Cell-attached Configuration Research Articles

Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na(+). A His residue within the extracellular domain of gammaENaC (gammaHis(239)) was found to have a critical role in Na(+) self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na(+) self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na(+) self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na(+) self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the gammaH239F mutant showed that, in the absence of Na(+) self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that gammaHis(239) functions within an extracellular allosteric regulatory subdomain of the gamma subunit that has an important role in conferring the response of the channel to external Na(+).

Probing Pore Constriction in a Ligand-gated Ion Channel by Trapping a Metal Ion in the Pore upon Agonist Dissociation

Eukaryotic pentameric ligand-gated ion channels (pLGICs) are receptors activated by neurotransmitters to rapidly transport ions across cell membranes, down their electrochemical gradients. Recent crystal structures of two prokaryotic pLGICs were interpreted to imply that the extracellular side of the transmembrane pore constricts to close the channel (Hilf, R. J., and Dutzler, R. (2009) Nature 457, 115-118; Bocquet, N., Nury, H., Baaden, M., Le Poupon, C., Changeux, J. P., Delarue, M., and Corringer, P. J. (2009) Nature 457, 111-114). Here, we utilized a eukaryotic acetylcholine (ACh)-serotonin chimeric pLGIC that was engineered with histidines to coordinate a metal ion within the channel pore, at its cytoplasmic side. In a previous study, the access of Zn(2+) ions to the engineered histidines had been explored when the channel was either at rest (closed) or active (open) (Paas, Y., Gibor, G., Grailhe, R., Savatier-Duclert, N., Dufresne, V., Sunesen, M., de Carvalho, L. P., Changeux, J. P., and Attali, B. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 15877-15882). In this study, the interactions of Zn(2+) with the pore were probed upon agonist (ACh) dissociation that triggers the transition of the receptor from the active conformation to the resting conformation (i.e. during deactivation). Application of Zn(2+) onto ACh-bound open receptors obstructed their pore and prevented ionic flow. Removing ACh from its extracellular binding sites to trigger deactivation while Zn(2+) is still bound led to tight trapping of Zn(2+) within the pore. Together with single-channel recordings, made to explore single pore-blocking events, we show that dissociation of ACh causes the gate to shut on a Zn(2+) ion that effectively acts as a "foot in the door." We infer that, upon deactivation, the cytoplasmic side of the pore of the ACh-serotonin receptor chimera constricts to close the channel.

Mechanism of verapamil action on wild‐type and slow‐channel mutant human muscle acetylcholine receptor

Verapamil, a Ca(2+) channel blocker widely used in clinical practice, also affects the properties of frog and mouse muscle acetylcholine receptor (AChR). Here, we examine the mechanism of action of verapamil on human wild-type and slow-channel mutant muscle AChRs harboring in any subunit a valine-to-alanine mutation of 13' residue of the pore-lining M2 transmembrane segment. Verapamil, after a pre-treatment of 0.5-10 s, accelerated the decay of whole-cell or macroscopic outside-out currents within milliseconds of ACh application even at clinically attainable doses. Recordings of unitary events in the cell-attached and outside-out configurations showed that verapamil does not alter single-channel conductance, but reduces channel open probability, by prolonging the dwell time into the closed state for wild-type and all mutant AChR. The duration of channel openings decreased only for the epsilonV265A-AChR, by shortening the longest exponential component of the open-time distribution. These results provide a rationale for the therapeutic use of verapamil in the slow-channel syndrome and emphasize the major role played by epsilon subunit in controlling the functional properties of human muscle AChR, as revealed by the peculiar alterations imparted by mutations in this subunit.

Interneurons of the cerebellar cortex toggle Purkinje cells between up and down states

We demonstrate that single interneurons can toggle the output neurons of the cerebellar cortex (the Purkinje cells) between their two states. The firing of Purkinje cells has previously been shown to alternate between an "up" state in which the cell fires spontaneous action potentials and a silent "down" state. We show here that small hyperpolarizing currents in Purkinje cells can bidirectionally toggle Purkinje cells between down and up states and that blockade of the hyperpolarization-activated cation channels (H channels) with the specific antagonist ZD7288 (10 microM) blocks the transitions from down to up states. Likewise, hyperpolarizing inhibitory postsnyaptic potentials (IPSPs) produced by small bursts of action potentials (10 action potentials at 50 Hz) in molecular-layer interneurons induce these bidirectional transitions in Purkinje cells. Furthermore, single interneurons in paired interneuron --> Purkinje cell recordings, produce bidirectional switches between the two states of Purkinje cells. The ability of molecular-layer interneurons to toggle Purkinje cells occurs when Purkinje cells are recorded under whole-cell patch-clamp conditions as well as when action potentials are recorded in an extracellular loose cell-attached configuration. The mode switch demonstrated here indicates that a single presynaptic interneuron can have opposite effects on the output of a given Purkinje cell, which introduces a unique type of synaptic interaction that may play an important role in cerebellar signaling.

Activity-independent intracellular signaling contributes to rate variability among neurons in the globus pallidus.

Synchrony among neurons is seen throughout the mammalian CNS, in particular within networks of GABAergic neurons. The globus pallidus (GP) consists of GABAergic neurons that project to neurons throughout the basal ganglia in addition to each other via local axon collaterals. The GP does not display synchronous rhythms and there are no clear correlations between neurons in healthy mammals. In contrast, strong correlations among GP neurons emerge in Parkinson’s disease patients and MPTP treated primates. Why GP neurons show no correlations in healthy animals is not known. GP neurons are autonomous pacemakers and are thought to discharge at a constant frequency. Theoretical work shows that the degree of synchrony in interconnected networks depends on the amount of heterogeneity in firing rate. We have observed in cell-attached recordings of GP neurons, in slices, that their firing rates are not stationary, but instead visit a range of rates over a period of one hour in a non-stereotypical fashion. The fluctuations in rate are stable for periods of minutes, can vary as much as 30 Hz, and appear to be unaffected by blocking synaptic inputs. HCN channels are a critical component of the GP neuron’s pacemaking mechanism, and their availability is positively correlated with the firing rate of GP neurons. Paired recordings revealed that these fluctuations in rate were neuron-independent. We hypothesized that these slow non-stereotypical fluctuations in firing rate could be attributed to regulation of HCN channels by cAMP. cAMP alters HCN availability by shifting the channel’s activation curve to depolarized potentials. Consistent with this, up-regulation of cAMP levels by bath application of 50 μM Forskolin increased the firing rate of GP neurons from 12.7 ± 1.3 to 20.5 ± 1.5 Hz. After the Forskolin induced increase in firing rate stabilized, there was a marked decrease in the amount of variability in interspike intervals (ISIs) observed over a one hour period of recording in the cell-attached configuration. In control conditions the average coefficient of variation of ISIs (CV), measured over one hour, was 0.43 ± 0.22 and was 0.09 ± 0.02 after exposure to Forskolin. Thus, fluctuations in cAMP can create heterogeneity in firing rate among GP neurons by altering the availability of the voltage-gated ion channels necessary to drive pacemaking. By this mechanism, activity independent intracellular signaling could potentially contribute to the absence of synchrony in the GP.

A Novel Way to Go Whole Cell in Patch-Clamp Experiments

With a conventional patch-clamp electrode, an Ag/AgCl wire sits stationary inside the pipette. To move from the gigaseal cell-attached configuration to whole-cell recording, suction is applied inside the pipette. We have designed and developed a novel Pushpen patch-clamp electrode, in which a W wire insulated and wound with Ag/AgCl wire can move linearly inside the pipette. The W wire has a conical tip, which can protrude from the pipette tip like a push pen, a procedure we call the Pushpen Operation. We use the Pushpen operation to impale the cell membrane in cell-attached configuration to go whole-cell without disruption of the gigaseal. We successfully recorded whole-cell currents from chinese hamster ovarian cells expressing influenza A virus protein A/M2, after obtaining whole-cell configuration with the Pushpen operation. This novel method of achieving whole-cell configuration may have a higher success rate than is the case with the conventional patch clamp technique.

Single mechano‐gated channels activated by mechanical deformation of acutely isolated cardiac fibroblasts from rats

Mechanosensitive conductances were reported in cardiac fibroblasts, but the properties of single channels mediating their mechanosensitivity remain uncharacterized. The aim of this work was to investigate single mechano-gated channels (MGCs) activated by mechanical deformations of cardiac fibroblasts. Currents through single MGCs and mechanosensitive whole-cell currents were recorded from isolated rat atrial fibroblasts using the cell-attached and whole-cell patch-clamp configurations respectively. Defined mechanical stress was applied via the patch pipette used for the whole-cell recordings. Under resting conditions occasional short openings of two types of single MGCs with conductances of 43 and 87 pS were observed. Both types of channels displayed a linear current-voltage relationship with the reversal potential around 0 mV. Small (1 microm) mechanical deformations affected neither single nor whole-cell mechano-gated currents. Cell compressions (2, 3 and 4 microm) augmented the whole-cell currents and increased the frequency and duration of single channel openings. Cell stretches (2, 3 and 4 microm) inactivated the whole-cell currents and abolished the activity of single MGCs. Gd(3+) (8 microm) blocked the whole-cell currents within 5 min. No single channel activity was observed in the cell-attached mode when Gd(3+) was added to the intrapipette solution. Cytochalasin D and colchicine (100 microm each) completely blocked both the whole-cell and single channel currents. These findings show that rat atrial fibroblasts express two types of MGCs whose activity is governed by cell deformation. We conclude that fibroblasts can sense the direction of applied stress and contribute to mechano-electrical coupling in the heart.

Voltage‐gated chloride channels in storage cells of the anhydrobiotic eutardigrade Richtersius coronifer

Tardigrades comprise polyextremophilic species that can survive in an anhydrobiotic state for years. Anhydrobiosis may be dependent on properties of the free floating storage cells found in the body cavity of all tardigrades. We studied ion channels in the storage cell membrane with patch clamp technique, using native cells from the eutardigrade Richtersius coronifer.In cell‐attached configuration the most frequently encountered ion channel had a linear i/V‐relationship with a reversal potential (Vrev) of ~0.4 mV (n ≥6). The single‐channel conductance was 82.4±0.0 pS (mean±SE, n≥6). Binomial analysis of patches with 2–4 active channels showed stochastic behavior. The open‐state probability increased with the pipette potential, indicating voltage‐gating. In whole‐cell configuration the currents showed outward rectification with Vrev = −18.2±1.1 mV (mean±SE, n=24), closest to the equilibrium potential for Cl−. Perfusion with Na‐gluconate Ringer right‐shifted Vrev from −19.5±1.9 to 4.6±1.3 mV, and the current decreased significantly (mean±SE, n=5), confirming that a significant component of the whole‐cell current is attributed to Cl− channels. This was verified by perfusion with KCl Ringer (n=5) and addition of BaCl2 (n=7), neither of which significantly changed Vrev. Future studies are needed to firmly establish the identity and function of the demonstrated ion channel in storage cells.

Anion conductance of the human red cell is carried by a maxi-anion channel

Historically, the anion transport through the human red cell membrane has been perceived to be mediated by Band 3, in the two-component concept with the large electroneutral anion exchange accompanied by the conductance proper, which dominated the total membrane conductance. The status of anion channels proper has never been clarified, and the informations obtained by different groups of electrophysiologists are rather badly matched. This study, using the cell-attached configuration of the patch-clamp technique, rationalizes and explains earlier confusing results by demonstrating that the diversity of anionic channel activities recorded in human erythrocytes corresponds to different kinetic modalities of a unique type of maxi-anion channel with multiple conductance levels and probably multiple gating properties and pharmacology, depending on conditions. It demonstrates the role of activator played by serum in the recruitment of multiple new conductance levels showing very complex kinetics and gating properties upon serum addition. These channels, which seem to be dormant under normal physiological conditions, are potentially activable and could confer a far higher anion conductance to the red cell than the ground leak mediated by Band 3.

Biophysical characterisation of the persistent sodium current of the Nav1.6 neuronal sodium channel: a single-channel analysis

Na(v)1.6 is the major voltage-gated sodium channel at nodes of Ranvier. This channel has been shown to produce a robust persistent inward current in whole-cell experiments. Na(v)1.6 plays an important role in axonal conduction and may significantly contribute to the pathophysiology of the injured nervous system through this persistent current. However, the underlying molecular mechanisms and regulation of the persistent current are not well understood. Using the whole-cell configuration of the patch-clamp technique, we investigated the Na(v)1.6 transient and persistent currents in HEK-293. Previous studies have shown that the persistent current depended on the content of the patch electrode. Therefore, we characterised the single-channel properties of the persistent current with an intact intracellular medium using the cell-attached configuration of the patch-clamp technique. In HEK-293 cells, the Na(v)1.6 persistent current recorded in the whole-cell configuration was 3-5% of the peak transient current. In single-channel recording, the ratio between peak and persistent open probability confirmed the magnitude of the persistent current observed in the whole-cell configuration. The cell-attached configuration revealed that the molecular mechanism of the whole-cell persistent current is a consequence of single Na(v)1.6 channels reopening.

Ethanol Stabilizes the Open State of Single 5-Hydroxytryptamine3A(QDA) Receptors

Ethanol enhancement of 5-hydroxytryptamine (5-HT)(3A) receptor-mediated responses may have important consequences in the intoxicating and addictive properties of ethanol. Although the exact mechanism is unknown, ethanol-mediated enhancement of 5-HT(3) receptor current has been proposed to occur due to stabilization of the open-channel state. It has not been possible to directly measure the open state of the channel due to the extremely low single-channel conductance of 5-HT(3A) channels. Recently, three arginine residues within the large intracellular loop of the 5-HT(3A) subunit were substituted by their equivalent residues (glutamine, aspartate, and alanine) of the 5-HT(3B) subunit to produce a 5-HT(3A)(QDA) subunit that forms functional homomeric channels exhibiting a measurable single-channel conductance. Using whole-cell rapid-agonist application techniques and the cell-attached single-channel recording configuration, we examined human 5-HT(3A)(QDA) receptors expressed in human embryonic kidney 293 cells. The agonist sensitivity, macroscopic kinetics, and modulation by ethanol were similar between mutant and wild-type channels, suggesting the substitutions had not altered these channel structure-function properties. The open time histogram for single-channel events mediated by 5-HT(3A)(QDA) receptors in the presence of maximal 5-HT was best fit by three exponentials, but in the presence of ethanol a fourth open state was evident. In summary, the QDA substitution greatly enhanced single-channel conductance with little effect on 5-HT(3A) channel's kinetic properties and ethanol enhances agonist action on 5-HT(3A) receptors by inducing a new, long-lived open-channel state. Furthermore, the 5-HT(3A)(QDA) receptor appears to be suitable for pharmacological studies of 5-HT(3A) receptor modulation at a single-channel level.

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

BackgroundThe mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.Methodology/Principal FindingsThe cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents.Conclusions/SignificanceThe stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.

Blocking actions of glibenclamide on ATP-sensitive K+ channels in pig urethral myocytes.

The inhibitory effects of glibenclamide on the levcromakalim-induced ATP-sensitive K+ (K(ATP)) channels were investigated with cell-attached configuration in pig proximal urethra. Application of 10 microM glibenclamide reversibly inhibited the activity of the 100 microM levcromakalim-induced K(ATP) channel, decreasing not only the channel open probability but also the amplitude of unitary current. The inhibitory concentration-response curve of the glibenclamide-induced sublevel conductance of K(ATP) channel was shifted to the right (IC50 = 4.7 microM), compared with the levcromakalim-induced K(ATP) channel (full conductance, IC50 = 0.5 microM). Glibenclamide is the first reported sulphonylurea to selectively block K(ATP) channel, not only by decreasing the channel activity but also by reducing the unitary amplitude in smooth muscle.

The Activation Mechanism of the Rat Homomeric alpha2 Glycine Receptor

The alpha2 glycine receptor subunit, abundant in embryonic neurons, is replaced by alpha1 in the adult nervous system. Glycine-activated single-channel currents were recorded in the cell-attached configuration at +100 mV pipette holding potential from alpha2 homomeric receptors expressed in HEK293. At all glycine concentrations (0.02 - 10 mM), openings were grouped in long (> 300 ms) bursts/clusters with high open probability (0.99). The mean duration of individual apparent openings was c. 30 ms. Shut-time intervals within groups of openings were dominated by short shuttings of 5-10 μs. The properties of these groups of openings appeared to have an unusually steep concentration dependence and it is unclear whether they represent receptor activations (i.e. bursts) or clusters. Several mechanisms were fitted by maximising the likelihood of the entire sequence of open and shut times, with exact allowance for missed events (program HJCFIT, ref.1). Several records obtained at different glycine concentrations were fitted simultaneously. Good fits were obtained with several qualitatively different mechanisms incorporating 2 or 3 binding sites. In fits with the flip mechanism (2), we found that the closing rate constant of alpha2 glycine receptors is slow (about 500 s-1) and thus the efficacy for the final opening step in the activation is >10 fold higher than that of alpha1 beta channels, the receptor in adult synapses. On the other hand, flipping efficacy and binding affinity were lower for alpha2 than for alpha1 beta channels. These differences confirm that the alpha2 glycine receptor properties make it less suitable than alpha1 beta channels to mediate fast synaptic transmission (3). 1. Colquhoun et al. (2003) J Physiol 547, 699-728. 2. Burzomato et al. (2004) J Neurosci 24, 10924-10940. 3. Mangin et al. (2003) J Physiol 553, 369-386.

Opposing effects of tenidap on the volume-regulated anion channel and K ATP channel activity in rat pancreatic β-cells

Tenidap (5-chloro-2-hydroxy-3-(thiophene-2-carbonyl)indole-1-carboxamide) is a non-steroidal anti-inflammatory and anti-rheumatic drug with several cellular actions including inhibition of anion transport processes. Since other anion transport inhibitors have been shown to inhibit activity of the volume-regulated anion channel (VRAC), the present study investigated the effects of tenidap on activity of this channel in pancreatic β-cells. Membrane potential, VRAC currents and input conductance were recorded from single rat β-cells in primary culture using perforated patch, conventional whole-cell and cell-attached configurations of the patch-clamp technique. Relative cell volume was measured using a video-imaging method. Tenidap (0.1 mM) was found to rapidly hyperpolarise the β-cell membrane potential and terminate glucose-induced electrical activity. This effect was associated with a pronounced outward current shift at a holding potential of − 65 mV. Tenidap was found to inhibit activity of the volume-regulated anion channel with IC 50 values of 31 and 43 µM for outward and inward currents respectively. Tenidap also markedly increased β-cell input conductance, representing an activation of the K ATP conductance. β-cell regulatory volume decrease following hypotonically-induced cell swelling was sensitive to inhibition by 50 µM tenidap. Tenidap is a potent inhibitor of the volume-regulated anion channel and K ATP channel activator in rat pancreatic β-cells. These actions could at least in part explain the recently reported inhibitory actions of the drug on electrical and secretory activity in the β-cell, and could also underlie other pharmacological actions of the drug.

Multiple levels of the single L-type Ca2+ channel conductance in adult mammalian ventricular myocytes

In cardiac muscle, Ca2+ entry through the L-type Ca2+ channel during excitation triggers Ca2+ release from sarcoplasmic reticulum (SR) via the ryanodine receptor, causing muscle contraction. This Ca2+ induced Ca2+ release (CICR) is locally controlled by openings of the single L-type Ca2+ channel that triggers the opening of ryanodine receptors located within the dyadic junction. The unitary current through the single Ca2+ channel determines the open probability of the ryanodine receptors, and hence the efficacy of CICR. Here, we report findings of multiple conductance levels of L-type Ca2+ channels in freshly isolated rat ventricular myocytes. When 10mM Ca2+ was used in the pipette solution as the charge carrier in the cell-attached configuration, the most frequently occurring conductance levels are 6.9pS and 2.9pS. Three distinct conductance levels were also observed, although infrequently, in the same cell, corresponding to unitary currents of 0.33pA, 0.24pA, and 0.17pA upon depolarization to −10mV. In conclusion, our data demonstrate the existence of multiple single L-type Ca2+ channel conductance levels in the adult mammalian ventricular myocytes with Ca2+ as the charge carrier. The multiple conductance levels present heterogeneity in the Ca2+ trigger signal strength at local dyadic junctions. The physiological significance of this heterogeneity on affecting the efficacy of CICR in the cardiac muscle is discussed.

Mechanisms of Impaired Glucose Tolerance and Insulin Secretion during Isoflurane Anesthesia

Volatile anesthetics impair insulin secretion and glucose utilization; however, the precise mechanism of action that underlies these effects is unknown. The authors hypothesized that isoflurane inhibits glucose-induced inhibition of adenosine triphosphate-sensitive potassium channel activity in pancreatic beta cells, which could result in impaired insulin secretion and glucose tolerance. Intravenous glucose tolerance tests were performed on 28 male Japanese White rabbits anesthetized with sodium pentobarbital. Glibenclamide (50 microg/kg + 33.5 microg x kg x h) or vehicle was administered 75 min before intravenous administration of 0.6 g/kg glucose. Half of the animals (n = 7) in the vehicle and glibenclamide groups received isoflurane at 1.0 minimum alveolar concentration 30 min before administration of glucose, and the other half received a vehicle control. Hemodynamics, blood glucose, and plasma insulin were measured. A cell-attached patch clamp configuration was used to record single channel currents in the pancreas from male Swiss-Webster mice. Isoflurane alone or a combination of isoflurane and glibenclamide inhibited the insulinogenic index to a greater extent than in the vehicle and glibenclamide groups. In the patch clamp experiments, channel activity was significantly decreased as the glucose concentration was increased from 0 to 10 mm. The subsequent application of 0.5 mm isoflurane reversed the effects of glucose on channel activity. These results show that isoflurane impairs insulin secretion and glucose utilization. The mechanism of action responsible for these effects may involve a decrease in glucose-induced inhibition of adenosine triphosphate-sensitive potassium channel activity in pancreatic beta cells.

Augmented bladder urothelial polyamine signaling and block of BK channel in the pathophysiology of overactive bladder syndrome

Overactive bladder syndrome (OAB) is an idiopathic condition characterized by urinary urgency and urge incontinence. Detrusor overactivity has been traditionally described as the physiologic mechanism for OAB. However, the bladder urothelium (BU) may also be involved in the pathophysiology. This study measured polyamine signaling and its downstream effects on membrane conductivity in bladder urothelial cells (BUC) obtained from asymptomatic and OAB subjects. Immunohistofluorescence was used to measure ornithine decarboxylase (ODC) expression in BU. BUC, cultured from BU biopsies, were used for electrophysiologic studies. dl-alpha-Difluoromethylornithine (DFMO), spermine, or spermidine was used to modulate polyamine signaling in BUC. Results showed ODC overexpression in OAB BU. In OAB BUC, whole cell and cell-attached configuration showed significantly decreased currents. Using inside-out patches, outward currents increased significantly, suggesting a cytoplasmic source of the outward current block in OAB BUC. In control BUC, outward currents were mediated by the large-conductance calcium-activated potassium (BK) channel due to calcium dose-dependence and block by iberiotoxin. Spermidine and spermine blocked the outward current in normal BUC in dose-dependent fashion. Conversely, DFMO significantly increased (P < 0.01) outward currents in OAB BUC both in cell-attached and in whole cell configuration. The outward currents in DFMO-treated-OAB BUC could be significantly reduced (P < 0.05) by adding back spermidine and spermine. These data suggest that polyamine signaling is upregulated in OAB urothelium and OAB BUC. Furthermore, polyamines in BUC block the BK channel. Targeting of bladder urothelial polyamine signaling may represent a novel approach for OAB treatment based on pathophysiologic mechanisms.

Enhancement of BK Ca channel activity induced by hydrogen peroxide: Involvement of lipid phosphatase activity of PTEN

Large-conductance calcium and voltage-dependent potassium (BK Ca) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H 2O 2) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hβ1 were cotransfected with the PTEN wt, PTEN C124S, PTEN G129E in HEK 293 cells. Typical BK Ca channel currents could be recorded in cell-attached configurations. We found that PTEN wt reduced the H 2O 2-induced BK Ca channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTEN C124S/PTEN G129E mutants that lack lipid phosphatase activity produced no regulation on the H 2O 2-induced BK Ca channel activation. These results demonstrated that PTEN regulated the H 2O 2-induced BK Ca channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H 2O 2-induced BK Ca channel activation was attenuated when cells were treated with H 2O 2 at concentrations higher than 100 μM or at 100 μM for long-term treatment. In addition, the p-AKT expression level in PTEN wt overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca 2+] i) induced by H 2O 2 was also inhibited. These findings may elucidate a new mechanism for H 2O 2-induced BK Ca channel activation and provide some evidences for the role of PTEN on vasodilation induced by H 2O 2.

Activity-Dependent Control of Neuronal Output by Local and Global Dendritic Spike Attenuation

Neurons possess elaborate dendritic arbors which receive and integrate excitatory synaptic signals. Individual dendritic subbranches exhibit local membrane potential supralinearities, termed dendritic spikes, which control transfer of local synaptic input to the soma. Here, we show that dendritic spikes in CA1 pyramidal cells are strongly regulated by specific types of prior input. While input in the linear range is without effect, supralinear input inhibits subsequent spikes, causing them to attenuate and ultimately fail due to dendritic Na(+) channel inactivation. This mechanism acts locally within the boundaries of the input branch. If an input is sufficiently strong to trigger axonal action potentials, their back-propagation into the dendritic tree causes a widespread global reduction in dendritic excitability which is prominent after firing patterns occurring in vivo. Together, these mechanisms control the capability of individual dendritic branches to trigger somatic action potential output. They are invoked at frequencies encountered during learning, and impose limits on the storage and retrieval rates of information encoded as branch excitability.