Single-channel Events Research Articles

Two gates mediate NMDA receptor activity and are under subunit-specific regulation

Kinetics of NMDA receptor (NMDAR) ion channel opening and closing contribute to their unique role in synaptic signaling. Agonist binding generates free energy to open a canonical gate at the M3 helix bundle crossing. Single channel activity is characterized by clusters, or periods of rapid opening and closing, that are separated by long silent periods. A conserved glycine in the outer most transmembrane helices, the M4 helices, regulates NMDAR function. Here we find that the GluN1 glycine mainly regulates single channel events within a cluster, whereas the GluN2 glycine mainly regulates entry and exit from clusters. Molecular dynamics simulations suggest that, whereas the GluN2 M4 (along with GluN2 pre-M1) regulates the gate at the M3 helix bundle crossing, the GluN1 glycine regulates a ‘gate’ at the M2 loop. Subsequent functional experiments support this interpretation. Thus, the distinct kinetics of NMDARs are mediated by two gates that are under subunit-specific regulation.

Two gates mediate NMDAR activity and are under subunit-specific regulation.

NMDA receptors (NMDAR) are ionotropic glutamate receptors that mediate fast synaptic transmission and synaptic plasticity. Dysfunction of NMDARs has been found in numerous neurological diseases including Alzheimer's disease, Intellectual disability, epilepsy, autism, and schizophrenia. The NMDARs broad role in neuronal function is reflected in the complexity of its opening and closing kinetics, which can be broadly divided into periods of high opening activity, or clusters, interspersed by periods of no activity, or inter clusters. The initial step in ion channel opening is binding of agonists to the ligand-binding domain (LBD), which generates free energy that propagates to the ion channel to open ‘gate(s)’. NMDARs are heterotetramers composed of the obligate GluN1 and typically some combination of GluN2 subunits. The ion channel pore consists of two transmembrane segments (M1 and M3), a reentrant pore loop (M2) and a peripheral M4. A conserved glycine in the outer most transmembrane helix, the M4 helix, plays important roles in regulating NMDAR function. Here we find that these glycines play different roles in regulating channel opening. The GluN1 glycine mainly regulates single channel events within a cluster or bursts of activity, whereas GluN2A glycine mainly regulates opening into and out of clusters. Molecular dynamic simulations suggest that the GluN1 M4 regulates a ‘gate’ at the M2 loop, whereas the GluN2 M4 (along with GluN2 pre-M1) regulates a gate at the M3 helix bundle crossing. Subsequent functional experiments support this interpretation, leading to the hypothesis that the distinct kinetics of NMDARs are mediated by two gates that are under subunit-specific regulation. These results indicate that the GluN2A subunit regulates early steps in the gating process whereas the GluN1 regulate later steps including transitions form short to long-lived open states.

The speed of the hair cell mechanotransducer channel revealed by fluctuation analysis.

Although mechanoelectrical transducer (MET) channels have been extensively studied, uncertainty persists about their molecular architecture and single-channel conductance. We made electrical measurements from mouse cochlear outer hair cells (OHCs) to reexamine the MET channel conductance comparing two different methods. Analysis of fluctuations in the macroscopic currents showed that the channel conductance in apical OHCs determined from nonstationary noise analysis was about half that of single-channel events recorded after tip link destruction. We hypothesized that this difference reflects a bandwidth limitation in the noise analysis, which we tested by simulations of stochastic fluctuations in modeled channels. Modeling indicated that the unitary conductance depended on the relative values of the channel activation time constant and the applied low-pass filter frequency. The modeling enabled the activation time constant of the channel to be estimated for the first time, yielding a value of only a few microseconds. We found that the channel conductance, assayed with both noise and recording of single-channel events, was reduced by a third in a new deafness mutant, Tmc1 p.D528N. Our results indicate that noise analysis is likely to underestimate MET channel amplitude, which is better characterized from recordings of single-channel events.

Patch-clamp technique for studying ion channels in activated platelets

In this study, we have revisited the existing and suggested new approaches to the use of patch-clamp methodology for measuring the activity of single ion channels and membrane potential of human platelets in the cell-attached configuration. We recorded single-channel events of platelets in the cell-attached configuration after activation with potent agonists: thrombin and ionomycin. The feasibility of platelet membrane potential measurement in cell-attached mode was investigated both experimentally and with the help of simple electrical circuits, revealing that the single-channel events can alter the recorded potential by invoking oscillations. Here, the simple approach to obtain inside-out configuration was described and calcium-dependent single-channel ion currents were recorded. Taken together, this study introduces new approaches for further investigations of the role of ion channels and membrane potential in platelet physiological and pathophysiological response.

Alternative Splicing at N Terminus and Domain I Modulates CaV1.2 Inactivation and Surface Expression

The CaV1.2 L-type calcium channel is a key conduit for Ca2+ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its α1C pore-forming subunit is known to undergo extensive alternative splicing to produce many CaV1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human CaV1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with CaVβ2a, displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V1/2inact of CaV1.2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V1/2act. However, the measured effects were β-subunit-dependent when comparing CaVβ2a with CaVβ3 coexpression. Notably, calcium-dependent inactivation mediated by local Ca2+-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing CaV1.2 as compared to exon-1a-containing CaV1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with CaVβ2a or CaVβ3 subunit. This finding correlated well with a higher channel surface expression for the exon 1-CaV1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human CaV1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.

The bovine TRPV3 as a pathway for the uptake of Na+, Ca2+, and NH4+

Absorption of ammonia from the gastrointestinal tract results in problems that range from hepatic encephalopathy in humans to poor nitrogen efficiency of cattle with consequences for the global climate. Previous studies on epithelia and cells from the native ruminal epithelium suggest functional involvement of the bovine homologue of TRPV3 (bTRPV3) in ruminal NH4+ transport. Since the conductance of TRP channels to NH4+ has never been studied, bTRPV3 was overexpressed in HEK-293 cells and investigated using the patch-clamp technique and intracellular calcium imaging. Control cells contained the empty construct. Divalent cations blocked the conductance for monovalent cations in both cell types, with effects higher in cells expressing bTRPV3. In bTRPV3 cells, but not in controls, menthol, thymol, carvacrol, or 2-APB stimulated whole cell currents mediated by Na+, Cs+, NH4+, and K+, with a rise in intracellular Ca2+ observed in response to menthol. While only 25% of control patches showed single-channel events (with a conductance of 40.8 ± 11.9 pS for NH4+ and 25.0 ± 5.8 pS for Na+), 90% of bTRPV3 patches showed much larger conductances of 127.8 ± 4.2 pS for Na+, 240.1 ± 3.6 pS for NH4+, 34.0 ± 1.7 pS for Ca2+, and ~ 36 pS for NMDG+. Open probability, but not conductance, rose with time after patch excision. In conjunction with previous research, we suggest that bTRPV3 channels may play a role in the transport of Na+, K+, Ca2+ and NH4+ across the rumen with possible repercussions for understanding the function of TRPV3 in other epithelia.

Frequency-Based Analysis of Gramicidin A Nanopores Enabling Detection of Small Molecules with Picomolar Sensitivity.

Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a C-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 103 times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays.

Cross-Talk between Cholesterol, PIP2 and Caveolin in Regulating Kir2 Channels

Multiple types of ion channels are regulated by two lipid modulators: phosphatidylinositol-4,5-bisphosphate (PIP2) and cholesterol, both modulators being enriched in caveolae. Our earlier studies identified cholesterol as a major negative regulator of Kir2 channels that are also well-known to be critically dependent on PIP2. Furthermore, it was proposed that cholesterol regulation of ion channels might be attributed to the disruption of their association with caveolin-1 and/or PIP2. In this study, we investigated the cross-talk between cholesterol, PIP2 and caveolin in the regulation of Kir2.1 channels. We show that cholesterol depletion strengthens the interaction between Kir2 channels and PIP2 and that a single-point mutation of Kir2.1 L222I that renders the channels cholesterol insensitive abrogates this effect. Furthermore, we present direct evidence that Kir2.1 is negatively regulated by Cav-1 and that Kir2.1-L222I mutation also abrogates the sensitivity of the channels to caveolin. Kinetic analysis of single-channel events also points to a common mechanism for cholesterol and Cav-1 regulation of the channels. However, neither Cav-1, nor intact caveolae are required for Kir2.1 channels to be cholesterol sensitive, thus challenging a general notion that cholesterol regulates ion channels by disruption of caveolae. Furthermore, we present first insights into the structural determinants of the cross-talk between the sensitivity of Kir2 channels to cholesterol, PIP2 and to caveolin: based on recent studies, we suggest that cholesterol, PIP2 and caveolin regulate the channels through distinct binding sites but that the signals generated by the binding of these modulators converge in regulating Kir2.1 gating.

Studying Clustering of KcsA Channels using Single-Channel Voltage-Clamp Fluorescence Imaging

Protein oligomerization lies at the core of numerous biological processes, from cellular signal transduction to muscle contraction and cellular metabolism. It also notably drives the function of ion channels, as several subunits are often required to stabilize and gate specific ions through the conducting pore. In addition to these protein-protein interactions within ion channels, oligomerization or “clustering” of several ion channels has been observed. Although ion channel clustering is not generally thought to be a prerequisite for their physiological function, it has been suggested to modify the function of several ion channels. In this study, we optically observed clustering of single KcsA (E71A mutant) channels in planar lipid bilayer using single molecule fluorescence, while simultaneously measuring single channel currents. We found that clustering was not caused by direct protein-protein interactions but was mediated via microdomains induced in the lipid matrix. Interestingly, while KcsA clusters remained in the lipid bilayer, cooperative gating events with conductance levels multiple to the “normal” single channel events were often recorded. These coupled events were also observed in absence of a negatively charged phospholipid, which is believed to be required for KcsA activity. To understand the role of microdomains in coupled activity, we explored the physical properties of the lipid which could promote channel opening. Our findings show that lipids able to produce negative curvature in the lamellar liquid crystal phase (Lα) can induce channel activity, even without any negative charged lipid. We propose that the lateral pressure distribution of such lipid on the channel supports channel opening. This idea is in line with the cooperative gating of KcsA in the presence of clusters. The assembly of oligomers helps to overcome the energy barrier for opening by distributing the lateral pressure more favorably.

Model Neural Membrane Droplet Interface Bilayers from Brain Total Lipid Extract for Studying Membrane-Peptide Interactions with Amyloid-β

ABSTRACTDroplet interface bilayers (DIBs) are formed using brain total lipid extract (BTLE) to create a synthetic bilayer whose lipid composition mimics that of neural cells. The electrical properties of BTLE DIBs, specifically membrane resistance, capacitance, and rupture potential, are determined and compared to the properties of bilayers formed using DPhPC, the most common lipid within the growing DIB field. There is no significant difference in the resistance or rupture potential of BTLE and DPhPC bilayers, for instance with average nominal resistance over 200 GΩ and rupture potential around 200 mV. In electrical measurements with either DPhPC or BTLE bilayers, applied voltages of up to ±150 mV yield low levels of leakage current. Upon interaction with the pore-forming amyloid-beta (Aβ) peptide, both bilayers display sudden significant voltage-dependent increases in conductance with characteristic threshold voltages well below 150 mV. Discrete single-channel type events are observed in the case of Aβ-BTLE whereas disordered fluctuating conductance is observed with Aβ-DPhPC. Circular dichroism is measured for Aβ incubated with BTLE and DPhPC liposomes, as well as pure Aβ, at a range of temperatures over a period of several weeks. Changes in secondary structure of liposome-bound and pure Aβ are significantly affected by both lipid type and temperature. A key finding includes the 100% conversion of Aβ to alpha-helical confirmation within 24 hours when incubated with liposomes (of either type) at physiologically relevant 37°C. The 100% alpha-helical Aβ is maintained for up to 2 weeks at 37°C when incubated with liposomes, although other structures begin to emerge after the 14 day mark. Between 14-31 days after reconstitution, Aβ incubated at 37C with BTLE bilayers displays longer lasting alpha-helical content than DPhPC. At the same temperature, pure Aβ is 100% alpha helical only at the 1 day mark with apparent restructuring from day 2 through day 31. Refrigerated Ab samples do not display 100% alpha-helical structure across the entire 31 day testing period. The differences observed between BTLE and DPhPC in both electrophysiological and spectroscopic experiments may be a result of phase separations or other variations in membrane fluidity that result from the use of a homogeneous total lipid extract. Time and temperature play essential roles in the aggregation and restructuring of potentially toxic Aβ oligomers, and there is motivation for further efforts to elicit the mechanistic differences in interactions of Ab with BTLE compared to DPhPC.

Microfabricated Si Chips Containing Human Ether-a-Go-Go-Related Gene Channels As a Platform for Drug Safety Screenings

Reconstitution of ion channel proteins in artificial bilayer lipid membranes (BLMs) provides an excellent system for drug screenings. However, the mechanical fragility of BLMs prevents them from being widely used. Recently, we have succeeded in the formation of mechanically stable BLMs by preparing membranes in microapertures fabricated in silicon (Si) chips (1). The key feature for BLM stabilization is probably the tapered shape of the aperture edge, which allows reducing the stress on the bilayer at the aperture edge. In this presentation, we report on an application of this stable BLM device to measure ion currents of human ether-a-go-go-related gene (hERG) channels (2), which have been found to be related to serious arrhythmic side effects following drug treatment. Microapertures (diameter: 20-60 micrometer) were fabricated in a silicon nitride (Si3N4) layer on a Si chip. The surface of the Si chip was coated with thermal oxide and Teflon-AF. After the chip was treated with a silane-coupling reagent, artificial BLMs were prepared in the microapertures by folding up two phospholipid monolayers in the apertures. Owing to the use of SiO2/Teflon dielectric layer, the current noise level of the BLM was reduced, and clear single-channel events with conductance of 11 ± 1.2 pS were observed for hERG channels. This conductance value is similar to a reported one (12 pS) by the patch-clamp method. The channel current was completely blocked by E-4031, a specific blocker for hERG, and astemizole, an antihistamine that has been withdrawn due to its side effect on hERG channels. Thus the sensitivity of the hERG channels to typical drugs was retained in the Si chips. The BLM with incorporated hERG channels was stable for 65 h and tolerant to repetitive solution exchanges. The next step is to extend this stable BLM device to a multisite array format. The realization of an ion channel array will open a variety of applications, including high-throughput drug screening procedures that can be a complement to the patch-clamp method.ReferencesA. Hirano-Iwata, K. Aoto, A. Oshima, T. Taira, R. Yamaguchi, Y. Kimura, M. Niwano, Langmuir, 26, 1949–1952 (2010).A. Oshima, A. Hirano-Iwata, H. Mozumi, Y. Ishinari, Y. Kimura, and M. Niwano, Anal. Chem., 85, 4363-4369 (2013).

Glycine transporter-1 controls nonsynaptic inhibitory actions of glycine receptors in the neonatal rat hippocampus.

Although functional glycinergic synapses have not been identified in the hippocampus, neurons in this area express Cl(-) permeable extrasynaptic glycine receptors (GlyRs). In experiments on CA3 pyramidal neurons on postnatal day 0-6 rat hippocampal slices, we detected robust GlyR activity as a tonic current and as single-channel events. Glycine release was independent of neuronal activity or extracellular Ca(2+). The endogenous GlyR activity was strongly enhanced by inhibition of the glycine-transporter-1 (GlyT1). Blockade of GlyT1 also caused a profound increase in the baseline current induced by exogenous glycine. Inhibition of GlyT1 reduced the frequency of spontaneous network events known as field giant depolarizing potentials (fGDPs) and of the unit activity in the absence of synaptic transmission. This inhibitory action on fGDPs was mimicked by applying 2 μm glycine or 0.1 μm isoguvacine, a GABAA-receptor agonist. Furthermore, 2 μm glycine suppressed unit spiking in the absence of synaptic transmission. Hence, despite the well known depolarizing Cl(-) equilibrium potential of neonatal hippocampal neurons, physiologically relevant extracellular glycine concentrations can exert an inhibitory action. The present data show that, akin to GABA uptake, GlyT1 exerts a powerful modulatory action on network events in the newborn hippocampus.

Gating modulation by heat of the polycystin transient receptor potential channel PKD2L1 (TRPP3)

Polycystic kidney disease 2-like 1 (PKD2L1), previously called transient receptor potential polycystin 3 (TRPP3), forms a voltage-dependent nonselective cation channel that exhibits large tail currents triggered by repolarization after depolarization. Since it has previously been proposed that temperature sensitivity of some TRP channels is linked to the voltage-dependent gating, we here investigated heating effects on PKD2L1 currents in human embryonic kidney HEK293T cells overexpressing mouse PKD2L1. Tail PKD2L1 currents were increased by heating to 32°C, but decreased at more than 36°C. Voltage dependency of the PKD2L1 channel was shifted by heating in a bimodal fashion: an increase in temperature to 32°C and to 36°C shifted the activation curves toward the left and the right, respectively. In addition, heating accelerated deactivation of tail PKD2L1 currents. To analyze the channel gating kinetics, single-channel events of the PKD2L1 channel were recorded at hyperpolarized potentials under whole-cell configurations. A rise in temperature decreased the open probability of the channel. Dwell-time analysis showed that both open and closed dwell times during heating were shorter than those at room temperature. Interestingly, a rapid temperature drop after heating markedly enhanced the PKD2L1 currents at both single-channel and whole-cell levels. The rebound activation of the PKD2L1 channel was due to an increase in the open probability but not in the single-channel conductance. These results suggest that heating opens but subsequently inactivates PKD2L1 channels, which is essential for the rebound activation of the channel after heating.

TARP‐associated AMPA receptors display an increased maximum channel conductance and multiple kinetically distinct open states

Fast excitatory synaptic transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPARs), whose biophysical properties are dramatically modulated by the presence of transmembrane AMPAR regulatory proteins (TARPs). To help construct a kinetic model that will realistically describe native AMPAR/TARP function, we have examined the single-channel properties of homomeric GluA1 AMPARs in combination with the TARPs, γ-2, γ-4 and γ-5. In a saturating concentration of agonist, each of these AMPAR/TARP combinations gave rise to single-channel currents with multiple conductance levels that appeared intrinsic to the receptor-channel complex, and showed long-lived subconductance states. The open time and burst length distributions of the receptor complexes displayed multiple dwell-time components. In the case of γ-2- and γ-4-associated receptors, these distributions included a long-lived component lasting tens of milliseconds that was absent from both GluA1 alone and γ-5-associated receptors. The open time distributions for each conductance level required two dwell-time components, indicating that at each conductance level the channel occupies a minimum of two kinetically distinct open states. We have explored how these data place novel constraints on possible kinetic models of TARP-associated AMPARs that may be used to define AMPAR-mediated synaptic transmission.

Communicating with Our External and Internal Environments

The following reviews illustrate how, to be successful, life must communicate and interact with both the external and internal worlds. In his review ([3][1]), Dietmar Kultz explores the evolution and physiological mechanisms of adaptations to osmotic stress in fish that tolerate high salinity. The

Imposing Resolution on Single Channel Data Containing Sublevels

Several classes of ion channel, including AMPA-type glutamate receptors, exhibit single channel events of different amplitudes (‘sublevels’). In some cases, sublevel behaviour might reflect fundamental principles of channel gating (e.g. ROSenmund et al., Science 1998). Such observations provide motivation for developing analysis methods that treat sublevels correctly. All idealisation methods (including threshold crossing, time course fitting and segmental k-means) have a limited temporal resolution for detecting brief events. To account for this, a fixed resolution or ‘dead time’ is normally imposed post hoc on the idealised data, on the basis of a low frequency of false event detection (‘error rate’). This error rate always varies steeply with the signal to noise ratio (SNR). In records with sublevels, SNR - and thus the error rate - varies in a discrete way from event to event. Small events have a lower SNR and thus a dramatically higher error rate. By inverting the relation between error rate and SNR, I propose a simple method to take account of the amplitude dependence of the error rate in these records, by varying the resolution in order to hold the error rate constant across the data set. Using simulated data, I show the distortions of the apparent duration of events that are introduced by imposing conservative, cavalier and variable resolutions. Although this approach complicates data display, the correct treatment of missed events in subsequent mechanism fitting should now be possible. Thus, extensions of missed event correction to data containing sublevels should consider explicitly accounting for event-dependent resolution.

Swelling-Activated Anion Channels Are Essential for Volume Regulation of Mouse Thymocytes

Channel-mediated trans-membrane chloride movement is a key process in the active cell volume regulation under osmotic stress in most cells. However, thymocytes were hypothesized to regulate their volume by activating a coupled K-Cl cotransport mechanism. Under the patch-clamp, we found that osmotic swelling activates two types of macroscopic anion conductance with different voltage-dependence and pharmacology. At the single-channel level, we identified two types of events: one corresponded to the maxi-anion channel, and the other one had characteristics of the volume-sensitive outwardly rectifying (VSOR) chloride channel of intermediate conductance. A VSOR inhibitor, phloretin, significantly suppressed both macroscopic VSOR-type conductance and single-channel activity of intermediate amplitude. The maxi-anion channel activity was largely suppressed by Gd3+ ions but not by phloretin. Surprisingly, [(dihydroindenyl)oxy] alkanoic acid (DIOA), a known antagonist of K-Cl cotransporter, was found to significantly suppress the activity of the VSOR-type single-channel events with no effect on the maxi-anion channels at 10 μM. The regulatory volume decrease (RVD) phase of cellular response to hypotonicity was mildly suppressed by Gd3+ ions and was completely abolished by phloretin suggesting a major impact of the VSOR chloride channel and modulatory role of the maxi-anion channel. The inhibitory effect of DIOA was also strong, and, most likely, it occurred via blocking the VSOR Cl− channels.

Analysis of Glycoprotein Interactions with Multichannel Membranes

We investigate the interaction of glycoproteins (including ovalbumin and alpha 1 antitrypsin) with lipid membranes by using a number of physical methods including light spectroscopy and ion current measurements. Glycoproteins are a class of proteins that are decorated with sugar (glycan) chains. Most known glycoproteins have been shown to play a role in intercellular interactions but the exact role of the glycan chains is still under investigation. The ion-current measurements involve the channel forming peptide gramicidin A (gA) which is used as a reporter. Alteration of gA activity indicates an interaction of the added glycoprotein with the membrane. For the analysis of such events, single channel recordings are preferable because the calculation of channel on and off times is straightforward. Obtaining single channel events however depends critically not only on the amount of gA present in the sample but also on the amount of the added glycoprotein. Therefore, the majority of the recordings exhibit multichannel events. We show how the multichannel events can be used (rather than discared) to calculate single channel parameters. This analysis allows us to characterize the interaction of glycoproteins with lipid bilayers which can help elucidate the role of glycosylation in cell signaling.

CFTR Mediates Noradrenaline-Induced ATP Efflux from DRG Neurons

In our earlier study, noradrenaline (NA) stimulated ATP release from dorsal root ganglion (DRG) neurons as mediated via β3 adrenoceptors linked to Gs protein involving protein kinase A (PKA) activation, to cause allodynia. The present study was conducted to understand how ATP is released from DRG neurons. In an outside-out patch-clamp configuration from acutely dissociated rat DRG neurons, single-channel currents, sensitive to the P2X receptor inhibitor PPADS, were evoked by approaching the patch-electrode tip close to a neuron, indicating that ATP is released from DRG neurons, to activate P2X receptor. NA increased the frequency of the single-channel events, but such NA effect was not found for DRG neurons transfected with the siRNA to silence the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the immunocytochemical study using acutely dissociated rat DRG cells, CFTR was expressed in neurons alone, but not satellite cells, fibroblasts, or Schwann cells. It is concluded from these results that CFTR mediates NA-induced ATP efflux from DRG neurons as an ATP channel.

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.