Sensitive Nonselective Cation Research Articles

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.

Matrix stiffness regulates myocardial differentiation of human umbilical cord mesenchymal stem cells.

Myocardial infarction is a cardiovascular disease with high mortality. Human umbilical cord mesenchymal stem cells (hUC-MSCs) with strong self-renewal capacity and multipotency, provide the possibility of replacing injured cardiomyocytes. hUC-MSCs were cultured on polyacrylamide hydrogels with stiffnesses corresponding to Young's modulus of 13-16kPa and 62-68kPa which mimic the stiffnesses of healthy heart tissue and fibrotic myocardium. The expression of early myocardial markers Nkx2.5, GATA4, Mesp1 and the mature myocardial markers cTnT, cTnI, α-actin were detected by RT-PCR and Western Blot, which showed that soft matrix (13-16 kPa) tended to induce the differentiation of hUC-MSCs into myocardium, compared with stiff matrix (62-68 kPa). Piezos are mechanically sensitive non-selective cation channels. The expression of Piezo1 increased with the stiffness gradient of 1-10kPa, 13-16kPa, 35-38kPa and 62-68kPa on the 1st day, but Piezo2 expression was irregular. The expression of integrin β1 and calcium ions were also higher on stiff substrate than on soft substrate. hUC-MSCs tend to differentiate into myocardium on the matrix stiffness of 13-16 kPa. The relationship among matrix stiffness, Piezo1 and myocardial differentiation needs further validation.

Fluid Pressure-Activated Non-Selective Cation Current and Cl- Current in Rat Atrial Myocytes

When intact atrial muscle is exposed to turbulant flow or high fluid pressure during valve diseases, it produces arrhythmias. Here we characterized a novel fluid pressure (FP)-gated ionic current (IFP) in single rat atrial myocytes using a whole-cell patch clamp. A flow of pressurized (∼16 dyn/cm2) fluid was applied onto single rat atrial myocytes using a microperfusion method. The application of FP with a normal bath solution elicited a transient inward current (∼1 pA/pF at −80 mV). The magnitude of IFP was increased in a pressure-dependent manner. The removal of extracellular Ca2+ largely enhanced the IFP and eliminated the current adaptation. Under physiological ionic gradients, the IFP displayed an inwardly- and outwardly-rectifying current-voltage relationship with a reversal potential (Erev) of approximately −52 mV. The Cl− channel blockers, DIDS and 9-AC, suppressed inward and outward IFP by about 50% and 70-80%, respectively. In symmetrical Cl− solutions, the Erev was shifted rightward (≌−18 mV) and the outwardly rectifying IFP was attenuated. In the symmetrical Cl− conditions, removal of extracellular Na+ largely reduced inward IFP, and produced a left shift of Erev (≌−64 mV). In addition, the elimination of internal K+ shifted Erev to ≌+8.4 mV and decreased outward IFP. Although low concentrations of extracellular Ca2+ blocked IFP with a negative shift of Erev, high concentrations of extracellular Ca2+ produced a right shift of Erev. Gadolinium ion (Gd3+), the stretch-activated channel blocker, partially blocked the inward IFP. In current-clamped cells, FP of the same magnitude elicited spontaneous membrane depolarization with repetitive action potentials and prolonged action potential durations. These results indicate that FP may activate an outwardly rectifying Cl− channel and a Gd3+- and Ca2+-sensitive non-selective cation channel that carries Na+, K+, and Ca2+.

Structural changes during HCN channel gating defined by high affinity metal bridges

Hyperpolarization-activated cyclic nucleotide–sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4–S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd2+-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4–S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4–S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4–S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating.

Neuronal Ca2+-sensitive Non-selective Cation Channels and TRPC5

Neuronal Ca2+-sensitive Non-selective Cation Channels and TRPC5

Characterization of receptor‐ and store‐operated cation currents in rat pulmonary arterial smooth muscle cells (PASMCs)

Store‐ and receptor‐operated cation channels (SOC and ROC) in PASMCs have been implicated to play important roles in many physiological functions, including hypoxia‐ and agonist‐induced vasoconstriction, and pulmonary hypertension. Here we characterized the SOC and ROC currents in rat intralobar PASMCs. PASMCs were whole‐cell voltage‐clamped. K+ and Ca2+ currents were eliminated by substituting K+ with Cs+, and by nifedipine. Depletion of SR Ca2+ stores with cyclopiazonic acid activated a small inward current at −60 mV. The current was elicited with a time‐delay, exhibited typical bimodal dependence to extracellular Ca2+ due to the effect of anomalous mole fraction, and was blocked by La3+ or substitution of Na+ with N‐methyl‐D‐glucamine. I‐V relationship of La3+‐sensitive SOC current was quasi‐linear with a reverse potential close to 0 mM, indicative of its non‐selective nature. ROC agonist 1‐oleoyl‐2‐acetyl‐sn‐glycerol also activated a La3+ (1 mM) sensitive non‐selective cation current with slight outward rectification. Moreover, ROC currents recorded in PASMCs of chronic hypoxic rats (10% O2, 4 weeks) were significantly larger than those of normoxic rats. These results are consistent with the notion that functional SOC and ROC are present in PASMCs and their activities are up‐regulated in hypoxic pulmonary hypertension. (Supported by NIH R01HL074134, NSFC 30670772 and NSF(Fujian) C0620002)

Low concentrations of sphingosylphosphorylcholine enhance pulmonary artery vasoreactivity: the role of protein kinase C delta and Ca2+ entry.

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC(50) is approximately 100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (<or=1 micromol/L) modulated vasoreactivity of rat intrapulmonary arteries using myography and measurement of intracellular [Ca(2+)]. SPC (1 micromol/L) had no effect on force or intracellular [Ca(2+)] on its own, but dramatically potentiated constrictions induced by approximately 25 mmol/L [K(+)], such that at 40 minutes, force and intracellular [Ca(2+)] (Fura PE3 340/380 ratio) were increased by 429+/-96% and 134+/-26%, respectively. The potentiation was stereospecific, apparent at concentrations >100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane-sensitive Ca(2+) entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC delta inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC delta. The potentiation could be attributed to enhancement of Ca(2+) entry. SPC also potentiated the responses to prostaglandin F(2 alpha) and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 micromol/L) caused translocation of PKC delta to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca(2+) entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC delta. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.

Abstract 1289: Activation of the Transient Receptor Potential V4 Channel Causes Cardiovascular Collapse Mediated by Endothelial Failure

Transient receptor potential V4 (TRPV4), is an osmo/mechano-sensitive ligand-gated non-selective cationic channel that contributes to intracellular Ca 2+ homeostasis and regulation of cell volume. TRPV4 is highly expressed in endothelial and various epithelial cells and to a lesser extent in vascular smooth muscle. The purpose of the present study was to evaluate the cardiovascular effects of a potent novel small molecule TRPV4 activator (TRPV4A; EC 50 = 19, 8 or 1 nM in mouse, rat or dog, respectively) and to examine its mechanism of action. In the three species (mouse, rat and dog) examined, the intravenous administration of TRPV4A in anesthetized preparations induced a dose-dependent reduction in blood pressure, followed by cardiovascular collapse at doses ≤300 ug/kg. TRPV4A had no acute cardiovascular effects in the TRPV4 −/− null mouse (&gt;5 mg/kg, iv). Hemodynamic analysis in the dog suggested that the terminal event was related to a profound reduction in cardiac output (decreased SV followed by decreased HR). However, TRPV4A had no effect on rate or contractility in the isolated, buffer-perfused rat heart. In contrast, TRPV4A produced potent endothelial-dependent relaxation of rodent isolated vascular ring segments that was abolished by NOS inhibition (L-NAME) and eNOS gene deletion. Surprisingly, the in vivo cardiovascular collapse induced by TRPV4A was not altered by NOS inhibition (L-NAME), eNOS gene deletion or by treatment with anticoagulant or antiplatelet agents. Cardiovascular collapse induced by TRPV4A was correlated (concentration and time appropriate) with profound fluid extravasation (Evans Blue) and tissue hemorrhage in the lung, intestine and kidney. Consistent with these results, TRPV4A induced a ruthenium-red sensitive non-selective cation current, reduction in cell volume and subsequent cell death in cultured endothelial cells. These results provide the first characterization of the cardiovascular effects of a potent and selective TRPV4 channel activator and suggest that inappropriate activation of TRPV4 produces acute cardiovascular collapse associated with endothelial activation/injury and failure of the microvascular permeability barrier.

Dynamics of α‐synuclein aggregation and inhibition of pore‐like oligomer development by β‐synuclein

Accumulation of alpha-synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinson's disease and Lewy body dementia. In contrast, beta-synuclein (beta-syn), a close homologue, does not aggregate and reduces alpha-synuclein (alpha-syn)-related pathology. Although considerable information is available about the conformation of alpha-syn at the initial and end stages of fibrillation, less is known about the dynamic process of alpha-syn conversion to oligomers and how interactions with antiaggregation chaperones such as beta-synuclein might occur. Molecular modeling and molecular dynamics simulations based on the micelle-derived structure of alpha-syn showed that alpha-syn homodimers can adopt nonpropagating (head-to-tail) and propagating (head-to-head) conformations. Propagating alpha-syn dimers on the membrane incorporate additional alpha-syn molecules, leading to the formation of pentamers and hexamers forming a ring-like structure. In contrast, beta-syn dimers do not propagate and block the aggregation of alpha-syn into ring-like oligomers. Under in vitro cell-free conditions, alpha-syn aggregates formed ring-like structures that were disrupted by beta-syn. Similarly, cells expressing alpha-syn displayed increased ion current activity consistent with the formation of Zn(2+)-sensitive nonselective cation channels. These results support the contention that in Parkinson's disease and Lewy body dementia, alpha-syn oligomers on the membrane might form pore-like structures, and that the beneficial effects of beta-synuclein might be related to its ability to block the formation of pore-like structures.

TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells

Stretch- and swelling-activated cation (SSAC) channels play essential roles not only in sensing and transducing external mechanical stresses but also in regulating cell volume in living cells. However, the molecular nature of the SSAC channel has not been clarified. In human epithelial HeLa cells, single-channel recordings in cell-attached and inside-out patches revealed expression of a Mg(2+)- and Gd(3+)-sensitive nonselective cation channel that is exquisitely sensitive to membrane stretch. Whole cell recordings revealed that the macroscopic cationic currents exhibit transient receptor potential (TRP) melastatin (TRPM)7-like properties such as outward rectification and sensitivity to Mg(2+) and Gd(3+). The whole cell cation current was augmented by osmotic cell swelling. RT-PCR and Western blotting demonstrated molecular expression of TRPM7 in HeLa cells. Treatment with small interfering RNA (siRNA) targeted against TRPM7 led to abolition of single stretch-activated cation channel currents and of swelling-activated, whole cell cation currents in HeLa cells. The silencing of TRPM7 by siRNA reduced the rate of cell volume recovery after osmotic swelling. A similar inhibition of regulatory volume decrease was also observed when extracellular Ca(2+) was removed or Gd(3+) was applied. It is thus concluded that TRPM7 represents the SSAC channel endogenously expressed in HeLa cells and that, by serving as a swelling-induced Ca(2+) influx pathway, it plays an important role in cell volume regulation.

Intracellular pH Modulates Taste Receptor Cell Volume and the Phasic Part of the Chorda Tympani Response to Acids

The relationship between cell volume and the neural response to acidic stimuli was investigated by simultaneous measurements of intracellular pH (pHi) and cell volume in polarized fungiform taste receptor cells (TRCs) using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in vitro and by rat chorda tympani (CT) nerve recordings in vivo. CT responses to HCl and CO2 were recorded in the presence of 1 M mannitol and specific probes for filamentous (F) actin (phalloidin) and monomeric (G) actin (cytochalasin B) under lingual voltage clamp. Acidic stimuli reversibly decrease TRC pHi and cell volume. In isolated TRCs F-actin and G-actin were labeled with rhodamine phalloidin and bovine pancreatic deoxyribonuclease-1 conjugated with Alexa Fluor 488, respectively. A decrease in pHi shifted the equilibrium from F-actin to G-actin. Treatment with phalloidin or cytochalasin B attenuated the magnitude of the pHi-induced decrease in TRC volume. The phasic part of the CT response to HCl or CO2 was significantly decreased by preshrinking TRCs with hypertonic mannitol and lingual application of 1.2 mM phalloidin or 20 μM cytochalasin B with no effect on the tonic part of the CT response. In TRCs first treated with cytochalasin B, the decrease in the magnitude of the phasic response to acidic stimuli was reversed by phalloidin treatment. The pHi-induced decrease in TRC volume induced a flufenamic acid–sensitive nonselective basolateral cation conductance. Channel activity was enhanced at positive lingual clamp voltages. Lingual application of flufenamic acid decreased the magnitude of the phasic part of the CT response to HCl and CO2. Flufenamic acid and hypertonic mannitol were additive in inhibiting the phasic response. We conclude that a decrease in pHi induces TRC shrinkage through its effect on the actin cytoskeleton and activates a flufenamic acid–sensitive basolateral cation conductance that is involved in eliciting the phasic part of the CT response to acidic stimuli.

Identification of a protein that interacts with the vanilloid receptor

The vanilloid receptor (VR1 or TRPV1) is a capsaicin (CAP)-sensitive non-selective cation channel. Although its channel activity is reportedly modulated through protein–protein interactions, to date very few VR1 interacting proteins have been identified. To address this issue, a yeast two-hybrid screening technique using the C-terminus of rVR1 as bait was employed. Upon interrogation of a mouse brain library, one gene product that interacts with VR1 and is highly homologous to human eferin was found. Its interaction with VR1 was confirmed by GST-pull-down and co-immunoprecipitation. When cotransfected into HEK cells, VR1 and eferin largely colocalize. Furthermore, in rat dorsal root ganglion cells, the rat eferin homologue also colocalizes with rVR1. However, this protein had no significant effect on VR1 channel activity in response to CAP. This was determined by two-electrode recording of oocytes and whole cell recording of HEK cells that were cotransfected with VR1 and human eferin.

Hydroxyl radical activation of a Ca(2+)-sensitive nonselective cation channel involved in epithelial cell necrosis.

In a previous work the involvement of a fenamate-sensitive Ca(2+)-activated nonselective cation channel (NSCC) in free radical-induced rat liver cell necrosis was demonstrated (5). Therefore, we studied the effect of radical oxygen species and oxidizing agents on the gating behavior of a NSCC in a liver-derived epithelial cell line (HTC). Single-channel currents were recorded in HTC cells by the excised inside-out configuration of the patch-clamp technique. In this cell line, we characterize a 19-pS Ca(2+)-activated, ATP- and fenamate-sensitive NSCC nearly equally permeable to monovalent cations. In the presence of Fe(2+), exposure of the intracellular side of NSCC to H(2)O(2) increased their open probability (P(o)) by approximately 40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (.OH) scavenger MCI-186 inhibited the effect of H(2)O(2), indicating that the increase in P(o) was mediated by.OH. Exposure of the patch membrane to the oxidizing agent 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) had a similar effect to.OH. The increase in P(o) induced by.OH or DTNB was not reverted by preventing formation or by DTNB washout, respectively. However, the reducing agent dithiothreitol completely reversed the effects on P(o) of both.OH and DTNB. A similar increase in P(o) was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca(2+). The effect of GSSG was fully reversed by GSH. These results suggest an intracellular site(s) of action of oxidizing agents on cysteine targets on the fenamate-sensitive NSCC protein implicated in epithelial cell necrosis.

Cell Swelling and a Nonselective Cation Channel Regulated by Internal Ca2+and ATP in Native Reactive Astrocytes from Adult Rat Brain

Hypoxia-ischemia and ATP depletion are associated with glial swelling and blebbing, but mechanisms involved in these effects remain incompletely characterized. We examined morphological and electrophysiological responses of freshly isolated native reactive astrocytes (NRAs) after exposure to NaN(3), which depletes cellular ATP. Here we report that NaN(3) caused profound and sustained depolarization attributable to activation of a novel 35 pS Ca(2+)-activated, [ATP](i)-sensitive nonselective cation (NC(Ca-ATP)) channel, found in >90% of excised membrane patches. The channel was impermeable to Cl(-), was nearly equally permeable to monovalent cations, with permeabilities relative to K(+) being P(Cs)+/P(K)+(1.06) approximately P(Na)+/P(K)+(1.04) approximately P(Rb)+/P(K)+(1.02) approximately P(Li)+/P(K)+(0.96), and was essentially impermeable to Ca(2+) and Mg(2+) (P(Ca)2+/P(K)+ approximately P(Mg)2+/P(K)+ < 0.001), with intracellular Mg(2+) (100 microm to 1 mm) causing inward rectification. Pore radius, estimated by fitting relative permeabilities of organic cations to the Renkin equation, was 0.41 nm. This channel exhibited significantly different properties compared with previously reported NC(Ca-ATP) channels, including different sensitivity to block by various adenine nucleotides (EC(50) of 0.79 microm for [ATP](i), with no block by AMP or ADP), and activation by submicromolar [Ca](i). The apparent dissociation constant for Ca(2+) was voltage dependent (0.12, 0.31, and 1.5 microm at -40, -80, and -120 mV, respectively), with a Hill coefficient of 1.5. Channel opening by [ATP](i) depletion was accompanied by and appeared to precede blebbing of the cell membrane, suggesting participation of this channel in cation flux involved in cell swelling. We conclude that NRAs from adult rat brain express a 35 pS NC(Ca-ATP) channel that may play an important role in the pathogenesis of brain swelling.

Ionic basis for membrane potential changes induced by hypoosmotic stress in guinea-pig ventricular myocytes.

Causal relation between changes in action potentials and activation of several ionic currents during hypoosmotic challenge was investigated. We recorded changes in membrane potentials and currents during hypotonic stress in guinea-pig ventricular myocytes using whole-cell patch-clamp technique. Exposure of ventricular myocytes to hypotonic solution (0.6 T) caused initial prolongation ( approximately 107% of control) of action potential duration at 90% repolarization (APD(90)) in 65% of examined myocytes. Later shortening (approximately 75% of control) of APD(90) and depolarization of resting potential (RP) (approximately 4 mV) developed in all cells. Initial prolongation of APD(90) in hypotonic solution was mainly caused by transient activation of Gd(3+)-sensitive non-selective cation (NSC) current. Late changes after approximately 180 s in hypotonic solution were sustained increase in slow component of delayed rectifier K(+) current (I(Ks)) in all cells, and activation of I(Clswell) in 40% of cells. Prevention of APD(90) shortening by chromanol, a selective blocker of I(Ks), was seen in about 40% of myocytes due to short APD in our experimental conditions. Application of 1 mM anthracene-9-carboxylic acid (9-AC) partially inhibited APD shortening in three of seven cells. Depolarization of RP was unaffected by the above-mentioned drugs, but was dependent on [K(+)](o). Initial prolongation followed by later shortening of APD in hypotonic solution are mostly caused by different sequences of NSC, I(Ks) and I(Clswell) currents activation. Depolarization of RP in hypotonic solution is probably due to dilution of subsarcolemmal K(+) concentration and/or change in permeability ratio for Na(+) and K(+).

S14-1 - Expression of Putative Stretch sensitive Nonselective Cation Channels of mammal

S14-1 - Expression of Putative Stretch sensitive Nonselective Cation Channels of mammal

A role for Ca2+-sensitive nonselective cation channels in regulating the membrane potential of pancreatic beta-cells.

The incretin hormones, glucagon-like peptide 1 and pituitary adenylyl cyclase-activating polypeptide, are proposed to activate a maitotoxin (MTX)-sensitive, Ca2+-dependent nonselective cation current in pancreatic beta-cells and insulinoma cells. This MTX-sensitive current is present in human beta-cells as well as in mouse and rat beta-cells, and is accompanied by a rise in cytosolic Ca2+ in voltage-clamped cells in which the activation of voltage-dependent Ca2+ channels is prevented. Activation of the nonselective cation current is inhibited by reduction of disulfide bonds with intracellular, but not extracellular, dithiothreitol, and is also abolished by intracellular dialysis with trypsin. The nonselective cation channels that carry this current have a conductance of about 30 pS, with Na+ as the major extracellular cation. We estimate that these cation channels are expressed on beta-cells at a density similar to that of ATP-sensitive potassium channels (K(ATP) channels) and exhibit spontaneous activity at basal glucose concentrations. We propose that this spontaneous cation channel activity constitutes at least part of the depolarizing background conductance that permits changes in the activity of K(ATP) channels to regulate the resting potential of beta-cells.

Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+ concentration in the store lumen.

The mechanism by which agonist-evoked cytosolic Ca2+ signals are terminated has been investigated. We measured the Ca2+ concentration inside the endoplasmic reticulum store of pancreatic acinar cells and monitored the cytoplasmic Ca2+ concentration by whole-cell patch-clamp recording of the Ca2+-sensitive currents. When the cytosolic Ca2+ concentration was clamped at the resting level by a high concentration of a selective Ca2+ buffer, acetylcholine evoked the usual depletion of intracellular Ca2+ stores, but without increasing the Ca2+-sensitive currents. Removal of acetylcholine allowed thapsigargin-sensitive Ca2+ reuptake into the stores, and this process stopped when the stores had been loaded to the pre-stimulation level. The apparent rate of Ca2+ reuptake decreased steeply with an increase in the Ca2+ concentration in the store lumen and it is this negative feedback on the Ca2+ pump that controls the Ca2+ store content. In the absence of a cytoplasmic Ca2+ clamp, acetylcholine removal resulted in a rapid return of the elevated cytoplasmic Ca2+ concentration to the pre-stimulation resting level, which was attained long before the endoplasmic reticulum Ca2+ store had been completely refilled. We conclude that control of Ca2+ reuptake by the Ca2+ concentration inside the intracellular store allows precise Ca2+ signal termination without interfering with store refilling.

A calcium and ATP sensitive nonselective cation channel in the antiluminal membrane of rat cerebral capillary endothelial cells

The patch-clamp technique was applied to the antiluminal membrane of freshly isolated capillaries of rat brain (blood-brain barrier). With 1.3 mM Ca 2+ in the bath, excision of membrane patches evoked ion channels, which could not be observed in cell-attached mode. The channel was about equally permeable to Na + and K + ions, but not measurable permeable to Cl − and and the single-channels conductance was 31 + 2 pS ( n = 22). The channel open probability was not dependent on the applied potential. Lowering of Ca 2+ to 1 μM or below on the cytosolic side inactivated the channels, whereas addition of cytosolic ATP (1 mM) inhibited channel activity completely and reversibly. The channel was blocked by the inhibitor of nonselective cation channels in rat exocrine pancreas 3′,5-dichlorodiphenylamine-2-car☐ylic acid (DCDPC, 10 μM) and by the antiinflammatory drugs flufenamic acid (> 10 μM) and tenidap (100 μM), as well as by gadolinium (10 μM). Thus, these nonselective cation channels have many properties in common with similar channels observed in fluid secreting epithelia. The channel could be involved in the transport of K + ions from brain to blood side.