5-10 nM U46619, a stable agonist of TP, was essential for the contraction of isolated rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K + channel (Kv). Whole-cell voltage clamp experiments with rat PASMCs revealed that U46619 induced a weakly outward-rectifying NSC current (I NSC,TXA2 ), which was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid (TRPV) subfamily. In addition, 2-aminoethoxydiphenyl borate, an agonist for TRPV1-3, activated NSC channels. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (4α-PDD) rarely increased the membrane conductance. RT-PCR analysis showed the expression of transcripts for TRPV2 and TRPV4 in rat PASMCs. Finally, it was confirmed that the pretreatment with RR and tranilast (a TRPV2 inhibitor) both significantly inhibited HPV in the presence of U46619. Taken together, these data suggest that the concerted effects of I NSC,TXA2 increase and Kv inhibition during under hypoxia are required to induce membrane depolarization sufficient for eliciting HPV. Our results also imply that TXA 2 -activated NSC might be formed by TRPV2 in rat PASMCs and contribute to the hypoxia-induced membrane depolarization in PASMCs.

Plasma membrane insertion of TRPC5 channels contributes to the cholinergic plateau potential in hippocampal CA1 pyramidal neurons

In cultured hippocampal neurons, transient receptor potential 5 (TRPC5) channels are translocated and inserted into plasma membranes of hippocampal neurons to generate nonselective cation (NSC) currents. We investigated whether TRPC5 channel translocation also contributes to the generation of NSC currents underlying the afterdepolarizations and plateau potentials (PPs) in hippocampal pyramidal cells that are induced by muscarinic receptor activation. Using a biotinylation assay to quantify the change in surface membrane proteins in acute hippocampal slices, we found that muscarinic stimulation significantly enhanced the levels of TRPC5 protein on the membrane surface but not those of TRPC1 or TRPC4 channels. We then investigated the pharmacological sensitivity of the cation current observed during muscarinic stimulation to determine if a component could be due to TRPC5 channels. The TRPC channel antagonists 2-APB and SKF96365 strongly depressed the generation of PPs, the underlying tail currents (I(tail)) and the associated dendritic Ca(2+) influx induced by muscarinic receptor activation in pyramidal neurons. High intracellular concentrations of ATP, which specifically inhibit TRPC5 channels, depressed I(tail). In addition, pretreatment with the calmodulin (CaM) inhibitor W-7, which depresses recombinant TRPC5 currents, inhibited both the cation current (I(tail)) and the surface insertion of TRPC5 channels. Finally, the phosphatidylinositide 3-kinase (PI(3)K) inhibitor wortmannin, which blocks translocation of TRPC5 channels in cell culture, also inhibited both the I(tail) and the surface insertion of TRPC5 channels. Therefore, we conclude that insertion of TRPC5 channels contributes to the generation of the prolonged afterdepolarizations following muscarinic stimulation. This altered plasma membrane expression of TRPC5 channels in pyramidal neurons may play an important role in the generation of prolonged neuronal depolarization and bursting during the epileptiform seizure discharges of epilepsy.

Mechanical control of cation channels in the myogenic response

Microcirculatory vessel response to changes in pressure, known as the myogenic response, is a key component of a tissue's ability to regulate blood flow. Experimental studies have not clearly elucidated the mechanical signal in the vessel wall governing steady-state reduction in vessel diameter upon an increase in intraluminal pressure. In this study, a multiscale computational model is constructed from established models of vessel wall mechanics, vascular smooth muscle (VSM) force generation, and VSM Ca(2+) handling and electrophysiology to compare the plausibility of vessel wall stress or strain as an effective mechanical signal controlling steady-state vascular contraction in the myogenic response. It is shown that, at the scale of a resistance vessel, wall stress, and not stretch (strain), is the likely physiological signal controlling the steady-state myogenic response. The model is then used to test nine candidate VSM stress-controlled channel variants by fitting two separate sets of steady-state myogenic response data. The channel variants include nonselective cation (NSC), supplementary Ca(2+) and Na(+), L-type Ca(2+), and large conductance Ca(2+)-activated K(+) channels. The nine variants are tested in turn, and model fits suggest that stress control of Ca(2+) or Na(+) influx through NSC, supplementary Ca(2+) or Na(+), or L-type Ca(2+) channels is sufficient to produce observed steady-state diameter changes with pressure. However, simulations of steady-state VSM membrane potential, cytosolic Ca(2+), and Na(+) with pressure show only that Na(+) influx through NSC channel also generates known trends with increasing pressure, indicating that stress-controlled Na(+) influx through NSC is sufficient to generate the myogenic response.