Whole-cell Current Recordings Research Articles

Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Cav1.2 channels.

Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits β2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor β2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

Cell-Flow Technique

Various devices have been used to flow neurotransmitter solutions over cells containing receptors (e.g., ligand-gated ion channels) for whole-cell current recordings. With many of the devices, the orientation between the porthole of the flow device and the cell is not maintained absolutely constant. Orientation is critical for reproducibility in kinetic experiments. To be able to change the composition of the flowing solution during an experiment and still maintain a constant orientation, we use the cell-flow device described here. A peristaltic pump, a stainless steel U-tube, two different sizes of peristaltic tubing, and a solenoid valve are required to create a simple solution exchange system that can rapidly apply and remove solutions over the surface of a cell in tens of milliseconds. This system allows one to test multiple conditions on a cell containing the receptor of interest while constantly "washing" the cell with extracellular buffer solution between experimental applications. The use of the solenoid valve allows for the application of solutions to be precisely timed and controlled by a computer during electrophysiological current recording.

Voltage-gated sodium channels were differentially expressed in human normal prostate, benign prostatic hyperplasia and prostate cancer cells.

Voltage-gated sodium channels (VGSCs) are expressed not only in excitable cells but also in numerous metastatic cells, particularly in certain types of cancer cells. In some types of cancer, including prostate cancer, the expression of VGSCs is associated with cancer migration, invasion and metastasis in vivo. However, the detailed expression profiles of VGSC α subunits in normal human prostate, in prostatic hyperplasia and prostatic cancer remain controversial. In the present study, quantitative polymerase chain reaction was used to systematically detect all subtypes of VGSC α subunits in normal human prostate, benign prostatic hyperplasia (BPH) and prostate cancer cells. The expression profile of VGSC α subunits was observed to differ between these cell types. Nav1.5 was the major isoform expressed in normal human prostate tissue, while Nav1.5 and Nav1.2 were the predominant isoforms in BPH tissue. However, in PC-3 and LNCaP cells, two typical prostate cancer cell lines, Nav1.6 and Nav1.7 were abundantly expressed. By comparing the relative expression levels of Nav1.5, Nav1.6 and Nav1.7 in these cells, the mRNA levels of Nav1.6 and Nav1.7 were identified to be 6- to 27-fold higher in PC-3 and LNCaP cells than in either normal or BPH samples (P<0.05); however, Nav1.5 mRNA levels were relatively lower compared with those of Nav1.6 or Nav1.7 in all cells analyzed. To confirm whether Nav1.6 and Nav1.7 expression in cancer cells was functional, a patch-clamp technique was used to record whole-cell currents. A tetrodotoxin-sensitive sodium current was successfully recorded in PC-3 cells, but not in LNCaP cells. It was concluded that although all types of VGSC α subunits exhibited low expression levels in normal prostate and BPH cells, both Nav1.6 and Nav1.7 were significantly upregulated in the prostate cancer cell lines, suggesting these subtypes may be potential diagnostic markers and therapeutic targets for certain types of prostate cancer in humans.

The potent and selective α4β2*/α6*-nicotinic acetylcholine receptor partial agonist 2-[5-[5-((S)Azetidin-2-ylmethoxy)-3-pyridinyl]-3-isoxazolyl]ethanol demonstrates antidepressive-like behavior in animal models and a favorable ADME-tox profile.

Preclinical and clinical studies demonstrated that the inhibition of cholinergic supersensitivity through nicotinic antagonists and partial agonists can be used successfully to treat depressed patients, especially those who are poor responders to selective serotonin reuptake inhibitors (SSRIs). In our effort to develop novel antidepressant drugs, LF-3-88 was identified as a potent nicotinic acetylcholine receptor (nAChR) partial agonist with subnanomolar to nanomolar affinities for β2-containing nAChRs (α2β2, α3β2, α4β2, and α4β2*) and superior selectivity away from α3β4 − (Ki > 104 nmol/L) and α7-nAChRs (Ki > 104 nmol/L) as well as 51 other central nervous system (CNS)-related neurotransmitter receptors and transporters. Functional activities at different nAChR subtypes were characterized utilizing 86Rb+ ion efflux assays, two-electrode voltage-clamp (TEVC) recording in oocytes, and whole-cell current recording measurements. In mouse models, administration of LF-3-88 resulted in antidepressive-like behavioral signatures 15 min post injection in the SmartCube® test (5 and 10 mg/kg, i.p.; about 45-min session), decreased immobility in the forced swim test (1–3 mg/kg, i.p.; 1–10 mg/kg, p.o.; 30 min pretreatment, 6-min trial), and decreased latency to approach food in the novelty-suppressed feeding test after 29 days chronic administration once daily (5 mg/kg but not 10 mg/kg, p.o.; 15-min trial). In addition, LF-3-88 exhibited a favorable profile in pharmacokinetic/ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) assays. This compound was also shown to cause no mortality in wild-type Balb/CJ mice when tested at 300 mg/kg. These results further support the potential of potent and selective nicotinic partial agonists for use in the treatment of depression.

Deciphering the Kinetic and Gating Properties of Purinergic P2X7 Receptor Channels

Purinergic P2X7 receptors (P2X7Rs) are trimeric nonselective cation channels possessing three intrasubunit orthosteric binding sites and unidentified number of allosteric binding sites. In previous work, we showed that the same receptor is capable of exhibiting sensitization and pore dilation (leading to the formation of biphasic currents and cell death) and/or desensitization (leading to a decline in current amplitude and cell life signaling) during sustained application of orthosteric agonists, depending on their concentrations. This was done in part by developing a 12-state Markov model consisting of naive (not previously exposed to orthosteric agonist), desensitized, and sensitized/dilated states that reproduced whole-cell current recordings generated experimentally. In this model, we assumed that the occupancy of one or two ATP-binding sites of naive receptors favored transition from open to desensitized states, whereas the occupancy of the third binding site favored receptor sensitization. Our goal here is to extend this modeling study by examining how these model cells behave under various ionic conditions in the medium, including those that contain large organic cation N-methylD-glucamine (NMDG + ) or divalent cation such as Ca 2+ . We illustrate how the two main gating patterns behave under these ionic conditions and determine why the shift in reversal potential and the dilation of the channels are accompanied paradoxically by a decrease in the total conductance during voltage ramp protocols. The model adds more evidence to our previous hypothesis, suggesting that dilation is masking desensitization. Our results also indicate that the allosteric sites through which divalent cations inhibit P2X7R should be extracellularly located.

Mechanism of Inhibition of Recombinant GABAA Receptors by Pentylenetetrazole and Its Alleviation by Anticonvulsants

Despite the wide prescription of drugs targeting postsynaptic γ-aminobutyric acid receptors (GABAARs) for seizure and epilepsy, mechanisms by which those drugs exert their effects on GABAAR are poorly understood. This is mainly attributed to the unavailability of crystal structure of the receptor and the various assembly of 19 different subunits (α1- α6, β1-β3, γ1-γ3, δ, e, π, θ, ρ1-ρ3). Here, we studied the effects of two anticonvulsants on the recombinant α1β3γ2 GABAAR using whole-cell current recordings combined with rapid kinetic techniques. Our results showed that pentylenetetrazole (PTZ), a compound used to artificially induce convulsive effect in animal models, is a mixed inhibitor of the receptor. Anticonvulsant ethosuximide (ES) and the major metabolite (α-methyl-α-pheny-succinimide, MPS) of another anticonvulsant methsuximide (MS) were found to alleviate the inhibition of the receptor by PTZ, while MS itself doesn't. Those data suggest that the enhancement of postsynaptic GABAAR response is one of the mechanisms of the anticonvulsant effect of ES and MS. T-type calcium channel was suggested to be one of the targets of ES.Acknowledgement of support: This project was supported by grants from the National Center for Research Resources (5P20RR016467-11) and the National Institute of General Medical Sciences (8P20GM103466-11) from the National Institutes of Health. The content is solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

Rescue of Amyloid-Beta-Induced Inhibition of Nicotinic Acetylcholine Receptors by a Peptide Homologous to the Nicotine Binding Domain of the Alpha 7 Subtype

Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.

Functional Properties of Five Dictyostelium discoideum P2X Receptors

The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.

Virtual neurophysiology laboratories for life science education: action potentials and voltage-/patch-clamp recordings

Understanding neurophysiology is for many students a particularly difficult task, especially the interpretation of different measures their physiological meaning and their interrelations. It could be of great help if the students could do the recordings themselves, according to the didactically advantageous strategy learning by doing. When such experiments are not possible in regular students' courses, for whatever reasons (too difficult, no resources etc.), virtual laboratories can become valuable alternatives. During recent years, we have developed a series of virtual laboratories (Virtual Physiology), among them three neurophysiology tools. There is SimNerv for clinically still most relevant extracellular recordings of compound actions potentials. A second program, SimPatch, is for whole-cell current recordings from different types of neurons, also allowing the application of channel blockers or changing ion concentrations. These programs are providing realistically appearing laboratories on the computer screen that allow the students performing physiological and pharmacological experiments almost as in real life. All stimulus and recording parameters of the virtual devices are freely adjustable and a mathematical algorithm guarantees for the appropriate reaction of the virtual preparations. A third series of applications, SimNeuron, has been designed in a more simple, easy to overlook form to demonstrate the relations between current-clamp, whole-cell voltage-clamp and single-channel patch-clamp recordings. This program also allows changing the model neuron's parameters to examine how altered membrane properties are reflected in the different recordings. All these programs are designed with the major goal to train the students' understanding and know-how, although factual knowledge is a prerequisite for systematic experimentation in the virtual labs. With this respect, detailed tutorial and protocol form are provided, supplemented by additional simulations and animation of specific features. Figure 1 SimPatch, the Virtual Lab Figure 2 SimNeuron menu and recording examples from the current-clamp, whole-cell voltage-clamp lab, single channel patch-clamp lab (closed and open states with gating) with I-V curves for K+- (blue), Na+- (red) and unspecific cation (black) ion channels

In Intact Islets Interstitial GABA Activates GABAA Receptors That Generate Tonic Currents in α-Cells

In the rat islets γ-aminobutyric acid (GABA) is produced by the β-cells and, at least, the α-cells express the GABAA receptors (GABAA channels). In this study, we examined in intact islets if the interstitial GABA activated the GABAA receptors. We used the patch-clamp technique to record whole-cell and single-channel currents and single-cell RT-PCR to identify the cell-type we recorded from, in the intact rat islets. We further identified which GABAA receptor subunits were expressed. We determined the cell-type of 43 cells we recorded from and of these 49%, 28% and 7% were α, β and δ-cells, respectively. In the remaining 16% of the cells, mRNA transcripts of more than one hormone gene were detected. The results show that in rat islets interstitial GABA activates tonic current in the α-cells but not in the β-cells. Seventeen different GABAA receptor subunits are expressed with high expression of α1, α2, α4, α6, β3, γ1, δ, ρ1, ρ2 and ρ3 subunits whereas no expression was detected for α5 or ε subunits. The abundance of the GABAA receptor subunits detected suggests that a number of GABAA receptor subtypes are formed in the islets. The single-channel and tonic currents were enhanced by pentobarbital and inhibited by the GABAA receptor antagonist SR-95531. The single-channel conductance ranged from 24 to 105 pS. Whether the single-channel conductance is related to subtypes of the GABAA receptor or variable interstitial GABA concentrations remains to be determined. Our results reveal that GABA is an extracellular signaling molecule in rat pancreatic islets and reaches concentration levels that activate GABAA receptors on the glucagon-releasing α-cells.

Sphingosine and FTY720 are potent inhibitors of the transient receptor potential melastatin 7 (TRPM7) channels

Transient receptor potential melastatin 7 (TRPM7) is a unique channel kinase which is crucial for various physiological functions. However, the mechanism by which TRPM7 is gated and modulated is not fully understood. To better understand how modulation of TRPM7 may impact biological processes, we investigated if TRPM7 can be regulated by the phospholipids sphingosine (SPH) and sphingosine-1-phosphate (S1P), two potent bioactive sphingolipids that mediate a variety of physiological functions. Moreover, we also tested the effects of the structural analogues of SPH, N,N-dimethyl-D-erythro-sphingosine (DMS), ceramides and FTY720 on TRPM7. HEK293 cells stably expressing TRPM7 were used for whole-cell, single-channel and macropatch current recordings. Cardiac fibroblasts were used for native TRPM7 current recording. SPH potently inhibited TRPM7 in a concentration-dependent manner, whereas S1P and other ceramides did not produce noticeable effects. DMS also markedly inhibited TRPM7. Moreover, FTY720, an immunosuppressant and the first oral drug for treatment of multiple sclerosis, inhibited TRPM7 with a similar potency to that of SPH. In contrast, FTY720-P has no effect on TRPM7. It appears that SPH and FTY720 inhibit TRPM7 by reducing channel open probability. Furthermore, endogenous TRPM7 in cardiac fibroblasts was markedly inhibited by SPH, DMS and FTY720. This is the first study demonstrating that SPH and FTY720 are potent inhibitors of TRPM7. Our results not only provide a new modulation mechanism of TRPM7, but also suggest that TRPM7 may serve as a direct target of SPH and FTY720, thereby mediating S1P-independent physiological/pathological functions of SPH and FTY720.

Two open states of P2X receptor channels

The occupancy of the orthosteric ligand binding sites of P2X receptor (P2XR) channels causes the rapid opening of a small cation-permeable pore, followed by a gradual dilation that renders the pore permeable to large organic cations. Electrophysiologically, this phenomenon was shown using whole-cell current recording on P2X2R-, P2X2/X5R-, P2X4R- and P2X7R-expressing cells that were bathed in N-methyl-D-glucamine (NMDG+)-containing buffers in the presence and/or absence of small monovalent and divalent cations. The pore dilation of P2X4R and P2X7R caused a secondary current growth, whereas that of P2X2R showed a sustained kinetic coupling of dilation and desensitization, leading to receptor channel closure. The pore size of the P2X7R open and dilated states was estimated to be approximately 0.85 nm and greater than 1 nm, respectively. The P2XR pore dilation was also observed in intact cells by measurement of fluorescent dye uptake/release, application of polyethylene glycols of different sizes, and atomic force microscopy. However, pore dilation was not observed at the single channel level. Structural data describing the dilated state are not available, and the relevance of orthosteric and allosteric ligand interactions to pore dilation was not studied.

Regulation of reactive oxygen species-mediated abscisic acid signaling in guard cells and drought tolerance by glutathione

The phytohormone abscisic acid (ABA) induces stomatal closure in response to drought stress, leading to reduction of transpirational water loss. A thiol tripeptide glutathione (GSH) is an important regulator of cellular redox homeostasis in plants. Although it has been shown that cellular redox state of guard cells controls ABA-mediated stomatal closure, roles of GSH in guard cell ABA signaling were largely unknown. Recently we demonstrated that GSH functions as a negative regulator of ABA signaling in guard cells. In this study we performed more detailed analyses to reveal how GSH regulates guard cell ABA signaling using the GSH-deficient Arabidopsis mutant cad2-1. The cad2-1 mutant exhibited reduced water loss from rosette leaves. Whole-cell current recording using patch clamp technique revealed that the cad2-1 mutation did not affect ABA regulation of S-type anion channels. We found enhanced activation of Ca2+ permeable channels by hydrogen peroxide (H2O2) in cad2-1 guard cells. The cad2-1 mutant showed enhanced H2O2-induced stomatal closure and significant increase of ROS accumulation in whole leaves in response to ABA. Our findings provide a new understanding of guard cell ABA signaling and a new strategy to improve plant drought tolerance.

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets(1). The dopamine signal is terminated by diffusion(2-3), extracellular enzymes(4), and membrane transporters(5). The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine(5). More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)(5). The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis(6). Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume(7). Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

Incorporation of DPP6a and DPP6K Variants in Ternary Kv4 Channel Complex Reconstitutes Properties of A-type K Current in Rat Cerebellar Granule Cells

Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (ISA). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of ISA found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native ISA channels. Differential expression of DPP6 variants was detected in rat CG cells, with DPP6K (41±3%)>DPP6a (33±3%)>>DPP6S (18±2%)>DPP6L (8±3%). To better understand how DPP6 variants shape native neuronal ISA, we focused on studying interactions between the two dominant variants, DPP6K and DPP6a. Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve. By contrast, DPP6a uses its distinct N-terminus to directly confer rapid N-type inactivation independently of KChIP3a. When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function. The more rapid inactivation from DPP6a dominates during strong depolarization; however, DPP6K produces a negative shift in the steady-state inactivation curve and introduces a slow phase of recovery from inactivation. A direct comparison to the native CG cell ISA shows that these mixed effects are present in the native channels. Our results support the hypothesis that the precise expression and co-assembly of different auxiliary subunit variants are important factors in shaping the ISA functional properties in specific neuronal populations.

Regulation of polysynaptic subthalamonigral transmission by D2, D3 and D4 dopamine receptors in rat brain slices

Dopamine depletion in experimental models of Parkinson's disease promotes burst firing of neurons in the subthalamic nucleus (STN) and substantia nigra zona reticulata (SNR). A synaptically generated form of burst firing has been shown to arise from complex excitatory postsynaptic currents (EPSCs) that are evoked in SNR neurons by STN stimulation. The present experiments were designed to characterize actions of dopamine on complex EPSCs in slices of rat brain. Using patch pipettes to record whole-cell currents under voltage clamp, dopamine (30 μm) caused a reversible 64% reduction in complex EPSC charge. This effect was partially mimicked by D(2), D(3) and D(4) receptor agonists, and the action of dopamine could be nearly completely blocked by the combined effects of the D(2/3) antagonist sulpiride and the D(4) antagonist L-745,870. Local application of dopamine to the STN caused a larger inhibition of the complex EPSC (55% reduction) than did dopamine application to the SNR (15% reduction). Simple, monophasic EPSCs, which were evoked in SNR neurons by stimulating the SNR close to the recording pipette, were inhibited to a smaller extent compared to complex EPSCs. Bursts of action potentials evoked in SNR neurons by STN stimulation were inhibited by dopamine to a greater extent than was spontaneous firing. These results show that dopamine D(2)-like receptors inhibit complex EPSCs and burst discharges in the SNR by acting within the STN to suppress transmission in the subthalamonigral pathway. Dopamine receptor-mediated inhibition of polysynaptic connections in the STN might be beneficial in the treatment of Parkinson's disease.

Intramembrane Proton Binding Site Linked to Activation of Bacterial Pentameric Ion Channel

Prokaryotic orthologs of eukaryotic Cys-loop receptor channels recently emerged as structural and mechanistic surrogates to investigate this superfamily of intercellular signaling proteins. Here, we examine proton activation of the prokaryotic ortholog GLIC using patch clamp electrophysiology, mutagenesis, and molecular dynamics (MD) simulations. Whole-cell current recordings from human embryonic kidney (HEK) 293 cells expressing GLIC show half-maximal activation at pH 6, close to the pK(a) of histidine, implicating the three native His residues in proton sensing linked to activation. The mutation H235F abolishes proton activation, H277Y is without effect, and all nine mutations of His-127 prevent expression on the cell surface. In the GLIC crystal structure, His-235 on transmembrane (TM) α-helix 2, hydrogen bonds to the main chain carbonyl oxygen of Ile-259 on TM α-helix 3. MD simulations show that when His-235 is protonated, the hydrogen bond persists, and the channel remains in the open conformation, whereas when His-235 is deprotonated, the hydrogen bond dissociates, and the channel closes. Mutations of the proximal Tyr-263, which also links TM α-helices 2 and 3 via a hydrogen bond, alter proton sensitivity over a 1.5 pH unit range. MD simulations show that mutations of Tyr-263 alter the hydrogen bonding capacity of His-235. The overall findings show that His-235 in the TM region of GLIC is a novel proton binding site linked to channel activation.

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-μM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.

Characterization of a novel, dominant negative KCNJ2 mutation associated with Andersen-Tawil syndrome

Andersen-Tawil syndrome is characterized by periodic paralysis, ventricular ectopy, and dysmorphic features. Approximately 60% of patients exhibit loss-of-function mutations in KCNJ2, which encodes the inwardly rectifying K+ channel pore forming subunit Kir2.1. Here, we report the identification of a novel KCNJ2 mutation (G211T), resulting in the amino acid substitution D71Y, in a patient presenting with signs and symptoms of Andersen-Tawil syndrome. The functional properties of the mutant subunit were characterized using voltage-clamp experiments on transiently transfected HEK-293 cells and neonatal mouse ventricular myocytes. Whole-cell current recordings of transfected HEK-293 cells demonstrated that the mutant protein Kir2.1-D71Y fails to form functional ion channels when expressed alone, but co-assembles with wild-type Kir2.1 subunits and suppresses wild-type subunit function. Further analysis revealed that current suppression requires at least two mutant subunits per channel. The D71Y mutation does not measurably affect the membrane trafficking of either the mutant or the wild-type subunit or alter the kinetic properties of the currents. Additional experiments revealed that expression of the mutant subunit suppresses native IK1 in neonatal mouse ventricular myocytes. Simulations predict that the D71Y mutation in human ventricular myocytes will result in a mild prolongation of the action potential and potentially increase cell excitability. These experiments indicate that the Kir2.1-D71Y mutant protein functions as a dominant negative subunit resulting in reduced inwardly rectifying K+ current amplitudes and altered cellular excitability in patients with Andersen-Tawil syndrome.

Molecular determinants of hERG potassium channel inhibition by disopyramide

The Class Ia antiarrhythmic drug disopyramide (DISO) causes QT interval prolongation that is potentially dangerous in acquired Long QT Syndrome but beneficial in short QT syndrome, through inhibition of the hERG-encoded channels responsible for rapid delayed rectifier K+ current (IKr). In this study, alanine mutants of hERG S6 and pore helix residues and MthK-based homology modelling and ligand docking were used to investigate molecular determinants of DISO binding to hERG. Whole-cell hERG current (IhERG) recordings were made at 37°C from HEK-293 cells expressing WT or mutant hERG channels. WT outward IhERG tails were inhibited with an IC50 of 7.3μM, whilst inward IhERG tails in a high [K+]e of 94mM were blocked with an IC50 of 25.7μM. The IC50 for the Y652A mutation was ~55-fold that of WT IhERG; this mutation also abolished a leftward shift in voltage-dependent IhERG activation present for WT hERG. The IC50 for F656A IhERG was ~51 fold its corresponding WT control. In contrast to previously studied methanesulphonanilide hERG inhibitors, neither the G648A S6 nor the T623A and S624A pore helical mutations modified DISO IC50. Computational docking with the hERG model showed that DISO did not exhibit a single unique binding pose; instead several low energy binding poses at the lower end of the pore cavity favoured interactions with Y652 and F656. In the WT hERG model DISO did not interact directly with residues at the base of the pore helix, consistent with the minimal effect of mutation of these residues on drug block.