Patch-clamp Amplifier Research Articles

Pathogenic Serum Amyloid A 1.1 Shows a Long Oligomer-rich Fibrillation Lag Phase Contrary to the Highly Amyloidogenic Non-pathogenic SAA2.2

Serum amyloid A (SAA) is best known for being the main component of amyloid in the inflammation-related disease amyloid A (AA) amyloidosis. Despite the high sequence identity among different SAA isoforms, not all SAA proteins are pathogenic. In most mouse strains, the AA deposits mostly consist of SAA1.1. Conversely, the CE/J type mouse expresses a single non-pathogenic SAA2.2 protein that is 94% identical to SAA1.1. Here we show that SAA1.1 and SAA2.2 differ in their quaternary structure, fibrillation kinetics, prefibrillar oligomers, and fibril morphology. At 37 °C and inflammation-related SAA concentrations, SAA1.1 exhibits an oligomer-rich fibrillation lag phase of a few days, whereas SAA2.2 shows virtually no lag phase and forms small fibrils within a few hours. Deep UV resonance Raman, far UV-circular dichroism, atomic force microscopy, and fibrillation cross-seeding experiments suggest that SAA1.1 and SAA2.2 fibrils possess different morphology. Both the long-lived oligomers of pathogenic SAA1.1 and the fleeting prefibrillar oligomers of non-pathogenic SAA2.2, but not their respective amyloid fibrils, permeabilized synthetic bilayer membranes in vitro. This study represents the first comprehensive comparison between the biophysical properties of SAA isoforms with distinct pathogenicities, and the results suggest that structural and kinetic differences in the oligomerization-fibrillation of SAA1.1 and SAA2.2, more than their intrinsic amyloidogenicity, may contribute to their diverse pathogenicity.

Development of a Nav1.5 Assay for Prolonged Recording on an Automated Electrophysiology System

The IonWorks Barracuda system allows simultaneous and continuous measurement of both ligand-gated ion channels (LGICs) and voltage-gated ion channels (VGICs) in 384 individual recording sites. To achieve this, IonWorks Barracuda system is equipped with 384 individual patch-clamp amplifiers together with a 384-channel fluidic pipettor. Similar to its predecessor, IonWorks Quattro, IonWorks Barracuda system measures cell membrane currents using the perforated patch clamp techniques on a polyimide substrate. Currents are measured using a single hole at each recording site or an array of 64 holes at each site (Population Patch Clamp or PPC, Finkel et al. 2006). In this study, we investigated a new technique to increase the assay window for ionic currents on the IonWorks Barracuda instrument. using Nav1.5 channel as an example, we demonstrated stable recording of Nav1.5 currents elicited by repetitive scans for 30 minutes or longer. This prolonged assay window, which is about twice of that of IonWorks Quattro, allows the researchers not only to precisely control the state of the channel by voltage clamp; but also to accurately analyze compounds with slow binding rate to the channel. In a drug discovery environment, this allows for the use of more sophisticated voltage protocols to explore state-dependent blockers of ion channels.

Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held

Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle (N(Glu)) and its diffusion coefficient (D(Glu)) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of N(Glu) and D(Glu) with an optimum of 7000 and 0.3 μm(2) ms(-1) reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals.

Activation of metabotropic glutamate receptors improves the accuracy of coincidence detection by presynaptic mechanisms in the nucleus laminaris of the chick

Interaural time difference (ITD) is a major cue for localizing a sound source and is processed in the nucleus laminaris (NL) in birds. Coincidence detection (CD) is a crucial step for processing ITD and critically depends on the size and time course of excitatory postsynaptic potentials (EPSPs). Here, we investigated a role of metabotropic glutamate receptors (mGluRs) in the regulation of EPSP amplitude and CD in the NL of chicks. A non-specific agonist of mGluRs ((±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) reduced the amplitude and extent of depression of excitatory postsynaptic currents (EPSCs) during a stimulus train, while the paired pulse ratio and coefficient of variation of EPSC amplitude were increased. In contrast, the amplitudes of spontaneous EPSCs were not affected, but the frequency was reduced. Thus, the effects of t-ACPD were presynaptic and reduced the release of neurotransmitter from terminals in the NL. Expression of group II mGluRs was graded along the tonotopic axis and was stronger towards the low frequency region in the NL. Both group II (DCG-IV) and group III (l-AP4) specific agonists reduced EPSC amplitude by presynaptic mechanisms, and the reduction was larger in the low frequency region; however, we could not find any effects of group I-specific agonists on EPSCs. The reduced EPSP amplitude in DCG-IV improved CD. A specific antagonist of group II mGluRs (LY341495) increased the amplitude of both EPSCs and EPSPs and enhanced the depression during a stimulus train, indicating constitutive activation of mGluRs in the NL. These observations indicate that mGluRs may work as autoreceptors and regulate EPSP size to improve CD in the NL.

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

Kernel Based Non-Iterative Automatic Fast Capacitance Compensation in Patch-Clamp Experiments

Patch-clamp recording is a widely used technique for studying ion channel functions and cellular responses. During patch-clamp experiments, the fast capacitance (CFast) transient is a detrimental artifact and must be eliminated with CFast compensation circuit. When applying the conventional computer-aided CFast compensation procedure, the patch-clamp amplifier faces the risk of saturation due to large square-wave responses, and compensation errors may be caused due to inherent disadvantages of iterative algorithm. Here, we introduce a novel non-iterative automatic CFast compensation method, namely K-method, based on kernel estimation with white noise excitation. The kernel was estimated with cross-correlation technique and captured dynamic properties of the CFast and its related hardware. To achieve the optimal settings of the CFast compensation, the kernel of CFast was fitted to two calibrated kernels indicating two distinct types of compensation effect: the “instantaneous” and “delay” effect. The fitted coefficients were used to adjust compensation circuit. White noise excitation significantly reduced the possibility of saturation, and the K-method suffered from no typical disadvantage of iterative method. We performed compensation experiments on a model circuit and HEK293 cells. The results demonstrated a good accuracy of the K-method and the membrane capacitance measurement could benefit from it.

Requirement of neuronal connexin36 in pathways mediating presynaptic inhibition of primary afferents in functionally mature mouse spinal cord

Electrical synapses formed by gap junctions containing connexin36 (Cx36) promote synchronous activity of interneurones in many regions of mammalian brain; however, there is limited information on the role of electrical synapses in spinal neuronal networks. Here we show that Cx36 is widely distributed in the spinal cord and is involved in mechanisms that govern presynaptic inhibition of primary afferent terminals. Electrophysiological recordings were made in spinal cord preparations from 8- to 11-day-old wild-type and Cx36 knockout mice. Several features associated with presynaptic inhibition evoked by conditioning stimulation of low threshold hindlimb afferents were substantially compromised in Cx36 knockout mice. Dorsal root potentials (DRPs) evoked by low intensity stimulation of sensory afferents were reduced in amplitude by 79% and in duration by 67% in Cx36 knockouts. DRPs were similarly affected in wild-types by bath application of gap junction blockers. Consistent with presynaptic inhibition of group Ia muscle spindle afferent terminals on motoneurones described in adult cats, conditioning stimulation of an adjacent dorsal root evoked a long duration inhibition of monosynaptic reflexes recorded from the ventral root in wild-type mice, and this inhibition was antagonized by bicuculline. The same conditioning stimulation failed to inhibit monosynaptic reflexes in Cx36 knockout mice. Immunofluorescence labelling for Cx36 was found throughout the dorsal and ventral horns of the spinal cord of juvenile mice and persisted in mature animals. In deep dorsal horn laminae, where interneurones involved in presynaptic inhibition of large diameter muscle afferents are located, cells were extensively dye-coupled following intracellular neurobiotin injection. Coupled cells displayed Cx36-positive puncta along their processes. Our results indicate that gap junctions formed by Cx36 in spinal cord are required for maintenance of presynaptic inhibition, including the regulation of transmission from Ia muscle spindle afferents. In addition to a role in presynaptic inhibition in juvenile animals, the persistence of Cx36 expression among spinal neuronal populations in the adult mouse suggests that the contribution of electrical synapses to integrative processes in fully mature spinal cord may be as diverse as that found in other areas of the CNS.

Intracellular Proton-mediated Activation of TRPV3 Channels Accounts for the Exfoliation Effect of α-Hydroxyl Acids on Keratinocytes

α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.

High-Bandwidth Solid-State Nanopore Sensors

Nanopores are attractive electrochemical sensors for their single-molecule sensitivity and simple experimental setup. Many of the electronic systems used for nanopore measurements are inherited from electrophysiology applications, and as a result they are primarily optimized for current levels of 50pA or less, at bandwidths below 10kHz. However, state-of-the-art solid-state nanopore sensors now commonly produce signals in the nanoampere range, for which common patch clamp amplifiers are no longer ideal. We present a new experimental platform which integrates a custom CMOS preamplifier with nanopores in thin silicon nitride membranes. The low parasitic capacitance of the integrated platform reduces noise at high frequencies, allowing nanopore measurements at finer temporal resolution than supported by popular instruments. We demonstrate the viability of this system with high-bandwidth electrical recordings of the passage of short DNA molecules through nanopores. This new platform will improve the signal-to-noise ratios of nanopore sensors, enabling further single-molecule studies of fast biophysical processes.

“Clip Detecting” with Series Resistance Compensation using an Automated Patch Clamp System

One of the significant challenges for automated patch clamp has been to incorporate amplifiers with the ability of adjusting to changes in membrane capacitance, series resistance (Rs) and voltage-clamp time constants without user intervention. If these adjustments are not made correctly there is a risk of generating un-precise data or even losing data due to oscillations in the recorded trace. Rs compensation capability of the patch clamp amplifier of QPatch is presented. A new “clip detector feature” allowing Rs to be temporarily turned off avoiding loss of cells due to fatal oscillations, and thereby increasing the data throughput while maintaining high quality recordings, is presented.

Voltage Dependence of Calcium Spike Characteristics in Rat Cardiac Myocytes

Integral calcium release fluxes in mammalian cardiac myocytes increase in amplitude not only due to the increased recruitment of individual release sites but also due to their increased synchronization (1). Here we characterize the voltage dependence of synchronization of individual release sites at high temporal resolution. Calcium spikes and calcium currents elicited in isolated rat ventricular myocytes (2) by 80 ms voltage pulses from −50 to −40 - +50 mV were recorded using Leica TCS AOBS confocal microscope (2 kHz line scan mode) and Axopatch 200B patch-clamp amplifier, respectively. Calcium spikes were identified and analyzed by a new Matlab-based software that highly increased the productivity and reliability of approximation of calcium spikes by the kinetic model of local calcium release (2). Calcium spikes were described by their amplitude, onset latency, time-to-peak, FDHM, time constants of activation and termination, and fractional probability of activation. The synchrony of calcium spikes increased with depolarization and could be fully explained by the voltage dependence of their onset latency that reached a lower limit of about 4 ms at depolarizations between 0 and 40 mV. The rate of calcium spike activation after its onset was independent of the applied voltage or of calcium current amplitude, while the probability of calcium spike activation was related to the voltage dependence of the integral of calcium current. We conclude that both the synchrony of local calcium release activation and recruitment of individual release sites are fully controlled by the activation probability of DHPR calcium channels and by the DHPR-RYR coupling fidelity.Supported by APVV-0721-10, VEGA 2/0203/11, VEGA 2/0917/11.1. Song LS, Sham JS, Stern MD, Lakatta EG, Cheng H. J Physiol. 512:677-91, 1998.2. Zahradnikova A Jr, Polakova E, Zahradnik I, Zahradnikova A. J Physiol. 578:677-91, 2007.

Electronics Configuration to Co-Trap Single DNA Molecules in Dual Solid-State Nanopores - Solving the DNA Translocation Rate Problem

Nanopore sequencing aims to identify DNA bases by detecting base-specific signatures in the nanopore current signal, which are generated in principle when a DNA molecule passes through the nanopore channel. Nanopore channels can be formed by employing a protein channel in a lipid membrane, or by fabricating a “solid-state” pore in a substrate such as silicon nitride. Among several obstacles for nanopore sequencing, one challenge is that the DNA passes too quickly through the nanopore. One conventional solution is to reduce the command voltage, which is related to the intensity of the electric field. However, the decreased voltage leads to a reduction in the measured current, resulting in a loss in signal-to-noise ratio (SNR). We present a novel dual-nanopore concept that can permit high SNR while simultaneously controlling the DNA's speed through the nanopores. The configuration achieves this by electrophoretically trapping DNA between the two pores. The primary element of the proposed dual-nanopore architecture is an electrical configuration to independently control the voltage across each nanopore and simultaneously sense variations in each nanopore current signal. In the implementation presented, the dual-nanopore channels are fabricated in silicon-nitride membranes where the pores are serially and concentrically aligned within 0.4μm. We implement a pair of fully integrated low-noise CMOS patch-clamp amplifiers developed in our lab for independent and simultaneous voltage control/current measurement in the dual-nanopore device. Finite state machine logic running on a field-programmable gate array (FPGA) controls each amplifier and logs data. We will present our preliminary results on capturing a single DNA in both pores, and controlling the DNA's motion by competing voltage control.

A Novel Degradation Signal Derived from Distal C-terminal Frameshift Mutations of KCNQ2 Protein Which Cause Neonatal Epilepsy

Benign familial neonatal convulsions is an autosomal-dominant idiopathic form of epilepsy primarily caused by gene mutations of the voltage-gated Kv7.2/KCNQ2/M-channel that exert only partial dominant-negative effects. However, the mechanism underlying the incomplete dominance of channel mutations, which cause epilepsy in infancy, remains unknown. Using mutagenesis and biochemistry combined with electrophysiology, we identified a novel degradation signal derived from distal C-terminal frameshift mutations, which impairs channel function. This degradation signal, transferable to non-channel CD4, can lead to accelerated degradation of mutant proteins through ubiquitin-independent proteasome machinery but does not affect mRNA quantity and protein trafficking. Functional dissection of this signal has revealed a key five-amino acid (RCXRG) motif critical for degradation. Taken together, our findings reveal a mechanism by which proteins that carry this signal are subject to degradation, leading to M-current dysfunction, which causes epilepsy.

Masked excitatory crosstalk between the ON and OFF visual pathways in the mammalian retina

A fundamental organizing feature of the visual system is the segregation of ON and OFF responses into parallel streams to signal light increment and decrement. However, we found that blockade of GABAergic inhibition unmasks robust ON responses in OFF α-ganglion cells (α-GCs). These ON responses had the same centre-mediated structure as the classic OFF responses of OFF α-GCs, but were abolished following disruption of the ON pathway with L-AP4. Experiments showed that both GABA(A) and GABA(C) receptors are involved in the masking inhibition of this ON response, located at presynaptic inhibitory synapses on bipolar cell axon terminals and possibly amacrine cell dendrites. Since the dendrites of OFF α-GCs are not positioned to receive excitatory inputs from ON bipolar cell axon terminals in sublamina-b of the inner plexiform layer (IPL), we investigated the possibility that gap junction-mediated electrical synapses made with neighbouring amacrine cells form the avenue for reception of ON signals. We found that the application of gap junction blockers eliminated the unmasked ON responses in OFF α-GCs, while the classic OFF responses remained. Furthermore, we found that amacrine cells coupled to OFF α-GCs display processes in both sublaminae of the IPL, thus forming a plausible substrate for the reception and delivery of ON signals to OFF α-GCs. Finally, using a multielectrode array, we found that masked ON and OFF signals are displayed by over one-third of ganglion cells in the rabbit and mouse retinas, suggesting that masked crossover excitation is a widespread phenomenon in the inner mammalian retina.

Common Molecular Determinants of Tarantula Huwentoxin-IV Inhibition of Na+ Channel Voltage Sensors in Domains II and IV

The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxin-binding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.

Electrophysiological characteristics of medium spiny neurons in neocortex-striatum-substantia nigra brain slices of rats

To establish the neocortex-striatum-substantia nigra brain slices of rats and observe the medium spiny neurons of striatum under a visible condition so as to explore their electrophysiological characteristics. The brain slices containing the neocortex-striatum-substantia nigra were prepared from SD rats of postnatal 7 - 10 days. With infrared differential interference contrast (IR-DIC) microscope and patch clamp amplifier whole-cell recording technique, the medium spiny neurons were located in striatum and their spontaneous electrical activity was recorded in the current clamp mode. By infusing the step current, we observed the variation of membrane potentials. There were three types of conditions in the 92 medium spiny neurons successfully recorded. Among them, 14 were in persistent down state without action potential firing; 61 displayed persistent down state accompanied with interval temporal depolarizing to the threshold potential with action potential firing; and the remaining 17 showed persistent down state with the interval emergent up state. There was no statistical significance in the difference of mean resting and threshold potentials in three neuronal types (P > 0.05). When the neurons received an infused current, their membrane potentials displayed some delays. The variance of electric potentials showed a tendency of reduction along with the reinforcement of infusing current. The medium spiny neurons of striatum in parasagittal section brain slices reserve their in vivo electrophysiological characteristics. It establishes rationales for an in-depth study of the role of electrical generation and transmission of nigrostriatal access in the pathogenesis of Parkinson's disease.

Activity of BKCa Channel Is Modulated by Membrane Cholesterol Content and Association with Na+/K+-ATPase in Human Melanoma IGR39 Cells

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.

Direct and indirect control of orexin/hypocretin neurons by glycine receptors.

Hypothalamic hypocretin/orexin (hcrt/orx) neurons promote arousal and reward seeking, while reduction in their activity has been linked to narcolepsy, obesity and depression. However, the mechanisms influencing the activity of hcrt/orx networks in situ are not fully understood. Here we show that glycine, a neurotransmitter best known for its actions in the brainstem and spinal cord, elicits dose dependent postsynaptic Cl⁻ currents in hcrt/orx cells in acute mouse brain slices. The effect was blocked by the glycine receptor (GLyR) antagonist strychnine and mimicked by the GlyR agonist alanine. Postsynaptic GlyRs on hcrt/orx cells remained functional during both early postnatal and adult periods, and gramicidin-perforated patch-clamp recordings revealed that they progressively switch from excitatory to inhibitory during the first two postnatal weeks. The pharmacological profile of the glycine response suggested that developed hcrt/orx neurons contain α/β-heteromeric GlyRs that lack α2-subunits, whereas α2-subunits, whereas α2-subunits are present in early postnatal hcrt/orx neurons. All postsynaptic currents (PSCs) in developed hcrt/orx cells were blocked by inhibitors of GABA and glutamate receptors, with no evidence of GlyR-mediated PSCs. However, the frequency but not amplitude of miniature PSCs was reduced by strychnine and increased by glycine in ~50% of hcrt/orx neurons. Together, these results provide the first evidence for functional GlyRs in identified hcrt/orx circuits and suggest that the activity of developed hcrt/orx cells is regulated by two GlyR pools: inhibitory extrasynaptic GlyRs located on all hcrt/orx cells and excitatory GlyRs located on presynaptic terminals contacting some hcrt/orx cells.

Pannexin 1 Constitutes the Large Conductance Cation Channel of Cardiac Myocytes

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca(2+) release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.

Pores for thought

A team at the University of California Santa Cruz (UCSC) has developed the first amplifier that is optimised specifically for solid-state nanopore sensor applications. Their miniaturised patch-clamp amplifier, which is cheaper and smaller than the commercial bench top multi-purpose alternative, achieves a comparable performance and overcomes the dead time problem inherent in existing amplifiers.“Nanopores provide simple yet powerful single molecule detection and analysis capabilities without the need for particle labelling or amplification of the molecular samples,” said Professor William Dunbar from the Department of Computer Engineering at UCSC. “Our amplifier can be used and optimised for any solid-state nanopore. We see it as an advance for nanopore control instrumentation that will facilitate the use of multiple pores in single chips. This type of setup can provide alternative modes of nanopore-based single molecule sensing, and may also lead to new nanopore sequencing methods.”