Nonselective Cation Current Research Articles

Praziquantel activates a native cation current in Schistosoma mansoni

IntroductionPraziquantel (PZQ), an anthelmintic drug discovered in the 1970s, is still used to treat schistosomiasis and various other infections caused by parasitic flatworms. PZQ causes a triad of phenotypic effects on schistosome worms – rapid depolarization, muscle contraction, and damage throughout the worm tegument. The molecular target mediating these effects has been intimated as a Ca2+-permeable ion channel, but native currents evoked by PZQ have not been reported in any schistosome cell type. The properties of the endogenous PZQ activated conductance therefore remain unknown. MethodsHere, invasive electrophysiology was used to probe for responses to PZQ from different locales in a living schistosome worm.Results and discussionNo direct response was seen in tegument-derived vesicles, or from the sub-tegumental muscle layer despite the presence of voltage-operated currents. However, PZQ rapidly triggered a sustained, non-selective cation current in recordings from neuronal tissue, targeting both the anterior ganglion and the main longitudinal nerve cord. The biophysical signature of this PZQ-evoked current resolved at single channel resolution matched that of a transient receptor potential ion channel named TRPMPZQ, recently proposed as the molecular target of PZQ. The endogenous PZQ-evoked current was also inhibited by a validated TRPMPZQ antagonist. PZQ therefore is a neuroactive anthelmintic, causing a sustained depolarization through ion channels with the characteristics of TRPMPZQ.

The Molecular Heterogeneity of Store-Operated Ca2+ Entry in Vascular Endothelial Cells: The Different roles of Orai1 and TRPC1/TRPC4 Channels in the Transition from Ca2+-Selective to Non-Selective Cation Currents

Store-operated Ca2+ entry (SOCE) is activated in response to the inositol-1,4,5-trisphosphate (InsP3)-dependent depletion of the endoplasmic reticulum (ER) Ca2+ store and represents a ubiquitous mode of Ca2+ influx. In vascular endothelial cells, SOCE regulates a plethora of functions that maintain cardiovascular homeostasis, such as angiogenesis, vascular tone, vascular permeability, platelet aggregation, and monocyte adhesion. The molecular mechanisms responsible for SOCE activation in vascular endothelial cells have engendered a long-lasting controversy. Traditionally, it has been assumed that the endothelial SOCE is mediated by two distinct ion channel signalplexes, i.e., STIM1/Orai1 and STIM1/Transient Receptor Potential Canonical 1(TRPC1)/TRPC4. However, recent evidence has shown that Orai1 can assemble with TRPC1 and TRPC4 to form a non-selective cation channel with intermediate electrophysiological features. Herein, we aim at bringing order to the distinct mechanisms that mediate endothelial SOCE in the vascular tree from multiple species (e.g., human, mouse, rat, and bovine). We propose that three distinct currents can mediate SOCE in vascular endothelial cells: (1) the Ca2+-selective Ca2+-release activated Ca2+ current (ICRAC), which is mediated by STIM1 and Orai1; (2) the store-operated non-selective current (ISOC), which is mediated by STIM1, TRPC1, and TRPC4; and (3) the moderately Ca2+-selective, ICRAC-like current, which is mediated by STIM1, TRPC1, TRPC4, and Orai1.

Predictions and experimental tests of a new biophysical model of the mammalian respiratory oscillator.

Previously our computational modeling studies (Phillips et al., 2019) proposed that neuronal persistent sodium current (INaP) and calcium-activated non-selective cation current (ICAN) are key biophysical factors that, respectively, generate inspiratory rhythm and burst pattern in the mammalian preBötzinger complex (preBötC) respiratory oscillator isolated in vitro. Here, we experimentally tested and confirmed three predictions of the model from new simulations concerning the roles of INaP and ICAN: (1) INaP and ICAN blockade have opposite effects on the relationship between network excitability and preBötC rhythmic activity; (2) INaP is essential for preBötC rhythmogenesis; and (3) ICAN is essential for generating the amplitude of rhythmic output but not rhythm generation. These predictions were confirmed via optogenetic manipulations of preBötC network excitability during graded INaP or ICAN blockade by pharmacological manipulations in slices in vitro containing the rhythmically active preBötC from the medulla oblongata of neonatal mice. Our results support and advance the hypothesis that INaP and ICAN mechanistically underlie rhythm and inspiratory burst pattern generation, respectively, in the isolated preBötC.

Inspiratory rhythm generation is stabilized by Ih.

Cellular and network properties must be capable of generating rhythmic activity that is both flexible and stable. This is particularly important for breathing, a rhythmic behavior that dynamically adapts to environmental, behavioral, and metabolic changes from the first to the last breath. The pre-Bötzinger complex (preBötC), located within the ventral medulla, is responsible for producing rhythmic inspiration. Its cellular properties must be tunable, flexible as well as stabilizing. Here, we explore the role of the hyperpolarization-activated, nonselective cation current (Ih) for stabilizing PreBötC activity during opioid exposure and reduced excitatory synaptic transmission. Introducing Ih into an in silico preBötC network predicts that loss of this depolarizing current should significantly slow the inspiratory rhythm. By contrast, in vitro and in vivo experiments revealed that the loss of Ih minimally affected breathing frequency, but destabilized rhythmogenesis through the generation of incompletely synchronized bursts (burstlets). Associated with the loss of Ih was an increased susceptibility of breathing to opioid-induced respiratory depression or weakened excitatory synaptic interactions, a paradoxical depolarization at the cellular level, and the suppression of tonic spiking. Tonic spiking activity is generated by nonrhythmic excitatory and inhibitory preBötC neurons, of which a large percentage express Ih. Together, our results suggest that Ih is important for maintaining tonic spiking, stabilizing inspiratory rhythmogenesis, and protecting breathing against perturbations or changes in network state.NEW & NOTEWORTHY The Ih current plays multiple roles within the preBötC. This current is important for promoting intrinsic tonic spiking activity in excitatory and inhibitory neurons and for preserving rhythmic function during conditions that dampen network excitability, such as in the context of opioid-induced respiratory depression. We therefore propose that the Ih current expands the dynamic range of rhythmogenesis, buffers the preBötC against network perturbations, and stabilizes rhythmogenesis by preventing the generation of unsynchronized bursts.

A plasma membrane‐localized polycystin‐1/polycystin‐2 complex in endothelial cells elicits vasodilation

Polycystin‐1 (PC‐1, PKD1), a receptor‐like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms and physiological functions are poorly understood. Here, by studying tamoxifen‐inducible, endothelial cell (EC)‐specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC‐1‐mediated, Ca2+‐dependent cation currents in ECs. EC‐specific PC‐1 knockout attenuates flow‐mediated arterial hyperpolarization and vasodilation. PC‐1‐dependent vasodilation occurs over the entire functional shear stress range and via the activation of nitric oxide synthase (NOS) and small‐and intermediate‐conductance Ca2+‐activated K+ (SK/IK) channels. EC‐specific PC‐1 knockout increases systemic blood pressure without altering kidney anatomy. PC‐1 coimmunoprecipitates with polycystin‐2 (PC‐2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC‐1 or PC‐2 (Pkd2 ecKO mice) abolishes surface clusters of both PC‐1 and PC‐2 in ECs. Single knockout of PC‐1 or PC‐2 or double knockout of PC‐1 and PC‐2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow‐mediated vasodilation. Flow stimulates non‐selective cation currents in ECs that are similarly inhibited by either PC‐1 or PC‐2 knockout or by interference peptides corresponding to the C‐terminus coiled‐coil domains present in PC‐1 or PC‐2. In summary, we show that PC‐1 regulates arterial contractility and demonstrate that this occurs through the formation of an interdependent signaling complex with PC‐2 in endothelial cells. Flow stimulates PC‐1/PC‐2 clusters in the EC plasma membrane, leading to Ca2+ influx, NOS and SK/IK channel activation, vasodilation and a reduction in blood pressure.

Ultrashort nanosecond electric pulses activate a conductance in bovine adrenal chromaffin cells that involves cation entry through TRPC and NALCN channels

In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of −70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na+ that displayed inward rectification and a reversal potential near −3 mV, a voltage consistent with a non-selective cation current. The broad-spectrum inhibitors of transient receptor potential (TRP) channels, La3+ (10 μM), Gd3+ (10 μM), SKF-96365 (50 μM) and 2-aminoethoxydiphenyl borane (2-APB; 100 μM), inhibited the current similarly by ∼72%, ∼83%, ∼68% and ∼76%, respectively. Depleting membrane cholesterol with methyl-β-cyclodextrin (MβCD; 1–6 mg/ml) or inhibiting phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis with wortmannin (20 and 40 μM) produced a similar level of inhibition on the NEP-induced conductance as the broad spectrum TRP channel inhibitors. Moreover, no additive inhibitory effect was detected by combining MβCD (3 mg/ml), wortmannin (20 μM) and La3+ (10 μM), suggesting that each agent targeted different levels of the same pathway to exert a full effect. RT-PCR experiments revealed robust expression at the mRNA level of TRPC4, TRPC5 and TRPM7 channels for which specific blockers were available. Whereas the TRPM7 blocker FTY720 had no effect, the TRPC4/5 channel inhibitor M084 (20 μM) blocked the conductance by ∼50%, indicating that TRPC4 and/or TRPC5 channel(s) may be partially involved in mediating the NEP-induced current. CP-96345 (20 μM), a specific blocker of the sodium leak current channel (NALCN), also reduced the NEP-induced current. The inhibition was ∼30% and additive to that caused by the TRPC4/5 blocker M084. RT-PCR experiments confirmed the expression of this channel at the mRNA level. Taken as a whole, these data provide evidence that a large fraction of the current evoked by a 5 ns pulse in adrenal chromaffin cells may be carried by both TRPC4/5 channels and the NALCN channel. Understanding the biophysical properties of the NEP-elicited conductance in a neural-type cell will be extremely valuable for the future development of NEP stimulation approaches for neuromodulation.

Putting the theory into 'burstlet theory' with a biophysical model of burstlets and bursts in the respiratory preBötzinger complex.

Inspiratory breathing rhythms arise from synchronized neuronal activity in a bilaterally distributed brainstem structure known as the preBötzinger complex (preBötC). In in vitro slice preparations containing the preBötC, extracellular potassium must be elevated above physiological levels (to 7-9 mM) to observe regular rhythmic respiratory motor output in the hypoglossal nerve to which the preBötC projects. Reexamination of how extracellular K+ affects preBötC neuronal activity has revealed that low-amplitude oscillations persist at physiological levels. These oscillatory events are subthreshold from the standpoint of transmission to motor output and are dubbed burstlets. Burstlets arise from synchronized neural activity in a rhythmogenic neuronal subpopulation within the preBötC that in some instances may fail to recruit the larger network events, or bursts, required to generate motor output. The fraction of subthreshold preBötC oscillatory events (burstlet fraction) decreases sigmoidally with increasing extracellular potassium. These observations underlie the burstlet theory of respiratory rhythm generation. Experimental and computational studies have suggested that recruitment of the non-rhythmogenic component of the preBötC population requires intracellular Ca2+ dynamics and activation of a calcium-activated nonselective cationic current. In this computational study, we show how intracellular calcium dynamics driven by synaptically triggered Ca2+ influx as well as Ca2+ release/uptake by the endoplasmic reticulum in conjunction with a calcium-activated nonselective cationic current can reproduce and offer an explanation for many of the key properties associated with the burstlet theory of respiratory rhythm generation. Altogether, our modeling work provides a mechanistic basis that can unify a wide range of experimental findings on rhythm generation and motor output recruitment in the preBötC.

The role of activation of two different sGC binding sites by NO-dependent and NO-independent mechanisms in the regulation of SACs in rat ventricular cardiomyocytes.

The mechanoelectrical feedback (MEF) mechanism in the heart that plays a significant role in the occurrence of arrhythmias, involves cation flux through cation nonselective stretch‐activated channels (SACs). It is well known that nitric oxide (NO) can act as a regulator of MEF. Here we addressed the possibility of SAC’s regulation along NO‐dependent and NO‐independent pathways, as well as the possibility of S‐nitrosylation of SACs. In freshly isolated rat ventricular cardiomyocytes, using the patch‐clamp method in whole‐cell configuration, inward nonselective stretch‐activated cation current ISAC was recorded through SACs, which occurs during dosed cell stretching. NO donor SNAP, α1‐subunit of sGC activator BAY41‐2272, sGC blocker ODQ, PKG blocker KT5823, PKG activator 8Br‐cGMP, and S‐nitrosylation blocker ascorbic acid, were employed. We concluded that the physiological concentration of NO in the cell is a necessary condition for the functioning of SACs. An increase in NO due to SNAP in an unstretched cell causes the appearance of a Gd3+‐sensitive nonselective cation current, an analog of ISAC , while in a stretched cell it eliminates ISAC . The NO‐independent pathway of sGC activation of α subunit, triggered by BAY41‐2272, is also important for the regulation of SACs. Since S‐nitrosylation inhibitor completely abolishes ISAC , this mechanism occurs. The application of BAY41‐2272 cannot induce ISAC in a nonstretched cell; however, the addition of SNAP on its background activates SACs, rather due to S‐nitrosylation.ODQ eliminates ISAC , but SNAP added on the background of stretch increases ISAC in addition to ODQ. This may be a result of the lack of NO as a result of inhibition of NOS by metabolically modified ODQ. KT5823 reduces PKG activity and reduces SACs phosphorylation, leading to an increase in ISAC . 8Br‐cGMP reduces ISAC by activating PKG and its phosphorylation. These results demonstrate a significant contribution of S‐nitrosylation to the regulation of SACs.

A plasma membrane-localized polycystin-1/polycystin-2 complex in endothelial cells elicits vasodilation.

Polycystin-1 (PC-1, PKD1), a receptor-like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms, and physiological functions are poorly understood. Here, by studying tamoxifen-inducible, endothelial cell (EC)-specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC-1-mediated, Ca2+-dependent cation currents in ECs. EC-specific PC-1 knockout attenuates flow-mediated arterial hyperpolarization and vasodilation. PC-1-dependent vasodilation occurs over the entire functional shear stress range and via the activation of endothelial nitric oxide synthase (eNOS) and intermediate (IK)- and small (SK)-conductance Ca2+-activated K+ channels. EC-specific PC-1 knockout increases systemic blood pressure without altering kidney anatomy. PC-1 coimmunoprecipitates with polycystin-2 (PC-2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC-1 or PC-2 (Pkd2 ecKO mice) abolishes surface clusters of both PC-1 and PC-2 in ECs. Single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow-mediated vasodilation. Flow stimulates nonselective cation currents in ECs that are similarly inhibited by either PC-1 or PC-2 knockout or by interference peptides corresponding to the C-terminus coiled-coil domains present in PC-1 or PC-2. In summary, we show that PC-1 regulates arterial contractility through the formation of an interdependent signaling complex with PC-2 in ECs. Flow stimulates PC-1/PC-2 clusters in the EC plasma membrane, leading to eNOS, IK channel, and SK channel activation, vasodilation, and a reduction in blood pressure.

Abstract P334: Biophysical Properties Of A Novel Mitochondrial Large Ubiquitous Non-selective Amiloride Sensitive (luna) Current

Inner mitochondrial membrane (IMM) ion channels and transporters account for communication of the matrix with the intermembrane space (IMS) and the cytosol. Transport of solutes and ions is keep under strict regulation mainly because small changes in solute concentrations could generate changes in mitochondrial volume or membrane potential (ΔΨ m ), interrupting ATP synthesis and leading to mitochondrial damage. The list of recently discovered mitochondrial ion channels has been growing in the past decades. In this work, using the patch-clamp technique we observed the activity of a novel mitochondrial current, named here LUNA current, in mitoplasts (IMM striped of outer membrane) from mouse liver, spleen, brain and heart, as well as established cell lines. LUNA is a novel non-selective cation current (K + >Na + >NMDG + >H + ) active at depolarized membrane potentials. The basal activity of whole-mitoplast LUNA currents from wild type mice hearts changed from 445±106 pA/pF to 1232±287 pA/pF upon chelation of external divalent cations (Ca 2+ and Mg 2+ ). Moreover, the activity of LUNA is independent of the mitochondrial Ca 2+ uniporter and of the non-selective reactive oxygen species modulator channel (ROMO1). In the heart, the activity of LUNA was enhanced in both the Tfam -KO mice, which have impaired electron transport chain (ETC) activity and are a model for mitochondrial cardiomyopathies, and mice with cardiac pressure overload due to transverse aortic constriction (TAC) compared to sham-operated hearts (729±197; n=7 vs 283±137 pA/pF). LUNA current is reversibly inhibited by amiloride with no sensitivity to the vast majority of common K + , Na + and Ca 2+ channels and ETC inhibitors. The molecular identity of mitochondrial LUNA current remains to be determined.

Defective Function of KCa3.1 Channels in Lysosomal Storage Disorders

Background: The calcium-calmodulin-gated KCa3.1 is considered a key regulator of membrane hyperpolarization, maintenance of cellular electrolyte balance, Ca2+-signalling, secretion of pro-inflammatory cytokines, neurodegeneration, pathological tissue remodelling. Osteonecrosis/-penia, vasculopathies, and neurodegeneration are chronic and progressive inflammatory complications of several inherited lysosomal storage diseases, such as Gaucher Disease (GD1/GD3), Fabry Disease(FD), and Niemann-Pick disease type C1 (NPC1). Here we hypothesized that KCa3.1 functions are impaired in a LSD-related fashion in patients' fibroblasts or the monocyte/macrophages lineage.Methods: A total of25 subjects were analyzed (5 FD, 5 NPC, 5 GD1, 5 GD2, 5 healthy controls). Fibroblasts from patients' skin biopsies were cultured in DMEM supplemented with 10% FCS (Biochrom KG, Berlin, Germany) and cells were used at passages 1-4. For patch-clamp experiments, cells were seeded on coverslips and used within 24 h. Monocytes isolated and purified from peripheral blood from age-matched GD patients (GD1 and GD3) and healthy volunteers (Ctrl). Measurements were repeated in patient's and Ctrl's macrophages that were differentiated from monocyte precursors in vitro by exposure to patient's or Ctrl's erythrocyte lysates for 4 days, respectively.Ca2+-activated K+ currents were measured in the whole-cell configuration using an EPC10-USB amplifier (HEKA, Electronics, Lambrecht-Pfalz, Germany) and a K pipette solution (intracellular) containing 1 μM Ca2+ free (in mM): 140 KCl, 1 MgCl2, 2 EGTA, 1.71 CaCl2 (1 μM [Ca2+]free), and 5 HEPES (adjusted to pH 7.2 with KOH). For data acquisition and analysis we used the patch-master program (HEKA). Ohmic leak currents of up to 1 nS were subtracted where appropriate. We quantified outward currents at a 0 mV holding potential (to avoid contamination with non-selective cation currents and chloride currents). Capacitance values (a measure of cell surface and thus cell size)Results: Patch-clamp demonstrated lower fibroblast KCa3.1 membrane functions in NPC1 and male FD patients. KCa3.1-currents in monocytes were similar in GD-1/3 and Ctrls. In differentiated and activated macrophages, the overall larger KCa3.1 currents in GD-1 macrophages were reduced to 40% of controls while in GD-3 macrophages KCa3.1 currents were barely detectable. Glucosylceramide was found to produce KCa3.1-current desensitization. At the gene level, KCa3.1-mRNA expression was impaired in NPC1, GD1/3, and male FD patients.Conclusions: Defective KCa3.1 functions are a feature and biomarker of cellular dysfunction in the LSD, GD1/3, FD, and NPC1 and supports the concept that biased lipid metabolism harms ion channel functions. Since KCa3.1 is involved in the mechanism of cellular differentiation, neurodegeneration, and inflammation, the impaired KCa3.1 -activity could be of pathomechanistic relevance in LSD cellular malfunction by promoting differentiation into pro-inflammatory cell types. Pharmacological targeting of KCa3.1 could be therapeutic utility to achieve neuroprotection in GD3 and NPC1 patients and bone complications in GD1. DisclosuresNo relevant conflicts of interest to declare.

ICAN (TRPM4) Contributes to the Intrinsic Excitability of Prefrontal Cortex Layer 2/3 Pyramidal Neurons.

Pyramidal neurons in the medial prefrontal cortical layer 2/3 are an essential contributor to the cellular basis of working memory; thus, changes in their intrinsic excitability critically affect medial prefrontal cortex (mPFC) functional properties. Transient Receptor Potential Melastatin 4 (TRPM4), a calcium-activated nonselective cation channel (CAN), regulates the membrane potential in a calcium-dependent manner. In this study, we uncovered the role of TRPM4 in regulating the intrinsic excitability plasticity of pyramidal neurons in the mouse mPFC layer of 2/3 using a combination of conventional and nystatin perforated whole-cell recordings. Interestingly, we found that TRPM4 is open at resting membrane potential, and its inhibition increases input resistance and hyperpolarizes membrane potential. After high-frequency stimulation, pyramidal neurons increase a calcium-activated non-selective cation current, increase the action potential firing, and the amplitude of the afterdepolarization, these effects depend on intracellular calcium. Furthermore, pharmacological inhibition or genetic silencing of TRPM4 reduces the firing rate and the afterdepolarization after high frequency stimulation. Together, these results show that TRPM4 plays a significant role in the excitability of mPFC layer 2/3 pyramidal neurons by modulating neuronal excitability in a calcium-dependent manner.

Orexin-A directly depolarizes dorsomedial hypothalamic neurons, including those innervating the rostral ventrolateral medulla

The dorsomedial hypothalamus (DMH) receives dense orexinergic innervation. Intra-DMH application of orexins increases arterial pressure and heart rate in rats. We studied the effects of orexin-A on DMH neurons, including those innervating the medullary cardiovascular center, the rostral ventrolateral medulla (RVLM), by using whole-cell recordings in brain slices. In the presence of tetrodotoxin, orexin-A (30–1000 nM) depolarized 56% of DMH neurons (EC50 82.4 ± 4.4 nM). Under voltage-clamp recording, orexin-A (300 nM) induced three types of responses characterized by different current-voltage relationships, namely unchanged, increased, and decreased slope conductance in 68%, 14%, and 18% of orexin-A-responsive neurons, respectively. The reversal potential of the decreased-conductance response was near the equilibrium potential of K+ and became more positive in a high-K+ solution, suggesting that K+ conductance blockade is the underlying mechanism. In a low-Na+ solution, unchanged-, increased-, and decreased-conductance responses were observed in 56%, 11%, and 33% of orexin-A-responsive neurons, respectively, implying that a non-selective cation current (NSCC) underlies orexin-A-induced responses in a small population of DMH neurons. KBR-7943 (70 μM), an inhibitor of Na+-Ca2+ exchanger (NCX), suppressed orexin-A-induced depolarization in 7 of 10 neurons. In the presence of KBR-7943, the majority of orexin-A-responsive neurons exhibited decreased-conductance responses. These findings suggest that NCX activation may underlie orexin-A-induced depolarization in the majority of orexin-responsive DMH neurons. Of 19 RVLM-projecting DMH neurons identified by retrograde labeling, 17 (90%) were orexin-A responsive. In conclusion, orexin-A directly excited over half of DMH neurons, including those innervating the RVLM, through decreasing K+ conductance, activating NCX, and/or increasing NSCC.

Non-Selective Cation Currents Mediated by Cx43 Hemichannel-P2X4 Receptor Signaling Pathway in Rat Atrial Myocytes under Shear Stress

Cardiac myocytes are exposed to shear stress under physiological conditions and hemodynamic disturbances. Shear stress has been thought to trigger two distinct global Ca2+ waves in atrial myocytes through autocrine activation of P2X4 receptor (P2X4R) and P2Y1-receptor (P2Y1R) via connexin 43 (Cx43)-mediated ATP release. In this study, we examined whether such shear stress-triggered Cx43-ATP-P2 receptor signaling generates depolarizing currents in atrial myocytes. Whole-cell patch clamp and dye-flux assays were used with genetic and pharmacological interventions in rat atrial myocytes and HL-1 cells. We found that shear stress activated a non-selective inward cation (Cs+) current (IS,Cs) at resting potential which was eliminated by Cx43 blockade using siRNA, antibodies, carbenoxolone or La3+. IS,Cs was enhanced by removal of external Ca2+, whereas internal Ca2+ buffering suppressed IS,Cs. Hemichannel-mediated dye (calcein red-orange) flux was induced by shear stress. This response was augmented by removing external Ca2+ but was not affected by pannexin blocker (probenecid). The shear-activated current was significantly reduced when Cs+ was replaced with NMDG+, and this was enhanced by quinine and suppressed by La3+. P2X4R antagonists (5-BDBD and PSB-12054), P2XR-blocker (iso-PPADS) and anti-P2X4R antibodies each blocked IS,Cs by approximately 50%. About 25% of IS,Cs was suppressed by P2Y1R inhibitor (MRS2179) and/or transient receptor potential melastatin 4 blocker (9-phenanthrol). P2X4R, but not P2X5R, was co-localized with Cx43 at the cell ends. P2X4R-dependent IS,Cs was augmented in atrial myocytes from hypertrophied rat hearts by short-term aortic constriction. Our data suggest that shear stress triggers Cx43 hemichannels to gate P2X4Rs in their vicinity. This shear stress signaling may play an important role in membrane depolarization at negative potentials and in atrial remodeling under increased afterload.

Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells

The transient receptor potential ankyrin type 1 (TRPA1) channel belongs to the TRP superfamily of ion channels. TRPA1 is a membrane protein with multiple functions able to respond to noxious stimuli, reactive oxygen species, inflammatory cytokines or pungent substances, and it participates in pain signalling, taste, inflammation and various steps of the tumorigenic process. To date, no reports have addressed the expression and function of TRPA1 in pancreatic ductal adenocarcinoma (PDAC) cells. This work reports the endogenous expression of TRPA1 channels in human pancreatic adenocarcinoma cell lines and provides insights into the function of the TRPA1 protein in the Panc-1 cell line. This study reports that cell lines isolated from PDAC patients had different levels of TRPA1 expression. The channel activity in Panc-1 cells, as assessed with electrophysiological (whole-cell patch clamp) and microfluorimetry methods, showed that non-selective cationic currents were activated by allyl isothiocyanate (AITC) in Panc-1 cells and inhibited by the selective TRPA1 antagonist A-967079. The current elicited by the specific agonist was associated with a robust increase in intracellular Ca2+. Furthermore, siRNA-induced downregulation of TRPA1 enhanced cell migration in the wound healing assay, indicating a possible role of ion channels independent from pore function. Finally, TRPA1 activation changed the cell cycle progression. Taken together, these results support the idea of channel-dependent and independent role for TRPA1 in tumoral processes.

Functions of Muscarinic Receptor Subtypes in Gastrointestinal Smooth Muscle: A Review of Studies with Receptor-Knockout Mice.

Parasympathetic signalling via muscarinic acetylcholine receptors (mAChRs) regulates gastrointestinal smooth muscle function. In most instances, the mAChR population in smooth muscle consists mainly of M2 and M3 subtypes in a roughly 80% to 20% mixture. Stimulation of these mAChRs triggers a complex array of biochemical and electrical events in the cell via associated G proteins, leading to smooth muscle contraction and facilitating gastrointestinal motility. Major signalling events induced by mAChRs include adenylyl cyclase inhibition, phosphoinositide hydrolysis, intracellular Ca2+ mobilisation, myofilament Ca2+ sensitisation, generation of non-selective cationic and chloride currents, K+ current modulation, inhibition or potentiation of voltage-dependent Ca2+ currents and membrane depolarisation. A lack of ligands with a high degree of receptor subtype selectivity and the frequent contribution of multiple receptor subtypes to responses in the same cell type have hampered studies on the signal transduction mechanisms and functions of individual mAChR subtypes. Therefore, novel strategies such as genetic manipulation are required to elucidate both the contributions of specific AChR subtypes to smooth muscle function and the underlying molecular mechanisms. In this article, we review recent studies on muscarinic function in gastrointestinal smooth muscle using mAChR subtype-knockout mice.

Study of the Characteristics of the Pulmonary Vein and Superior Vena Cava of Rabbits

Background: This study aims to (1) investigate the characteristics of the action potential and triggering activity of cardiomyocytes in the pulmonary vein (PV) and superior vena cava (SVC) of rabbits and (2) study the features of cation currents in cardiomyocytes in rabbit PV and SVC-inward rectifier potassium current (IK1), transient outward potassium current (Ito), and non-selective cation currents (INSCC). Methods: The standard glass microelectrode and whole-cell patch-clamp techniques were used to record the action potential and various currents in the above cells. Results: (1) Cardiomyocytes in either PV or SVC had longer action potential durations than in the adjacent atrium, and spontaneous early after depolarization (EAD) could occur in both PV and SVC under normal physiological conditions. (2) The action potential in PV cardiomyocytes had a relative refractory period but did not have an absolute refractory period, and this characteristic enabled a premature beat that triggered a second plateau response, which led to EAD. (3) INSCC was found for the first time in the PV, SVC, and atria. (4) The current intensity of IK1, Ito, and INSCC was significantly lower in the PV and SVC than in the left and right atria, and the difference in the current intensity in INSCC could influence the action potential. Conclusions: PV and SVC can both initiate and maintain AF, but PV is the primary ectopic foci in initiating AF. The present study found that the second plateau response was easily induced in cardiomyocytes in PA shortly after depolarization. This was a specific characteristic of the action potential of PV. In addition, we preliminarily analyzed the differences in the main outward currents and noted a voltage-dependent INSCC in both PV and SVC rabbits’ cardiomyocytes. Furthermore, the current intensities of IK1, Ito, and INSCC were significantly lower in the PV and SVC than in the left and right atria, and the difference in the current intensity of INSCC influenced the action potential. The different permeability of INSCC for cations at different phases may play a role in inducing EAD.

Gastrointestinal motility modulation by stress is associated with reduced smooth muscle contraction through specific transient receptor potential channel.

Excessive stress response causes disability in social life. There are many diseases caused by stress, such as gastrointestinal motility disorders, depression, eating disorders, and cardiovascular diseases. Transient receptor potential (TRP) channels underlie non-selective cation currents and are downstream effectors of G protein-coupled receptors. Ca2+ influx is important for smooth muscle contraction, which is responsible for gastrointestinal motility. Little is known about the possible involvement of TRP channels in the gastrointestinal motility disorders due to stress. The purpose of this study was to measure the changes in gastrointestinal motility caused by stress and to elucidate the mechanism of these changes. The stress model used the water immersion restraint stress. Gastrointestinal motility, especially the ileum, was recorded responses to electric field stimulation (EFS) by isometric transducer. EFS-induced contraction was significantly reduced in the ileum of stressed mouse. Even under the conditions treated with atropine, EFS-induced contraction was significantly reduced in the ileum of stressed mouse. In addition, carbachol-induced, neurokinin A-induced, and substance P-induced contractions were all significantly reduced in the ileum of stressed mouse. Furthermore, the expression of TRPC3 was decreased in the ileum of stressed mouse. These results suggest that the gastrointestinal motility disorders due to stress is associated with specific non-selective cation channel.

Allyl isothiocyanate (AITC) activates nonselective cation currents in human cardiac fibroblasts: possible involvement of TRPA1

The effects of allyl isothiocyanate (AITC), transient receptor potential ankyrin 1 (TRPA1) agonist, on cultured human cardiac fibroblasts were examined by measuring intracellular Ca2+ concentration [Ca2+]i and whole-cell voltage clamp techniques. AITC (200 μM) increased Ca2+ entry in the presence of [Ca2+]i. Ruthenium red (RR) (30 μM), and La3+ (0.5 mM), a general cation channel blocker, inhibited AITC-induced Ca2+ entry. Under the patch pipette filled with Cs+- and EGTA-solution, AITC induced the current of a reversal potential (Er) of approximately +0 mV. When extracellular Na+ ion was changed by NMDG+, the inward current activated by AITC was markedly reduced. La3+ and RR inhibited the AITC-induced current. The conventional RT-PCR analysis, Western blot, and immunocytochemical studies showed TRPA1 mRNA and protein expression. The present study shows the first evidence for functional Ca2+-permeable nonselective cation currents induced by AITC, possibly via TRPA1 in human cardiac fibroblast.

TRPM4 Conductances in Thalamic Reticular Nucleus Neurons Generate Persistent Firing during Slow Oscillations.

During sleep, neurons in the thalamic reticular nucleus (TRN) participate in distinct types of oscillatory activity. While the reciprocal synaptic circuits between TRN and sensory relay nuclei are known to underlie the generation of sleep spindles, the mechanisms regulating slow (<1 Hz) forms of thalamic oscillations are not well understood. Under in vitro conditions, TRN neurons can generate slow oscillations in a cell-intrinsic manner, with postsynaptic Group 1 metabotropic glutamate receptor activation triggering long-lasting plateau potentials thought to be mediated by both T-type Ca2+ currents and Ca2+-activated nonselective cation currents (ICAN). However, the identity of ICAN and the possible contribution of thalamic circuits to slow rhythmic activity remain unclear. Using thalamic slices derived from adult mice of either sex, we recorded slow forms of rhythmic activity in TRN neurons, which were driven by fast glutamatergic thalamoreticular inputs but did not require postsynaptic Group 1 metabotropic glutamate receptor activation. For a significant fraction of TRN neurons, synaptic inputs or brief depolarizing current steps led to long-lasting plateau potentials and persistent firing (PF), and in turn, resulted in sustained synaptic inhibition in postsynaptic relay neurons of the ventrobasal thalamus (VB). Pharmacological approachesindicated that plateau potentials were triggered by Ca2+ influx through T-type Ca2+ channels and mediated by Ca2+- and voltage-dependent transient receptor potential melastatin 4 (TRPM4) channels. Together, our results suggest that thalamic circuits can generate slow oscillatory activity, mediated by an interplay of TRN-VB synaptic circuits that generate rhythmicity and TRN cell-intrinsic mechanisms that control PF and oscillation frequency.SIGNIFICANCE STATEMENT Slow forms of thalamocortical rhythmic activity are thought to be essential for memory consolidation during sleep and the efficient removal of potentially toxic metabolites. In vivo, thalamic slow oscillations are regulated by strong bidirectional synaptic pathways linking neocortex and thalamus. Therefore, in vitro studies in the isolated thalamus offer important insights about the ability of individual neurons and local circuits to generate different forms of rhythmic activity. We found that circuits formed by GABAergic neurons in the thalamic reticular nucleus and glutamatergic relay neurons in the ventrobasal thalamus generated slow oscillatory activity, which was accompanied by persistent firing in thalamic reticular nucleus neurons. Our results identify both cell-intrinsic and synaptic mechanisms that mediate slow forms of rhythmic activity in thalamic circuits.