Voltage Clamp

The effect of ethylenediamine derivatives of tetrodotoxin (enTTXI and enTTXn) on frog skeletal muscle was studied both electrophysiologically and biochemically. Electrophysiological experiments with one of these molecules (enTTX~) showed that the concentrations needed to block the early phase of the inward sodium current (Ko.5 = 7 nM) are much lower than those needed to block the late phase of inward current or muscle contraction (K0. 5 = 40nM). Conversely, tubular Na + channels are more sensitive to enTTXn than are surface Na + channels.

Cardiac contractile dysfunction is frequently reported in human patients and experimental animals with type-1 diabetes mellitus. The aim of this study was to investigate the voltage-dependence of contraction in ventricular myocytes from the streptozotocin (STZ)-induced diabetic rat. STZ-induced diabetes was characterised by hyperglycaemia and hypoinsulinaemia. Other characteristics included reduced body and heart weight and raised blood osmolarity. Isolated ventricular myocytes were patched in whole cell, voltage-clamp mode after correcting for membrane capacitance and series resistance. From a holding membrane potential of −40 mV, test pulses were applied at potentials between −30 and +50 mV in 10 mV increments. L-type Ca 2+ current (I Ca,L ) density and contraction was measured simultaneously using a video-edge detection system. Membrane capacitance was not significantly altered between control and STZ-induced diabetic myocytes. The I Ca,L density was significantly (p < 0.05) reduced throughout voltage ranges (−10 mV to +10 mV) in myocytes from STZ-treated rats compared to age-matched controls. Moreover, the amplitude of contraction was significantly reduced (p < 0.05) in myocytes from STZ-treated rats at all test potentials between −20 mV and +30 mV. However, in electrically field-stimulated (1 Hz) myocytes, the amplitude of contraction was not altered by STZ-treatment. It is suggested that in field-stimulated myocytes taken from STZ-induced diabetic hearts, prolonged action potential duration may promote increased Ca 2+ influx via the sodium-calcium exchanger (NCX), which may compensate for a reduction in Ca 2+ trigger through L-type-Ca 2+ -channels and lead to normalised contraction. (Mol Cell Biochem xxx: 1-9, 2004) 7

Rapid, sequence-specific DNA detection is essential for applications in medical diagnostics and genetic screening. Electrical biosensors that use immobilized nucleic acids are especially promising in these applications because of their potential for miniaturization and automation. Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available.

In this paper, the nonlinear dynamical phenomenon associated with a silicon neuron are described. The neuron has one transient sodium (activating and inactivating) channel and one activating potassium channel. These channels do not model specific equations; instead they directly mimic the desired voltage clamp responses. This allows us to create silicon structures that are very compact (six transistors and three capacitors) with activation and inactivation parameters being tuned by floating-gate (FG) transistors. Analysis of the bifurcation conditions allow us to identify regimes in the parameter space that are desirable for biasing the circuit. We show a subcritical Hopf-bifurcation which is characteristic of class 2 excitability in Hodgkin-Huxley (H-H) neurons. We also show a Hopf bifurcation at higher values of stimulating current, a phenomenon also observed in real neurons and termed excitation block. The phenomenon of post-inhibitory rebound and frequency preference are displayed and intuitive explanations based on the circuit are provided. The compactness and low-power nature of the circuit shall allow us to integrate a large number of these neurons on a chip to study complicated network behavior.

In this paper, a new computer software package, Yalzer, is introduced for simulating single-compartmental model of neurons. Passive or excitable membranes with voltage-gated ion channels can be modeled, and current clamp and voltage clamp experiments can be simulated. In the Yalzer, first-order differential equations used to define the dynamics of the gate variables and the membrane potential are solved by two separate integration methods with variable time steps: forward Euler and exponential Euler methods. Outputs of the simulation are shown on a spreadsheet template for allowing flexible data manipulation and can be graphically displayed. The user can define the model in detail, and examine the excitability of the model and the dynamics of voltage-gated ion channels. The software package addresses to ones who want to run simple simulations of neurons without need to any programming language skills or expensive software. It can also be used for educational purposes.

The specific membrane capacitance (Cm) of a neuron influences synaptic efficacy and determines the speed with which electrical signals propagate along dendrites and unmyelinated axons. The value of this important parameter remains controversial. In this study, Cm was estimated for the somatic membrane of cortical pyramidal neurons, spinal cord neurons, and hippocampal neurons. A nucleated patch was pulled and a voltage-clamp step was applied. The exponential decay of the capacitative charging current was analyzed to give the total membrane capacitance, which was then divided by the observed surface area of the patch. Cm was 0.9 μF/cm2 for each class of neuron. To test the possibility that membrane proteins may alter Cm, embryonic kidney cells (HEK-293) were studied before and after transfection with a plasmid coding for glycine receptor/channels. The value of Cm was indistinguishable in untransfected cells and in transfected cells expressing a high level of glycine channels, indicating that differences in transmembrane protein content do not significantly affect Cm. Thus, to a first approximation, Cm may be treated as a “biological constant” across many classes of neuron.

Neonicotinoid insecticides are compounds acting agonistically on insect nicotinic acetylcholine receptors (nAChR). They are especially active on hemipteran pest species such as aphids, whiteflies, and planthoppers, but also commercialized to control many coleopteran and some lepidopteran pest species. The most prominent member of this class of insecticides is imidacloprid. All neonicotinoid insecticides bind with high affinity (I 50 -values around 1 nM) to [ 3 H]imidacloprid binding sites on insect nAChRs. One notable ommission is the neonicotinoid thiamethoxam, showing binding affinities up to 10,000-fold less potent than the others, using housefly head membrane preparations. Electrophysiological whole cell voltage clamp studies using neurons isolated from Heliothis virescens ventral nerve cord showed no response to thiamethoxam when applied at concentrations of 0.3 mM, although the symptomology of poisoning in orally and topically treated noctuid larvae suggested strong neurotoxicity. Other neonicotinoids, such as clothianidin, exhibited high activity as agonists on isolated neurons at concentrations as low as 30 nM. There was no obvious correlation between biological efficacy of thiamethoxam against aphids and lepidopterans and receptor affinity in electrophysiological and binding assays. Pharmacokinetic studies using an LC-MS/MS approach to analyze haemolymph samples taken from lepidopteran larvae revealed that thiamethoxam orally applied to 5th instar Spodoptera frugiperda larvae was rapidly metabolized to clothianidin, an open-chain neonicotinoid. Clothianidin shows high affinity to nAChRs in both binding assays and whole cell voltage clamp studies. When applied to cotton plants, thiamethoxam was also quickly metabolized, with clothianidin being the predominant neonicotinoid in planta briefly after application, as indicated by LC-MS/MS analyses. Interestingly, the N-desmethylated derivative of thiamethoxam, N-desmethyl thiamethoxam, was not significantly produced in either lepidopteran larvae or in cotton plants, although it was often mentioned as a possible metabolite, being nearly as active as imidacloprid. In conclusion, our investigations show that thiamethoxam is likely to be a neonicotinoid precursor for clothianidin.

Preceded by a companion paper on Galvani's life, this article is written on the occasion of the bicentenary of the death of Luigi Galvani. From his studies on the effects of electricity on frogs, the scientist of Bologna derived the hypothesis that animal tissues are endowed with an intrinsic electricity that is involved in fundamental physiological processes such as nerve conduction and muscle contraction. Galvani's work swept away from life sciences mysterious fluids and elusive entities like "animal spirits" and led to the foundation of a new science, electrophysiology. Two centuries of research work have demonstrated how insightful was Galvani's conception of animal electricity. Nevertheless, the scholar of Bologna is still largely misrepresented in the history of science, because the importance of his researches seems to be limited to the fact that they opened the paths to the studies of the physicist Alessandro Volta, which culminated in 1800 with the invention of the electric battery. Volta strongly opposed Galvani's theories on animal electricity. The matter of the scientific controversy between Galvani and Volta is examined here in the light of two centuries of electrophysiological studies leading to the modern understanding of electrical excitability in nerve and muscle. By surveying the work of scientists such as Nobili, Matteucci, du Bois-Reymond, von Helmholtz, Bernstein, Hermann, Lucas, Adrian, Hodgkin, Huxley, and Katz, the real matter of the debate raised by Galvani's discoveries is here reconsidered. In addition, a revolutionary phase of the 18th century science that opened the way for the development of modern neurosciences is reevaluated.

Abstruct-Multilevel voltage source converters are emerging as a new breed of power converter options for high-power applications. The multilevel voltage source converters typically synthesize the staircase voltage wave from several levels of dc capacitor voltages. One of the major limitations of the multilevel converters is the voltage unbalance between different levels. The techniques to balance the voltage between different levels normally involve voltage clamping or capacitor charge control. There are several ways of implementing voltage balance in multilevel converters. Without considering the traditional magnetic coupled converters, this paper presents three recently developed multilevel voltage source converters: 1) diode-clamp, 2) flyingcapacitors, and 3) cascaded-inverters with separate dc sources. The operating principle, features, constraints, and potential applications of these converters will be discussed.

In this paper a methodology is presented for the purification of toxins acting on neuronal potassium channels. These neurotoxins are ideal tools for studying the physiological functioning of ion channels, for classifying them and for exploring them in several neuronal regions. Separation procedures leading to the identification of neurotoxins almost always include gel filtration chromatography as well as other chromatographic techniques such as ion exchange and reversed phase chromatography. The use of a Superdex 30 prep grade gel filtration column, which has the advantage of superior resolution in comparison with traditionally used gel filtration columns such as a Sephadex G-50 column, is reported here. The biological assay incorporates the use of Xenopus laevis oocytes, which express potassium channels. Screening of the collected venom fractions was performed by means of the whole-cell voltage clamp technique. The combination of these techniques represents a fast and efficient identification procedure in the search for new and selective neurotoxins for cloned channels and receptors.

This paper presents a cost effective edge-resonant soft-Besides, the soft-switching operating area in this type of switching PWM high frequency inverter with minimum circuit ZVS high frequency inverter is relatively narrow under a components. This inverter can achieve wider soft commutation, pulse frequency modulation strategy. On the other hand, an simpler circuit configuration, smaller volumetric size, lower cost active subsidiary power switch IGBT connected in series with and wider power regulation range, higher-efficiency as compared the capacitor for the voltage clamping of main power IGBT to single ended quasi-resonant ZVS-PFM and active voltage on the basis of conventional single ended ZCS-PFM HF clamped quasi-resonant ZVS-PWM inverters. The operation invte This invertionas the ende voltage HF principle of the proposed inverter is described on the basis of the inverter. This inverter is termed as the active voltage clamped simulation and experimental results. The operating performances ZVS PWM inverter and has been practically developed before of this unique proposed high frequency inverter based on ZVS by the authors for 200V AC utility interactive consumer high and ZCS arms-related soft commutation principle is evaluated power applications. However, further technological and discussed in steady state as compared to the previous improvement of the active voltage clamped ZVS PWM inverters. The extended bidirectional circuit topology of quasiinverter should be made from viewpoints of a constant resonant PWM high frequency inverter with minimum circuit frequency ZVS operation, physical size-based compactness in components is demonstrated, which operate as the direct high power appliance, and the power regulation range. frequency changer. This paper presents an improved prototype of cost effective switching, Consumer power electronics, Induction heating voltage source type quasi-resonant soft-switching HF inverter with minimum circuit components and devices. The inverter 0-7803-9296-5/05/$20.00 t 2005 IEEE

Xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins. A thorough understanding of the endogenous conductances and their regulation is paramount for proper characterization of expressed channel proteins. Here we detail a novel chloride current (Icl.swell) responsive to hypotonicity in Xenopus oocytes using the two-electrode voltage clamp technique. Reducing the extracellular osmolarity by 50% elicited a calcium-independent chloride current having an anion conductivity sequence identical with swelling-induced chloride currents observed in epithelial cells. The hypotonicity-activated current was blocked by chloride channel blockers, trivalent lanthanides, and nucleotides. G-protein, cAMP-PKA, and arachidonic acid signaling cascades were not involved in IcLswetl activation. Icl.swel I is distinct from both stretch-activated nonselective cation channels and the calcium-activated chloride current in oocytes and may play a critical role in volume regulation in Xenopus oocytes.

This paper presents a three-phase grid-connected photovoltaic generation system with unity power factor for any situation of solar radiation. The modelling of the PWM inverter and a control strategy using dq0 transformation are proposed. The system operates as an active filter capable of compensate harmonic components and reactive power, generated by the loads connected to the system. An input voltage clamping technique is proposed to control the power between the grid and photovoltaic system, where it is intended to achieve the maximum power point operation. Simulation and experimental results are presented to validate the proposed methodology for grid connected photovoltaic generation system.

We provide here detailed electrophysiological protocols to study voltage-gated sodium channels and to investigate how wild-type and mutant channels influence firing properties of transfected mammalian dorsal root ganglion (DRG) neurons. Whole-cell voltage-clamp recordings permit us to analyze kinetic and voltage-dependence properties of ion channels and to determine the effect and mode of action of pharmaceuticals on specific channel isoforms. They also permit us to analyze the role of individual sodium channels and their mutant derivatives in regulating firing of DRG neurons. Five to ten cells can be recorded daily, depending on the extent of analysis that is required. Because of different internal solutions that are used in voltage-clamp and current-clamp recordings, only limited information can be obtained from recording the same neuron in both modes. These electrophysiological studies help to elucidate the role of specific channels in setting threshold and suprathreshold responses of neurons, under normal and pathological conditions.

Active clamp topologies of low power dissipation have become a very attractive solution in order to limit overvoltages in flyback converters. Although many suitable topologies have been introduced for the case of discontinuous conduction mode (DCM), where the duty cycle value depends on the load level, in continuous conduction mode (CCM) it is more difficult to appropriately design such topologies so as to "sense" load changes-due to the small duty cycle divergence under wide load variation. Taking for granted that in order to achieve high power-factor correction in these converters, CCM is a more attractive mode of operation, a drastic solution for this case that will manage to eliminate voltage stresses under wide load changes has become very essential. For this purpose, this paper presents an active clamp topology with small power dissipation, suitable for flyback converters operating in CCM mode. Its main idea is the use of a load-dependent current source, consisting of an auxiliary converter operating in DCM mode. Experimental results highlight the effectiveness of the proposed topology under wide load changes, establishing it as an appropriate solution in order to develop flyback converters, even at the power range of 500 W.

The contractile responses of isolated Rana ridibunda frog sartorius muscle contractions evoked by electrical field stimulation (EFS) were studied at three temperature conditions of 17, 22 and 27 8C. Temperature-dependent increase of muscle contractility was found. ATP (10-100 AM) concentration dependently inhibited the electrical field stimulation-evoked contractions of sartorius muscle at all three temperatures; this effect was significantly more prominent at a temperature of 17 8C than at other two temperatures. Adenosine (100 AM) also caused inhibition of electrical field stimulation-evoked contractions which was statistically identical at all three temperature conditions tested. A P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2V,4V-disulphonic acid (PPADS, 10 AM) reduced the inhibitory effect of ATP at all three temperatures but did not affect inhibitory action of adenosine. In contrast, 8-( p-sulfophenyl)theophylline (8-SPT, 100 AM), a nonselective P1 receptor antagonist, abolished inhibitory effects of adenosine at all three temperature conditions but did not antagonize inhibition caused by ATP. In electrophysiological experiments, ATP (100 AM) and adenosine (100 AM) temperature dependently reduced end-plate currents recorded in sartorius neuromuscular junction by voltage-clamp technique. The inhibitory effects of both agonists were enhanced with the decrease of temperature. 8-SPT (100 AM) abolished the inhibitory effect of adenosine but not ATP on end-plate currents. Suramin (100 AM), a nonselective P2 receptor antagonist, inhibited the action of ATP but not adenosine, while PPADS (10 AM) had no influence on the effects of either ATP or adenosine. It is concluded from this study that the effectiveness of P2 receptor-mediated inhibition of frog skeletal muscle contraction in contrast to that of adenosine is dependent on the temperature conditions. D 2004 Published by Elsevier B.V.

Long-chain neurotoxins derived from the venom of the Buthidae scorpions, which affect voltage-gated sodium channels (VGSCs) can be subdivided according to their toxicity to insects into insect-selective excitatory and depressant toxins (b-toxins) and the a-like toxins which affect both mammals and insects.

5-Hydroxytryptamine (5-HT), a neurotransmitter and neuromodulator in the central nervous system of the leech Hirudo medicinalis hyperpolarizes the giant glial cell in the neuropil of segmental ganglia at micromolar concentrations. The 5-HTevoked glial response (EC 50 Ϸ2.5 M) is mediated by a non-desensitizing, G-proteincoupled receptor and due to activation of a Ca 2ϩ -independent K ϩ conductance. The adenylyl cyclase inhibitor SQ22,536 blocks the response to 5-HT; in the presence of 1 mM db-cAMP, but not of 1 mM db-cGMP, the glial response is suppressed. The 5-HTevoked response is reduced by Ba 2ϩ with half-maximal inhibition at 50 M Ba 2ϩ . The results suggest that release of 5-HT from serotonergic neurons, or the maintenance of micromolar levels of extracellular 5-HT in the ganglion, may help to set the glial membrane potential close to the K ϩ equilibrium potential.

The dopamine D 4 receptor (D 4 R) is predominantly expressed in the frontal cortex (FC), a brain region that receives dense input from midbrain dopamine (DA) neurons and is associated with cognitive and emotional processes. However, the physiological significance of this ...

Ionic (I i ) and gating currents (I g ) from noninactivating Shaker H4 K ϩ channels were recorded with the cut-open oocyte voltage clamp and macropatch techniques. Steady state and kinetic properties were studied in the temperature range 2-22 Њ C. The time course of I i elicited by large depolarizations consists of an initial delay followed by an exponential rise with two kinetic components. The main I i component is highly temperature dependent (Q 10 Ͼ 4) and mildly voltage dependent, having a valence times the fraction of electric field ( z ) of 0.2-0.3 e o . The I g On response obtained between Ϫ 60 and 20 mV consists of a rising phase followed by a decay with fast and slow kinetic components. The main I g component of decay is highly temperature dependent (Q 10 Ͼ 4) and has a z between 1.6 and 2.8 e o in the voltage range from Ϫ 60 to Ϫ 10 mV, and ‫ف‬ 0.45 e o at more depolarized potentials. After a pulse to 0 mV, a variable recovery period at Ϫ 50 mV reactivates the gating charge with a high temperature dependence (Q 10 Ͼ 4). In contrast, the reactivation occurring between Ϫ 90 and Ϫ 50 mV has a Q 10 ϭ 1.2. Fluctuation analysis of ionic currents reveals that the open probability decreases 20% between 18 and 8 Њ C and the unitary conductance has a low temperature dependence with a Q 10 of 1.44. Plots of conductance and gating charge displacement are displaced to the left along the voltage axis when the temperature is decreased. The temperature data suggests that activation consists of a series of early steps with low enthalpic and negative entropic changes, followed by at least one step with high enthalpic and positive entropic changes, leading to final transition to the open state, which has a negative entropic change. key words: Shaker K ϩ channel • gating and ionic current kinetics • charge movement • voltage-activation process • enthalpic and entropic changes Portions of this work were previously published in abstract form (Rodríguez, B.M., D. Sigg, and F. 1 Abbreviations used in this paper: G-V curve, conductance versus voltage curve; MES, methanosulphonate; NMG, N-methyl-glucamine; P or -V curve, relative open probability versus voltage curve; Q-V curve, charge versus voltage curve.

This review summarizes the structural and electrophysiological changes in the epileptic hippocampus in various animal models of epilepsy. Tornado hypothesis of epileptic hippocampus, states that there are many progressive detrimental changes in the hippocampus of the epileptic rats. PTZ, PTZ kindling, pilocarpine, electrical kindling, kainic acid, hyperthermia and in vitro models are taken as primary animal epilepsy models in this review. Paired pulse inhibition and GABAergic transmission is decreased in dentate gyrus (DG), CA3 and CA1 regions of the epileptic hippocampus. There is interneuron loss in the hilus, DG and CA3, while excitatory input from perforant path (PP) is increased and this is fed into the CA3 area by mossy fibers (MF) which have excessive sprouting that forms novel recurrent synapses with the pyramidal cells of CA3. Gate function of DG is either impaired or lost. CA3 area of hippocampus becomes an epileptic focus and sends ictal discharges. These ictal discharges are carried into CA1 by Schaffer collaterals and then into entorhinal cortex (EC). The input from EC is amplified in the hippocampal circuitry which is fed into hippocampus from EC into DG again successively. Thus, a weak electrical input into the hippocampal formation results in an amplified signal back into EC. Since the basic ultrastructural and electrophysiological feedback control mechanisms are impaired, this electrical tornado cannot be compensated for and an epileptic amplified ictal discharge spreads to the limbic system and other adjacent structures of the brain. Eventually the hippocampal circuitry, that has developed a vicious circle, becomes a bio-electrical amplifier which triggers an electrical tornado, under certain biochemical conditions. mossy fiber sprouting, in vitro model of epilepsy, perforant path, tonic clonic seizures, intractable temporal lobe epilepsy.

The voltage-clamp technique is applicable only to spherical cells. In nonspherical cells, such as neurons, the membrane potential is not clamped distal to the voltageclamp electrode. This means that the current recorded by the voltageclamp electrode is the sum of the local current and of axial currents from locations experiencing different membrane potentials. Furthermore, voltage-gated currents recorded from a nonspherical cell are, by definition, severely distorted due to the lack of space clamp. Justifications for voltage clamping in nonspherical cells are, first, that the lack of space clamp is not severe in neurons with short dendrites. Second, passive cable theory may be invoked to justify application of voltage clamp to branching neurons, suggesting that the potential decay is sufficiently shallow to allow spatial clamping of the neuron. Here, using numerical simulations, we show that the distortions of voltage-gated K ϩ and Ca 2ϩ currents are substantial even in neurons with short dendrites. The simulations also demonstrate that passive cable theory cannot be used to justify voltage clamping of neurons due to significant shunting to the reversal potential of the voltage-gated conductance during channel activation. Some of the predictions made by the simulations were verified using somatic and dendritic voltageclamp experiments in rat somatosensory cortex. Our results demonstrate that voltage-gated K ϩ and Ca 2ϩ currents recorded from branching neurons are almost always severely distorted.

G protein-activated K + channel (GIRK) subunits possess a conserved extracellular integrin-binding motif (RGD) and bind directly to L L1 integrins. We expressed GIRK1/GIRK4 channels labeled with green fluorescent protein in fibroblast cell lines expressing or lacking L L1 integrins. Neither plasma membrane localization nor agonist-evoked GIRK currents were affected by the absence of L L1 integrins or by incubation with externally applied RGD-containing peptide. Mutation of the aspartate (D) of RGD impaired currents, GIRK glycosylation, and membrane localization, but the interaction with L L1 integrins remained intact. Thus, L L1 integrins are not essential for functional GIRK expression; and the GIRK-integrin interactions involve structural elements other than the RGD motif.

Silicon controlled rectifiers (SCRs) are widely used ESD protection elements exhibiting extremely good voltage clamping and high failure current threshold. However, the turn-on behavior of the SCR is often a matter of concern. A detailed transient study of the 3-D current and temperature distribution in a SCR during a high current square pulse is presented leading to design options with shorter delay between trigger point and full clamp performance. [

Dystrophin-deficient muscle fibers from mdx mice are believed to suffer from increased calcium entry and elevated submembranous calcium level, the actual source and functional consequences of which remain obscure. Here we compare the properties of the dihydropyridine receptor as voltage sensor and calcium channel in control and mdx muscle fibers, using the silicone-voltage clamp technique. In control fibers charge movement followed a two-state Boltzmann distribution with values for maximal charge, midpoint voltage, and steepness of 23 6 2 nC/mF, ÿ37 6 3 mV, and 13 6 1 mV (n ¼ 7). Essentially identical values were obtained in mdx fibers and the time course of charge recovery from inactivation was also similar in the two populations (t ' 6 s). In control fibers the voltage dependence of the slow calcium current elicited by 100-ms-long pulses gave values for maximal conductance, apparent reversal potential, half-activation potential, and steepness factor of 156 6 15 S/F, 65.5 6 2.9 mV, ÿ0.76 6 1.2 mV, and 6.2 6 0.5 mV (n ¼ 17). In mdx fibers, the half-activation potential of the calcium current was slightly more negative (ÿ6.2 6 1.2 mV, n ¼ 16). Also, when using longer pulses, the time constant of calcium current decay was found to be significantly larger (by a factor of 1.5-2) in mdx than in control fibers. These changes in calcium current properties are unlikely to be primarily responsible for a dramatic alteration of intracellular calcium homeostasis. They may be speculated to result, at least in part, from remodeling of the submembranous cytoskeleton network due to the absence of dystrophin.

The role of tight junctions in modulating responses from chorda tympani (taste) and lingual (general sensory) nerves are clarified in regard to their responses to salts. Chorda tympani (CT) responses elicited by organic sodium salts require greater Na + concentrations to elicit the same magnitude of response as NaC1. These data can be understood in terms of the organic anions (compared with C1-) producing larger liquid-junction potentials across tight junctions between taste cells which, in turn, reduces Na + influx into taste cells via amiloride-inhibitable channels. The anion contribution to the CT response to different Na + salts can be eliminated (or enhanced) by voltage clamping the tongue with negative (with respect to the serosal solution) potentials.

Background: Two groups of gain-of-function mutations in sodium channel Na V 1.7, which are expressed in dorsal root ganglion (DRG) neurons, produce two clinically-distinct pain syndromes -inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). IEM is characterized by intermittent burning pain and skin redness in the feet or hands, triggered by warmth or mild exercise, while PEPD is characterized by episodes of rectal, ocular and mandibular pain accompanied with skin flushing, triggered by bowel movement and perianal stimulation. Most of the IEM mutations are located within channel domains I and II, while most of the PEPD mutations are located within domains III and IV. The structural dichotomy parallels the biophysical effects of the two types of mutations, with IEM mutations shifting voltage-dependence of Na V 1.7 activation in a hyperpolarized direction, and PEPD mutations shifting fast-inactivation of Na V 1.7 in a depolarized direction. While four IEM and four PEPD mutations are located within cytoplasmic linkers joining segments 4 and 5 (S4-S5 linkers) in the different domains (IEM: domains I and II; PEPD: domains III and IV), no S4-S5 linker has been reported to house both IEM and PEPD mutations thus far.

Despite intensive interest in under- standing the differentiation of vascular smooth mus- cle cells (VSMC), no information is available about differential regulation of ion channels in these cells. Since expression of the L-type Ca2/ channel can be influenced by differentiation in other cell types, we tested the hypothesis that the L-type (C class) channel is a specific differentiation marker of VSMC and that expression of these channels depends on the state of cell differentiation. We used rat aortic (A7r5) VSMC, which express functional L-type Ca2/ channels, and induced dedifferentiation by cell culture in different media. Treatment with retinoic acid was used to re- differentiate the VSMC. We characterized the dif- ferentiated state of the cells by using immunohisto- chemistry and Western blot analysis for smooth mus- cle (SM)a-actin and SM-myosin heavy chain (MHC). The number of functional Ca2/ channels was signifi-

In cortical neurons, synaptic "noise" is caused by the nearly random release of thousands of synapses. Few methods are presently available to analyze synaptic noise and deduce properties of the underlying synaptic inputs. We focus here on the power spectral density (PSD) of several models of synaptic noise. We examine different classes of analytically solvable kinetic models for synaptic currents, such as the "delta kinetic models," which use Dirac delta functions to represent the activation of the ion channel. We first show that, for this class of kinetic models, one can obtain an analytic expression for the PSD of the total synaptic conductance and derive equivalent stochastic models with only a few variables. This yields a method for constraining models of synaptic currents by analyzing voltage-clamp recordings of synaptic noise. Second, we show that a similar approach can be followed for the PSD of the the membrane potential (V m ) through an effective-leak approximation. Third, we show that this approach is also valid for inputs distributed in dendrites. In this case, the frequency scaling of the V m PSD is preserved, suggesting that this approach may be applied to intracellular recordings of real neurons. In conclusion, using simple mathematical tools, we show that V m recordings can be used to constrain kinetic models of synaptic currents, as well as to estimate equivalent stochastic models. This approach, therefore, provides a direct link between intracellular recordings in vivo and the design of models consistent with the dynamics and spectral structure of synaptic noise.

Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G 0 /G 1 phase. Furthermore, low expression of K v1.5 , a voltage-gated potassium channel known to alter myoblast proliferation during the G 1 phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between K v1.5 and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and K v1.5 within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of K v1.5 with FHL1C in Xenopus laevis oocytes markedly reduced K + currents when compared to oocytes expressing K v1.5 only. We here present the first evidence on a biological relevance of FHL1C. Citation: Poparic I, Schreibmayer W, Schoser B, Desoye G, Gorischek A, et al. (2011) Four and a Half LIM Protein 1C (FHL1C): A Binding Partner for Voltage-Gated Potassium Channel K v1.5 . PLoS ONE 6(10): e26524.

The electrical coupling of randomly migrating neurons from rat explant brain-stem slice cultures to the gates of non-metallized field-effect transistors (FETs) has been investigated. The objective of our work is the precise interpretation of extracellular recorded signal shapes in comparison to the usual patch-clamp protocols to evaluate the possible use of the extracellular recording technique in electrophysiology. The neurons from our explant cultures exhibited strong voltage-gated potassium currents through the plasma membrane. With an improved noise level of the FET set-up, it was possible to record individual extracellular responses without any signal averaging. Cells were attached by patchclamp pipettes in voltage-clamp mode and stimulated by voltage step pulses. The point contact model, which is the basic model used to describe electrical contact between cell and transistor, has been implemented in the electrical simulation program PSpice. Voltage and current recordings and compensation values from the patch-clamp measurement have been used as input data for the simulation circuit. Extracellular responses were identified as composed of capacitive current and active potassium current inputs into the adhesion region between the cell and transistor gate. We evaluated the extracellular signal shapes by comparing the capacitive and the slower potassium signal amplitudes. Differences in amplitudes were found, which were interpreted in previous work as enhanced conductance of the attached membrane compared to the average value of the cellular membrane. Our results suggest rather that additional effects like electrodiffusion, ion sensitivity of the sensors or more detailed electronic models for the small cleft between the cell and transistor should be included in the coupling model.

The role of tight junctions in modulating responses from chorda tympani (taste) and lingual (general sensory) nerves are clarified in regard to their responses to salts. Chorda tympani (CT) responses elicited by organic sodium salts require greater Na + concentrations to elicit the same magnitude of response as NaC1. These data can be understood in terms of the organic anions (compared with C1-) producing larger liquid-junction potentials across tight junctions between taste cells which, in turn, reduces Na + influx into taste cells via amiloride-inhibitable channels. The anion contribution to the CT response to different Na + salts can be eliminated (or enhanced) by voltage clamping the tongue with negative (with respect to the serosal solution) potentials. Whole nerve recordings from the lingual branch of the trigeminal nerve elicited by NaC1 (and other salts) were reversibly inhibited by the tight junction blocker, LaCI3. These data suggest that small hydrophilic molecules elicit responses from trigeminal fibers by diffusing across tight junctions between epithelial cells and altering the composition of the extracellular space.

Delayed neuroprotection against ischemic challenges is conferred by both ischemic preconditioning (IPC) and preconditioning by activation of the ε-isoform of protein kinase C (εPKC-PC). In vivo, ischemic preconditioning enhances GABA release and ameliorates glutamate release during lethal cerebral ischemia. We tested the hypothesis that IPC and εPKC-PC confer neuroprotection by GABA synapses in rat organotypic hippocampal slices. Ischemic preconditioning or εPKC-PC was induced with 15 mins oxygen-glucose deprivation (OGD) or ψεRACK, a selective εPKC activator; and test ischemia consisted of 40 mins OGD. At the time of peak neuroprotection (48 h after preconditioning), we recorded GABA A receptor-mediated miniature postsynaptic currents (GABA mPSCs) in vulnerable CA1 pyramidal neurons using whole-cell voltage clamp techniques. The frequency and amplitude of GABA mPSCs significantly increased 48 h after IPC. In contrast, εPKC-PC enhanced only the amplitude of GABA mPSCs with no effect on frequency. We next asked if neuroprotection depended on these changes in GABA synapses. Weak antagonism of the GABA A receptor with bicuculline (100 nmol/L) decreased the amplitude of GABA mPSCs by 20.9 ± 6.1%. When applied during test ischemia, 100 nmol/L bicuculline abolished neuroprotection conferred by either IPC or εPKC-PC. We conclude that neuroprotection conferred by preconditioning depends on functional modifications of GABA synapses.

Kv4.3 inactivation is a complex multiexponential process, which can occur from both closed and open states. The fast component of inactivation is modulated by the N-terminus, but the mechanisms mediating the other components of inactivation are controversial. We studied inactivation of Kv4.3 expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. Inactivation during 2000 ms pulses at potentials positive to the activation threshold was described by three exponents (46 6 3, 152 6 13, and 930 6 50 ms at 150 mV, n ¼ 7) whereas closed-state inactivation (at potentials below threshold) was described by two exponents (1079 6 119 and 3719 6 307 ms at ÿ40 mV, n ¼ 9). The fast component of openstate inactivation was dominant at potentials positive to ÿ20 mV. Negative to ÿ30 mV, the intermediate and slow components dominated inactivation. Inactivation properties were dependent on pulse duration. Recovery from inactivation was strongly dependent on voltage and pulse duration. We developed an 11-state Markov model of Kv4.3 gating that incorporated a direct transition from the open-inactivated state to the closed-inactivated state. Simulations with this model reproduced open-and closed-state inactivation, isochronal inactivation relationships, and reopening currents. Our data suggest that inactivation can proceed primarily from the open state and that multiple inactivation components can be identified.

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca 2 ϩ ] i in a Ca 2 ϩ -free medium followed by a secondary [Ca 2 ϩ ] i rise upon Ca 2 ϩ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca 2 ϩ -activated (I Cl(Ca) ) and volume-regulated (I Cl, swell ) chloride currents. NPPB and DIDS more efficiently inhibited I Cl(Ca) and I Cl, swell , respectively. Cell swelling caused by hypotonic solution invariably activated I Cl, swell while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist-and cell swelling-induced I Cl, swell , while its inactive analogue U-73343 had no effect. I Cl(Ca) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca 2 ϩ ] i in the pipette solution at 500 nM. In contrast, I Cl, swell could be directly activated by 1-oleoyl-2-acetyl-sn -glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP 3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced I Cl, swell in a nonadditive manner, suggesting their convergence on a common pathway. I Cl, swell and I Cl(Ca) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated I Cl(Ca) , but abolished I Cl, swell , thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that I Cl, swell can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a "selection" switch between closely localized channels.

Background: Lung epithelial Na + channels (ENaC) are regulated by cell Ca 2+ signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K + channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K + channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC) by up-regulating both apical and basolateral ion transport.