Inside-out Configuration Research Articles

Activation by Zonisamide, a Newer Antiepileptic Drug, of Large-Conductance Calcium-Activated Potassium Channel in Differentiated Hippocampal Neuron-Derived H19-7 Cells

Zonisamide (ZNS; 3-sulfamoylmethyl-1,2-benzisoxazole), as one of the newer antiepileptic drugs, has been demonstrated its broad-spectrum clinical efficacy on various neuropsychiatric disorders. However, little is known regarding the mechanism of ZNS actions on ion currents in neurons. We thus investigated its effect on ion currents in differentiated hippocampal 19-7 cells. In whole-cell configuration of patch-clamp technology, the ZNS (30 microM) reversibly increased the amplitude of K+ outward currents, and paxilline (1 microM) was effective in suppressing the ZNS-induced increase of K+ outward currents. In inside-out configuration, ZNS (30 microM) applied to the intracellular face of the membrane did not alter single-channel conductance; however, it did enhance the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels primarily by decreasing mean closed time. In addition, the EC50 value for ZNS-stimulated BK(Ca) channels was 34 microM. This drug caused a left shift in the activation curve of BK(Ca) channels, with no change in the gating charge of these channels. Moreover, ZNS at a concentration greater than 100 microM also reduced the amplitude of A-type K+ current in these cells. A simulation modeling based on hippocampal CA3 pyramidal neurons (Pinsky-Rinzel model) was also analyzed to investigate the inhibitory effect of ZNS on the firing of simulated action potentials. Taken together, this study suggests that, in hippocampal neurons during the exposure to ZNS, the ZNS-mediated effects on BK(Ca) channels and A-type K+ current could be potential mechanisms through which it affects neuronal excitability.

TRPM4, a Ca2+-activated nonselective cation channel in mouse sino-atrial node cells

A calcium-activated nonselective cation channel (NSC(Ca)) has been recently described in several cardiac preparations. This channel is over-expressed in models of ventricular hypertrophy showing electrophysiological perturbations of heart activity, including occurrence of spontaneous activity. While these perturbations are currently attributed to a modification of the pacemaker I(f) current activity, arguments are also in favor of participation of an NSC(Ca). Similarly, the NSC(Ca) may be expressed in specialized pacemaker cells, i.e. sino-atrial node (SAN) cells. The aim of the present study was to detect such current in mouse pacemaker cells. The inside-out configuration of the patch-clamp technique was used in freshly isolated SAN cells from adult mice. Also, RT-PCR and Western-blotting studies were used to probe for TRPM4 mRNA and protein expression. In these cells, an NSC(Ca) activity was detected. The channel is voltage dependant with a conductance of 20.9+/-0.5 pS (n = 11). It is equally permeable for Na+ and K+ but does not conduct Ca2+. It is activated by rise in intracellular calcium concentrations and blocked by intracellular ATP (0.5 mmol/L). Also, as a new property in cardiac cells, the channel is activated by internal application of phosphatidylinositol 4,5-bisphosphate (10 microM). It is reversibly inhibited by flufenamic acid and glibenclamide. This channel shows the hallmarks of the TRPM4 molecule, a member of the TRP melastatin subfamily. We confirm the expression of this TRP channel on SAN cells by Western blotting and RT-PCR and validate that TRPM4 is glibenclamide sensitive. TRPM4 is functionally expressed in SAN cells and may be a key player in the generation and/or perturbation of heart rhythm.

Regulation of Human Cone Cyclic Nucleotide-Gated Channels by Endogenous Phospholipids and Exogenously Applied Phosphatidylinositol 3,4,5-trisphosphate

Cyclic nucleotide-gated (CNG) channels are critical components of the vertebrate visual transduction cascade involved in converting light-induced changes in intracellular cGMP concentrations into electrical signals that can be interpreted by the brain as visual information. To characterize regulatory mechanisms capable of altering the apparent ligand affinity of cone channels, we have expressed heteromeric (CNGA3 + CNGB3) human cone CNG channels in Xenopus laevis oocytes and characterized the alterations in channel activity that occur after patch excision using patch-clamp recording in the inside-out configuration. We found that cone channels exhibit spontaneous changes in current at subsaturating cGMP concentrations; these changes are enhanced by application of ATP and seem to reflect alterations in channel gating. Similar to rod CNG channels, lavendustin A prevented this regulation, suggesting the involvement of a tyrosine phosphorylation event. However, the tyrosine residue in CNGB3 (Tyr545) that is equivalent to the critical tyrosine residues in rod and olfactory CNG channel subunits does not participate in cone channel regulation. Furthermore, the changes in ligand sensitivity of CNGA3 + CNGB3 channels were prevented by inhibition of phosphatidylinositol 3-kinase (PI3-kinase) using wortmannin or 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), which suggests that phospholipid metabolism can regulate the channels. Direct application of phosphatidylinositol 3,4,5-trisphosphate (PIP3) to the intracellular face of excised patches also resulted in down-regulation of channel activity. Thus, phospholipid metabolism and exogenously applied PIP3 can modulate heterologously expressed cone CNG channels.

Direct modulation of Ca 2+-activated K + current by H-89 in rabbit coronary arterial smooth muscle cells

The effects of H-89, a potent and selective inhibitor of protein kinase A (PKA) on Ca 2+-activated K + (BK Ca) channels in coronary arterial smooth muscle cells were examined using a patch-clamp technique. In inside-out configuration, H-89 increased the NP o of the BK Ca channel, but it reduced the dwell time of BK Ca currents. In whole-cell configuration, H-89 markedly increased BK Ca currents in a concentration-dependent manner. The EC 50 was 0.470 ± 0.0741 μM based on dwell time, 0.582 ± 0.0691 μM based on the NP o, and 0.519 ± 0.0295 μM based on the whole-cell current, respectively. H-85, which is an inactive form of H-89, increased BK Ca currents, similar to the result of H-89. The other PKA inhibitors (Rp-8-CPT-cAMPs and KT 5720) and protein phosphatase inhibitor (okadaic acid, 1 μM) had little effect on BK Ca currents and did not significantly alter the stimulatory effects of 1 μM H-89. These findings suggest that H-89 increases the BK Ca current independently of PKA.

The Opening Effect of Pregabalin on ATP‐Sensitive Potassium Channels in Differentiated Hippocampal Neuron–derived H19‐7 Cells

Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels can couple an intracellular metabolic state to an electrical activity, which is important in the control of neuronal excitability and seizure propagation. We investigated whether the newer antiepileptic drug, pregabalin (PGB), could exert effects on KATP channels in differentiated hippocampal neuron-derived H19-7 cells. The inside-out configuration of the patch-clamp technique was used to investigate KATP channel activities in H19-7 cells in the presence of PGB. Effects of various compounds known to alter KATP channel activities were compared. The activity of KATP channels in these cells was characterized. The single-channel conductance from a linear current-voltage relation was 78 +/- 2 pS (n = 8) with a reversal potential of 63 +/- 2 mV (n = 8), similar to that of KATP channels reported in pancreatic beta cells. 2,4-Dinitrophenol activated channel activity, but the further addition of glucose (20 mM) or glibenclamide (30 microM) could offset these increments. PGB significantly opened these KATP channel activities in a concentration-dependent fashion with a median effective concentration (EC50) value of 18 microM. A significant increase was noted in the mean open lifetime of KATP channels in the presence of PGB (1.71 +/- 0.04 to 5.62 +/- 0.04 ms). This study suggests that in differentiated hippocampal neuron-derived H19-7 cells, the opening effect on KATP channels could be one of the underlying mechanisms of PGB in the reduction of neuronal excitability.

The SLO-1 BK Channel of Caenorhabditis elegans is Critical for Muscle Function and is Involved in Dystrophin-dependent Muscle Dystrophy

The Caenorhabditis elegans SLO-1 channel belongs to the family of calcium-activated large conductance BK potassium channels. SLO-1 has been shown to be involved in neurotransmitter release and ethanol response. Here, we report that SLO-1 also has a critical role in muscles. Inactivation of the slo-1 gene in muscles leads to phenotypes similar to those caused by mutations of the dystrophin homologue dys-1. Notably, slo-1 mutations result in a progressive muscle degeneration when put into a sensitized genetic background. slo-1 localization was observed by gfp reporter gene in both the M-line and the dense bodies (Z line) of the C.elegans body-wall muscles. Using the inside-out configuration of the patch clamp technique on body-wall muscle cells of acutely dissected wild-type worms, we characterized a Ca2+-activated K+ channel that was identified unambiguously as SLO-1. Since neither the abundance nor the conductance of SLO-1 was changed significantly in dys-1 mutants compared to wild-type animals, it is likely that the inactivation of dys-1 causes a misregulation of SLO-1. All in all, these results indicate that SLO-1 function in C.elegans muscles is related to the dystrophin homologue DYS-1.

Mutations in the Amino Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Enhance Endocytosis

Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at approximately 50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype.

Ketanserin, a 5-HT2 Antagonist, Directly Inhibits the ATP-Sensitive Potassium Channel in Mouse Ventricular Myocytes

The effects of ketanserin, a 5-HT2 antagonist, on the ATP-sensitive K+ (K(ATP)) channels were studied in mouse ventricular myocytes using patch clamp technique. Under the whole-cell voltage clamp conditions, ketanserin (1-100 microM) reversibly inhibited pinacidil-induced K(ATP) current in a concentration-dependent fashion with a Ki value of 9.36 microM and the Hill coefficient was 0.67. This inhibition was developed even with the presence of 5-hydroxytryptamine (100 microM) in the bath. Prazosin, a selective alpha1-antagonist, also failed to mimic the effect of ketanserin. Ketanserin did not affect the channel activity in inside-out configuration under the ATP-free internal solution. Furthermore, ketanserin applied to the external solution did not affect the pinacidil-induced channel activity in the cell-attached patches, but did inhibit it when applied into the pipette. These results suggest that the inhibitory action of ketanserin observed in this study was probably due to a direct action on the K(ATP) channel rather than to an action through the 5-HT2 receptor or alpha1-adrenoceptor blockade, and that the antiarrhythmic activity of ketanserin against cardiac arrhythmias induced in the ischemic/reperfused heart is at least in part attributable to its inhibition of the K(ATP) channel.

Properties of a potassium channel in the basolateral membrane of renal proximal convoluted tubule and the effect of cyclosporine on it

We studied the potassium channel in the basolateral membrane of the rat proximal convoluted tubule as affected by cyclosporine A. Proximal convoluted tubules were dissected from the rat kidney under a stereoscopic microscope, without a preliminary enzyme treatment. The standard configuration for single-channel tight seal patch-clamp technique was used to record channel currents. A small conductance, stretch-sensitive potassium channel could be observed spontaneously in most of the cell-attached patches as the gigaohm seal was formed. In the inside-out configuration, channel activity was diminished. The K(+) channel appeared to be an inward rectifier. The limiting inward slope conductance was 28.3+/-1.7 pS (Vp was between 40 mV and 80 mV, n=6) and the outward chord conductance was 5.6+/-0.3 pS (Vp was between -40 and -60 mV, n=5). The open dwell time constants of the potassium channel were 0.524 ms and 5.087 ms, while the closed dwell time constants were 1.029 ms and 16.500 ms. The opening probability of the channel decreased when the extracellular fluid was acidified. Cyclosporine A had no significant effect on the potassium channel of the proximal tubular cell in the basolateral membrane at concentrations of 10 and 50 microg/ml, while at 100 microg/ml, it decreased the opening probability.

Direct inhibition of the pacemaker (If) current in rabbit sinoatrial node cells by genistein.

Genistein is a tyrosine kinase inhibitor which interferes with the activity of several ionic channels either by altering modulatory phosphorylating processes or by direct binding. In whole-cell conditions, genistein induces a partial inhibition of the pacemaker (I(f)) current recorded in cardiac sinoatrial and ventricular myocytes. We investigated the mechanism of action of genistein (50 microM) on the I(f) current in whole-cell, cell-attached, and inside-out configurations, and the measured fractional inhibitions were similar: 26.6, 27.2, and 33.6%, respectively. When ATP was removed from the whole-cell pipette solution no differences were revealed in the effect of the drug when compared to metabolically active cells. Genistein fully maintained its blocking ability even when herbimycin, a tyrosine kinase inhibitor, was added to the whole-cell ATP-free pipette solution. Genistein-induced block was independent of the gating state of the channel and did not display voltage or current dependence; this independence distinguishes genistein from all other f-channel blockers. When inside-out experiments were performed to test for a direct interaction with the channel, genistein, superfused on the intracellular side of the membrane, decreased the maximal I(f) conductance, and slightly shifted the current-activation curve to the left. Furthermore, the effect of genistein was independent of cAMP modulation. We conclude that, in addition to its tyrosine kinase-inhibitory properties, genistein also blocks I(f) by directly interacting with the channel, and thus cannot be considered a valuable pharmacological tool to investigate phosphorylation-dependent modulatory pathways of the I(f) current and of cardiac rhythm.

Expression and Modulation of the Intermediate- Conductance Ca2+-Activated K+ Channel in Glioblastoma GL-15 Cells

We report here the expression and properties of theintermediate-conductance Ca²⁺-activated K⁺ (IK_Ca)channel in the GL-15 human glioblastoma cell line.Macroscopic IK_Ca currents on GL-15 cells displayeda mean amplitude of 7.2±0.8 pA/pF at 0 mV, at day 1after plating. The current was inhibited by clotrimazole(CTL, IC₅₀=257 nM), TRAM-34 (IC₅₀=55 nM), andcharybdotoxin (CTX, IC₅₀=10.3 nM). RT-PCR analysisdemonstrated the expression of mRNA encodingthe IK_Ca channel in GL-15 cells. Unitary currents recordedusing the inside-out configuration had a conductanceof 25 pS, a K_D for Ca²⁺ of 188 nM at -100mV, and no voltage dependence. We tested whetherthe IKCa channel expression in GL-15 cells could bethe result of an increased ERK activity. Inhibition ofthe ERK pathway with the MEK antagonist PD98059(25 µM, for 5 days) virtually suppressed the IK_Ca currentin GL-15 cells. PD98059 treatment also increasedthe length of cellular processes and up-regulated theastrocytic differentiative marker GFAP. A significantreduction of the IKCa current amplitude was also observedwith time in culture, with mean currents of7.17±0.75 pA/pF at 1-2 days, and 3.11±1.35 pA/pFat 5-6 days after plating. This time-dependent downregulationof the IK_Ca current was not accompaniedby changes in the ERK activity, as assessed byimmunoblot analysis. Semiquantitative RT-PCR analysisdemonstrated a ~35% reduction of the IK_Ca channelmRNA resulting from ERK inhibition and a ~50%reduction with time in culture.

BAY 41‐2272, a potent activator of soluble guanylyl cyclase, stimulates calcium elevation and calcium‐activated potassium current in pituitary GH3 cells

The effects of BAY 41-2272, a nitric oxide-independent activator of soluble guanylyl cyclase, on Ca2+ signalling and ion currents were investigated in pituitary GH3 cells. Intracellular Ca2+ concentrations ([Ca2+]i) in these cells were increased by BAY 41-2272. Removing extracellular Ca2+ abolished the BAY 41-2272-induced increase in [Ca2+]i. After [Ca2+]i was elevated by BAY 41-2272 (300 nmol/L), subsequent application of 1-benzyl-3-(5'-hydroxymethyl-2'-furyl) indazole (YC-1; 1 micromol/L) did not increase [Ca2+]i further. In whole-cell recordings, BAY 41-2272 reversibly stimulated Ca2+-activated K+ current (I(K(Ca))) with an EC50 of 225 +/- 8 nmol/L. At 3 micromol/L, BAY 41-2272 slightly and significantly decreased L-type Ca2+ current. In the cell-attached configuration, BAY 41-2272 (300 nmol/L) enhanced the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels. After BK(Ca) channel activity was stimulated by spermine NONOate (30 micromol/L) or YC-1 (10 micromol/L) in cell-attached patches, subsequent application of BAY 41-2272 (300 nmol/L) further increased the channel open probability. In the inside-out configuration, BAY 41-2272 applied to the intracellular surface of excised patches enhanced BK(Ca) channel activity. Unlike 1 micromol/L paxilline, 1H-[1,2,4]oxadiazolol-[4,3a] quinoxalin-1-one (ODQ; 10 micromol/L) or heme (10 micromol/L) had no effect on BAY 41-2272-stimulated channel activity. BAY 41-2272 caused no shift in the activation curve of BK(Ca) channels; however, it did increase the Ca2+ sensitivity of these channels. At 300 nmol/L, BAY 41-2272 reduced the firing rate of spontaneous action potentials stimulated by thyrotropin-releasing hormone (10 micromol/L). The BK(Ca) channel activity was also enhanced by 300 nmol/L BAY 41-2272 in neuroblastoma IMR-32 cells. Therefore, the BAY 41-2272-induced increase in [Ca2+]i is primarily explained by an increase in Ca2+ influx. The BAY 41-2272-mediated simulation of IK(Ca) may result from direct activation of BKCa channels and indirectly as a result of elevated [Ca2+]i.

Glucocorticoids stimulate the activity of large-conductance Ca 2+-activated K + channels in pituitary GH 3 and AtT-20 cells via a non-genomic mechanism

The effects of glucocorticoids on ion currents were investigated in pituitary GH 3 and AtT-20 cells. In whole-cell configuration, dexamethasone, a synthetic glucocorticoid, reversibly increased the density of Ca 2+-activated K + current ( I K(Ca)) with an EC 50 value of 21 ± 5 μM. Dexamethasone-induced increase in I K(Ca) density was suppressed by paxilline (1 μM), yet not by glibenclamide (10 μM), pandinotoxin −Kα (1 μM) or mifepristone (10 μM). Paxilline is a blocker of large-conductance Ca 2+-activated K + (BK Ca) channels, while glibenclamide and pandinotoxin-Kα are blockers of ATP-sensitive and A-type K + channels, respectively. Mifepristone can block cytosolic glucocorticoid receptors. In inside-out configuration, the application of dexamethasone (30 μM) into the intracellular surface caused no change in single-channel conductance; however, it did increase BK Ca-channel activity. Its effect was associated with a negative shift of the activation curve. However, no Ca 2+-sensitiviy of these channels was altered by dexamethasone. Dexamethasone-stimulated channel activity involves an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, dexamethasone decreased the firing frequency of action potentials. In pituitary AtT-20 cells, dexamethasone (30 μM) also increased BK Ca-channel activity. Dexamethasone-mediated stimulation of I K(Ca) presented here that is likely pharmacological, seems to be not linked to a genomic mechanism. The non-genomic, channel-stimulating properties of dexamethasone may partly contribute to the underlying mechanisms by which glucocorticoids affect neuroendocrine function.

Stimulatory Actions of Thymol, a Natural Product, on Ca2+-Activated K+Current in Pituitary GH3Cells

Drugs that influence the opening of potassium (K(+)) channels and as a consequence cause hyperpolarization of cell membrane possess clinical potential. The large conductance Ca(2+)-activated K(+) (BK) channel is highly selective for K(+). Activation of this channel is Ca(2+)- and voltage-dependent. We have investigated the effects of thymol, a natural product, on ion currents in pituitary GH(3) cells. The patch-clamp technique was used to investigate the effect of thymol (100 microM) in these cells. Thymol reversibly stimulated the Ca(2+)-activated K(+) current with an EC (50) value of 75 microM. In a cell-attached configuration, application of thymol to the bath increased the activity of BK channels. BAPTA (1 mM) attenuated thymol-stimulated channel activity. In an experiment using the inside-out configuration, thymol exposed to the intracellular face of excised patches did not modify the single-channel conductance of these channels whereas it enhanced the channel activity. Neither menthol (100 microM) nor zingerone (100 microM) had an effect on BK-channel activity while AAPH (100 microM) suppressed it significantly. The stimulatory actions of thymol on Ca(2+)-activated K(+) currents may be associated with the underlying cellular mechanisms through which it affects neuronal or neuroendocrine functions.

Induction of Differentiation of Embryonic Stem Cells into Insulin-Secreting Cells by Fetal Soluble Factors

Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that the developing pancreas releases soluble factors responsible for in vitro endocrine pancreatic differentiation from embryonic stem cells (ESCs). A mouse D3 ESC line was transfected with a human insulin promoter/betageo/phosphoglycerate kinase-hygromycin-resistant construct. To direct differentiation, cells were cultured for 7 days to form embryoid bodies and then plated for an additional 7 days. During this 14-day period, besides eliminating leukemia inhibitory factor, cells were cultured in low serum concentration with the addition of conditioned media from embryonic day-16.5 pancreatic buds. Islet cell differentiation was studied by the following: (a) X-gal staining after neomycin selection, (b) BrdU (bro-modeoxyuridine) studies, (c) simple and double immunohistochemistry for insulin, C-peptide, and glucose transporter 2 (Glut-2), (d) reverse transcription-polymerase chain reaction for insulin and pancreas duodenum homeobox 1 (PDX-1), (e) insulin and C-peptide content and secretion assays, (f) intraperitoneal glucose tolerance test, (g) electrophysiology (patch-clamp studies in inside-out configuration), and (h) transplantation of differentiated cells under the kidney capsule of streptozotocin-diabetic mice. The differentiated ESCs showed the following: changes in the mRNA levels of insulin and PDX-1; coexpression of insulin, C-peptide, and Glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; and normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study, we establish a battery of techniques that could be used together to properly characterize islet cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental in engineering beta cells from stem cells.

In vivo targeting of ERG potassium channels in mice and dogs by a positron-emitting analogue of fluoroclofilium

The antiarrhythmic clofilium is an efficient blocker of hERG1 potassium channels that are strongly expressed in the heart. Therefore, derivatives of clofilium that emit positrons might be useful tools for monitoring hERG1 channels in vivo. Fluoro- clofilium (F-clofilium) was synthesized and its channel-blocking properties were determined for hERG1 and hEAG1 channels expressed in HEK?293 cells and in Xenopus oocytes. When applied extracellularly in the whole-cell patch-clamp configuration, F-cloflium exhibited a slower onset of block when compared with clofilium, presumably owing to its lower membrane permeability. When applied in the inside-out configuration at the intracellular membrane side, it blocked hEAG1 channels almost as efficiently as clofilium (IC50 1.37 nM and 0.83 nM, respectively). Similar results were obtained for hERG1, showing F-clofilium is a potent hERG1 and hEAG1 channel blocker once it has reached the intracellularly accessible target site at the channel. Using the (18)F-labeled analog we studied the in vivo binding and distribution of F-clofilium in mice and a dog. Greatest activity was found in kidneys and bones. A small but significant enrichment of activity in the dog myocardium known for its expression of cERG1 channels allowed to depict the myocardium of a living dog by PET. Thus, F-clofilium is a useful tool for imaging hERG channels in living organisms.

A store-operated nonselective cation channel in human lymphocytes.

1. Agonist interaction with phospholipase C-linked receptors at the plasma membrane can elicit both Ca2+ and Na+ influxes in lymphocytes. While Ca2+ influx is mediated by Ca2+ release-activated Ca2+ (CRAC) channels, the pathway responsible for Na+ influx is largely unknown. 2. We show that thapsigargin, ionomycin, ADP-ribose and IP3 activated a nonselective cation channel in lymphocytes that had a slightly outwardly rectifying I-V relationship, and a single channel conductance of 23.1 pS. We termed this channel a Ca2+ release-activated nonselective cation (CRANC) channel. 3. On activation in cell-attached configuration, switching to an inside-out configuration abolished CRANC channel activity. 4. Transfection of Jurkat T cells with antisense oligonucleotides for LTRPC2 reduced capacitative Ca2+ entry. 5. These results suggest that CRANC channels are responsible for the Na+ influx as well as a portion of the Ca2+ influx in lymphocytes induced by store depletion, that sustained activation of CRANC channels requires some property of the environment of a cell depleted of its Ca2+ stores; and that LTRPC2 protein is a likely component of the CRANC channel.

Mutation of the pore glutamate affects both cytoplasmic and external dequalinium block in the rat olfactory CNGA2 channel

Dequalinium has recently been reported to block CNGA1 and CNGA2 channels expressed in Xenopus laevis. Using the inside-out configuration of the patch-clamp technique, we examined the effects of dequalinium on rat olfactory CNGA2 channels expressed in human embryonic kidney (HEK293) cells and studied aspects of its molecular mechanism of action. We found that cytoplasmic dequalinium blocked wild-type (WT) CNGA2 channels in a voltage-dependent manner with an IC(50) of approximately 1.3 muM at a V(m) of + 60 mV, and an effective fractional charge, zdelta, of +0.8 (z=2, delta=+0.4), suggesting that cytoplasmic dequalinium interacts with a binding site that is about two fifths of the way along the membrane electric field (from the intracellular side). Neutralizing the negatively charged pore lining glutamate acid residue (E342Q) still allows effective channel block by cytoplasmic dequalinium with an IC(50) of approximately 2.2 muM at a V(m) of +60 mV but now having a zdelta of +0.1 (delta=+0.05), indicating a profoundly decreased level of voltage-dependence. In addition, by comparing the extent of block under different levels of channel activation, we show that the block by cytoplasmic dequalinium displayed clear state-dependence in WT channels by interacting predominantly with the closed channel, whereas the block in E342Q channels was state-independent. Application of dequalinium to the external membrane surface also blocked currents through WT channels and the E342Q mutation significantly increased the IC(50) for external block approximately fivefold. These results confirm dequalinium as a potent, voltage-dependent and state-dependent blocker of cyclic-nucleotide-gated channels, and show that neutralization of the E342 residue profoundly affects the block by both cytoplasmic and external application of dequalinium.

Microbial Passage in Low Pressure Membrane Elements with Compromised Integrity

The main objective of this study was to evaluate microbial passage through membrane elements with various levels of compromised integrity while installed within full-scale UF/MF systems. Challenge tests were performed for this purpose with the microbial contaminant surrogates Bacillus subtilis spores at two drinking water treatment plants. The experimental units used were one of several parallel racks part of each plant in which one of the elements was installed with various levels of compromised integrity achieved by precutting several hollow fibers and inserting removable pin-plugs atthe corresponding ends of the broken fiber segments. The UF rack was operated with 38 elements online (the rack has 50 elements) and was designed for an inside-out operation, and the MF rack included 50 elements and was operated in an outside-in mode (with a permeate outlet at one fiber end only). Spore removals observed for both the UF and MF racks with all precut fibers plugged were equal to or greater than 99.9992%, and as expected the removal efficiency deteriorated with an increasing number of unplugged fibers. Predictions made with a model based on the use of the Hagen-Poisseuille equation for laminar flow and the Darcy-Weisbach expression for turbulent flow inside broken fibers were found to provide an adequate conservative representation of experimental results. Additional simulations performed with the verified model revealed the occurrence of a greater microbial passage for an inside-out configuration compared to an outside-in mode. A lower microbial passage was predicted for the outside-in element configuration with one permeate outlet as compared to an element with permeate outlets at both fiber ends. The model offers a useful tool that together with other considerations such as membrane fouling, cleaning, and durability would assist in the selection of low-pressure membrane element configuration.

Regulation of the Ca2+ Sensitivity of the Nonselective Cation Channel TRPM4

TRPM4, a Ca(2+)-activated cation channel of the transient receptor potential superfamily, undergoes a fast desensitization to Ca(2+). The mechanisms underlying the alterations in Ca(2+) sensitivity are unknown. Here we show that cytoplasmic ATP reversed Ca(2+) sensitivity after desensitization, whereas mutations to putative ATP binding sites resulted in faster and more complete desensitization. Phorbol ester-induced activation of protein kinase C (PKC) increased the Ca(2+) sensitivity of wild-type TRPM4 but not of two mutants mutated at putative PKC phosphorylation sites. Overexpression of a calmodulin mutant unable to bind Ca(2+) dramatically reduced TRPM4 activation. We identified five Ca(2+)-calmodulin binding sites in TRPM4 and showed that deletion of any of the three C-terminal sites strongly impaired current activation by reducing Ca(2+) sensitivity and shifting the voltage dependence of activation to very positive potentials. Thus, the Ca(2+) sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation, and calmodulin binding at the C terminus.