Two-electrode Voltage Clamp Recordings Research Articles

The use of Xenopus oocytes in drug screening

Introduction: The physiological roles of ion channels are receiving increased interest in both basic research and drug discovery, and a demand for pharmacological approaches that can characterize or screen ion channels and their ligands with higher throughput has emerged. Traditionally, screening of compound libraries at ion channel targets has been performed using assays such as binding assays, fluorescence-based assays and flux assays that allow high-throughput, but sacrifice high data quality. The use of these assays with ion channel targets can also be problematic, emphasizing the usefulness of automated Xenopus oocyte electrophysiological assays in drug screening.Areas covered: This review summarizes the use of Xenopus oocytes in drug screening, presents the advantages and disadvantages of the use of Xenopus oocytes as expression system, and addresses the options available for automated two-electrode voltage-clamp recordings from Xenopus oocytes.Expert opinion: Automated and manual Xenopus oocyte two-electrode voltage-clamp recordings are useful and important techniques in drug screening. Although they are not compatible with high-throughput experimentation, these techniques are excellent in combination or as alternatives to fluorescence-based assays for hit validation, screening of focused compound libraries and safety screening on ion channels with their high flexibility for the choice of molecular targets, quality of data and reproducibility.

Quantification of the Mg2+-induced potency shift of amantadine and memantine voltage-dependent block in human recombinant GluN1/GluN2A NMDARs

Clinically, amantadine and memantine are drugs whose therapeutic utility is linked to their ability to block N-methyl-D-aspartate receptors (NMDARs) in a voltage-dependent manner. Nevertheless many studies that have characterized the pharmacological actions of amantadine and memantine have done so in the absence of physiological levels of Mg2+ ions. This study quantifies the extent to which Mg2+ alters the potency of the block produced by both amantadine and memantine at human recombinant GluN1/GluN2A NMDARs. Human recombinant GluN1/GluN2A NMDARs were expressed in Xenopus laevis oocytes and two-electrode voltage-clamp recordings were made at −80, −60 and −40mV to quantify amantadine and memantine block in the absence and presence of Mg2+. Amantadine and memantine blocked human GluN1/GluN2A NMDARs in a voltage-dependent manner with IC50 values (at −80mV) of 49±6μM (n=7) and 1.0±0.3μM (n=7), respectively. In the presence of Mg2+ (1mM) the equivalent IC50 values were 165±10μM (n=6) and 6.6±0.3μM (n=5). Similarly in the presence of amantadine or memantine the potency of Mg2+ in blocking GluN1/GluN2A NMDARs was reduced. The decrease in the potencies of both amantadine and memantine in the presence of physiological concentrations of Mg2+ indicates that other targets (e.g. α7-nicotinic acetylcholine receptors and 5-HT3 receptors) in addition to NMDARs may well be sites of the therapeutic action of these channel blockers.

Molecular determination of selectivity of the site 3 modulator (BmK I) to sodium channels in the CNS: a clue to the importance of Nav1.6 in BmK I-induced neuronal hyperexcitability

BmK I, a site-3-specific modulator of VGSCs (voltage-gated sodium channels) from the Chinese scorpion Buthus martensi Karsch, can induce spontaneous nociception and hyperalgesia and generate epileptiform responses in rats, which is attributed to the modulation of VGSCs in the neural system. However, which VGSC subtype is targeted by BmK I remains to be identified. Using two-electrode voltage-clamp recording, we studied the efficacy and selectivity of BmK I to three neuronal VGSCs co-expressed with the auxiliary β1 subunit in Xenopus oocytes. Results revealed that BmK I induced a large increase in both transient and persistent currents in mNav1.6α/β1 (where m indicates mouse), which correlated with a prominent reduction in the fast component of inactivating current. In comparison, BmK I-increased currents of rNav1.2α/β1 (where r indicates rat) and rNav1.3α/β1 were much smaller. The EC50 values of BmK I for rNav1.2α/β1 (252±60nM) and mNav1.6α/β1 (214±30nM) were similar and roughly half of that for rNav1.3α/β1 (565±16nM). Moreover, BmK I only accelerated the slow inactivation development and delay recovery of mNav1.6α/β1 through binding to the channel in the open state. Residue-swap analysis verified that an acidic residue (e.g. Asp1602 in mNav1.6) within the domain IV S3-S4 extracellular loop of VGSCs was crucial for the selectivity and modulation pattern of BmK I. Our findings thus provide the molecular determinant explaining the divergent and intriguing behaviour of neuronal VGSCs in response to site-3-specific modulators, indicating that these subtypes play different roles in BmK I-induced hyperexcitablity in rat models.

A potent potassium channel blocker from Mesobuthus eupeus scorpion venom

Scorpion venom-derived peptidyl toxins are valuable pharmacological tools for investigating the structure–function relationship of ion channels. Here, we report the purification, sequencing and functional characterization of a new K + channel blocker (MeuKTX) from the venom of the scorpion Mesobuthus eupeus. Effects of MeuKTX on ten cloned potassium channels in Xenopus oocytes were evaluated using two-electrode voltage-clamp recordings. MeuKTX is the orthologue of BmKTX (α-KTx3.6), a known Kv1.3 blocker from the scorpion Mesobuthus martensii, and classified as α-KTx3.13. MeuKTX potently blocks rKv1.1, rKv1.2 and hKv1.3 channels with 50% inhibitory concentration (IC 50) of 203.15 ± 4.06 pM, 8.92 ± 2.3 nM and 171 ± 8.56 pM, respectively, but does not affect rKv1.4, rKv1.5, hKv3.1, rKv4.3, and hERG channels even at 2 μM concentration. At this high concentration, MeuKTX is also active on rKv1.6 and Shaker IR. Our results also demonstrate that MeuKTX and BmKTX have the same channel spectrum and similar pharmacological potency. Analysis of the structure–function relationships of α-KTx3 subfamily toxins allows us to recognize several key sites which may be useful for designing toxins with improved activity on hKv1.3, an attractive target for T-cell mediated autoimmune diseases.

Pharmacological modulation of brain Nav1.2 and cardiac Nav1.5 subtypes by the local anesthetic ropivacaine

The present study was aimed to investigate the pharmacological modulatory effects of ropivacaine, an amide-type local anesthetic, on rat Nav1.2 (rNav1.2) and rNav1.5, the two Na(+) channel isoforms heterologously expressed in Xenopus oocytes and in HEK293t cell line, respectively. Two-electrode voltage-clamp (TEVC) and whole-cell patch-clamp recordings were employed to record the whole-cell currents. Ropivacaine induced tonic inhibition of peak Na(+) currents of both subtypes in a dose- and frequency-dependent manner. rNav1.5 appeared to be more sensitive to ropivacaine. In addition, for both Na(+) channel subtypes, the steady-state inactivation curves, but not the activation curves, were significantly shifted to the hyperpolarizing direction by ropivacaine. Use-dependent blockade of both rNav1.2 and rNav1.5 channels was induced by ropivacaine through a high frequency of depolarization, suggesting that ropivacaine could preferentially bind to the 2 inactivated Na(+) channel isoforms. The results will be helpful in understanding the pharmacological modulation by ropivacaine on Nav1.2 subtype in the central nervous system, and on Nav1.5 subtype abundantly expressed in the heart.

α-Conotoxin AuIB Isomers Exhibit Distinct Inhibitory Mechanisms and Differential Sensitivity to Stoichiometry of α3β4 Nicotinic Acetylcholine Receptors

Non-native disulfide isomers of alpha-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of "globular" and "ribbon" isomers of alpha-conotoxin AuIB have been characterized on alpha3beta4 nicotinic acetylcholine receptors (nAChRs) heterologously expressed in Xenopus oocytes. Using two-electrode voltage clamp recording, we showed that the inhibitory efficacy of the ribbon isomer of AuIB is limited to approximately 50%. The maximal inhibition was stoichiometry-dependent because altering alpha3:beta4 RNA injection ratios either increased AuIB(ribbon) efficacy (10alpha:1beta) or completely abolished blockade (1alpha:10beta). In contrast, inhibition by AuIB(globular) was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for the 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity alpha3beta4 nAChRs with corresponding EC(50) values of 22.6 and 176.9 microM, respectively. Increasing the agonist concentration antagonized the inhibition of LS alpha3beta4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS alpha3beta4 nAChRs by AuIB(globular) was unaffected. Inhibition of LS and HS alpha3beta4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both alpha3beta4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS alpha3beta4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS alpha3beta4 nAChRs competitively, whereas AuIB(globular) inhibits alpha3beta4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.

Fragrant Dioxane Derivatives Identify β1-Subunit-containing GABAA Receptors

Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

Implementation of a Fluorescence-Based Screening Assay Identifies Histamine H3 Receptor Antagonists Clobenpropit and Iodophenpropit as Subunit-Selective N-Methyl-d-Aspartate Receptor Antagonists

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.

Mutational Analysis of the Prokaryotic Pentameric Ligand-Gated Ion Channel GLIC

For pentameric ligand-gated ion channels (pLGICs), the movements involved in coupling neurotransmitter binding to channel activation are unclear. The crystal structures of the prokaryotic pLGICs homologs from E. chrysanthemi (ELIC) and G. violaceus (GLIC) in presumed closed and open channel conformations provide unparalleled opportunities to explore how ligand binding triggers channel opening. GLIC is a proton-gated channel. To test the hypothesis that the proton binding site in GLIC is located in an analogous position as the agonist binding sites of eukaryotic pLGICs and is formed, in part, by acidic amino acids, we neutralized acidic residues in Loops A (E74Q), C (E176Q, D177N, E180Q), and F (D144N, E146Q). Two-electrode voltage clamp recordings from wild-type and mutant GLIC channels expressed in Xenopus oocytes showed that the mutations had no effect on proton-mediated channel gating (pH50, pHmax) suggesting that titration of these carboxylates is not involved in the proton-dependent gating of GLIC and that the proton binding site is located elsewhere.We are using fluorescence recording of site-specific labels in GLIC expressed in Xenopus oocytes combined with two-electrode voltage clamping to monitor the motions in GLIC associated with channel opening.We made cysteine mutations in a cys-less GLIC (C26A) background in loop 2 (K32C), loop 9 (T157C), M2 helix (E242C, T243C, N244C) and M2-M3 loop (K247C, P249C). The cysteine mutant receptors were labeled with the sulfhydryl-reactive, environmentally-sensitive fluorescent probe Alexafluor 546 C5-maleimide. Combined voltage clamp and fluorometry monitor proton-induced channel activity and local protein movements simultaneously. Application of pH 5 buffer decreased fluorescence at T157C, K247C and P249C suggesting that these residues become more exposed to a hydrophilic or aqueous environment consistent with a movement of the M2-M3 loop upwards and outward on channel activation.

Functional Study of CLC-1 Mutants Expressed in Xenopus Oocytes Reveals that a C-terminal Region Thr891-Ser892-Thr893 is Responsible for the Effects of Protein Kinase C Activator

ClC-1 plays an important part in the maintenance of membrane potential in the mammalian skeletal muscle. To investigate the phosphorylation sites responsible for the effect of PKC (protein kinase C) activator, we constructed 21 different ClC-1 mutants with mutations at predicted phosphorylation sites for PKC. The functional experiments were performed on both wild-type and mutant proteins (17 point mutants and 4 double mutants) expressed in Xenopus oocytes with two-electrode voltage-clamp recording. PMA (12-myristate 13-acetate), a PKC activator, caused a right shift of half-maximum activation potential (V_1/2) significantly in the wild-type (from -42.9±4.4 to -13.7±1.7 mV; n = 8, P < 0.05) and most of the single mutants except the S892P (from -39.5±4.5 to -35.7±5.7 mV; n = 6) and S892D (from -10.2±4.9 to -9.6±3.5 mV; n = 4). S892D, a mutant mimicking PKC-mediated phosphorylation at position 892, can also mimic the effect of wild-type treated with PMA in V_1/2 value (-10.2±4.9 mV vs -13.7±1.7 mV, n = 4 - 8). However, S892A still had a significant response to PMA indicating that other sites responsible for PMA might exist. Thus double mutants are generated for the following analysis. The V_1/2 of double mutants, T891A/S892A, S892A/T893A and T891A/T893A, show no significant difference between before and after PMA treatment. We hypothesize that this structural modification results in the observed alteration of the gating properties of ClC-1 by PMA. In summary, our observations show that a C-terminal region Thr891-Ser892-Thr893, at least in part, responsible for the effect of PMA on ClC-1.

The Eag Domain Regulates Outward Current Density and Recovery from Inactivation of the hERG K+ Channel Through a Non-Covalent Interaction

The Human Ether-a-go-go Related Gene (hERG) encodes a voltage-activated K+ channel. hERG contributes to repolarization of the ventricular action potential as the primary component of the cardiac delayed rectifier K+ current (IKr) and has also been shown to modulate neuronal firing frequency. hERG gating is characterized by rapid inactivation upon depolarization and rapid recovery from inactivation and slow closing (deactivation) upon repolarization. These factors combine to create a resurgent hERG current, where the amplitude of the current is paradoxically larger with repolarization than with depolarization. These gating transitions also determine the timing and amplitude of the resurgent current. Previous data has suggested that the hERG N-terminus regulates gating kinetics, however the molecular mechanisms are not fully understood. Deletion of the N-terminus (amino acids 2-354) has been shown to speed channel deactivation and recovery from inactivation compared to that of wild-type hERG. Relative outward current amplitude is also increased during the depolarization phase of N-truncated channels, leading to reduced current rectification. A genetically encoded Eag domain fragment (amino acids 1-135) was shown to restore slow deactivation to N-truncated channels. Our present study sought to further investigate Eag domain contributions to hERG gating kinetics. We coexpressed the genetically encoded Eag domain fragment (N1-135) with hERG channels bearing a deletion of the N-terminus in Xenopus oocytes and measured current with two-electrode voltage-clamp recordings. Here we report that coexpression with the N1-135 peptide led to a reduction in relative outward current and slowed recovery from inactivation resulting in channels with properties similar to those measured in wild-type hERG. Through regulation of deactivation and inactivation gating, the Eag domain determines the physiologically critical resurgent component of hERG current via a non-covalent interaction with the channel.

Automated Two-Electrode Voltage-Clamp Recording with Additional Compensation Electrode

Two-electrode voltage-clamp (TEV) of Xenopus laevis oocytes is easily applied for the rapid screening of ion channel function, in particular in pharmacological experiments. However, conventional TEV hardware is not straightforwardly operated by technical personnel because adjustment of the electronics requires considerable practical experience. Moreover, the faithful interpretation of experimental data is often compromised by an incomplete control of all physical parameters determining the voltage-clamp performance. We therefore designed and implemented a hardware/software combination with a built-in 16-bit DA/AD USB interface board providing complete software control of a TEV amplifier featuring full digital calibration and tuning as well as automatic operation via electrophysiological data acquisition software. By means of automated features, such as offset compensation, filter setting, software adjustable amplifier controls (type of controller, gain, response time), and electrode resistance measurements, TEV experiments can be performed in a highly reproducible manner while monitoring the complete set of amplifier control settings. Direct software access to stimulation bandwidth, clamp mode, gain, and response time allows for the objective and automated optimization of voltage-clamp parameters. Two methods for obtaining optimized clamp control parameters will be discussed. An automatic hardware transient compensation increases the dynamic range, particularly important when assaying voltage-gated ion channels. The method is applied for the recording of currents mediated by voltage-gated potassium and sodium channels. In addition to the voltage recording electrode and the current injecting electrode, we implemented a third electrode that injects current in parallel to the second electrode. According to Baumgartner et al. (Biophys. J. 77:1980-1991, 1999), this additional compensation electrode placed in the extracellular space corrects for local current flows and helps improving the voltage clamp performance in big cells such as Xenopus oocytes. Additional operation modes of a third electrode will be presented.

N-linked glycosylation determines cell surface expression of two-pore-domain K + channel TRESK

Within the first external loop of mouse and human TRESK subunits one or two N-glycosylation consensus sites were identified, respectively. Using site directed mutagenesis and Western immunoblotting a single residue of both orthologues was found to be glycosylated upon heterologous expression. Two-electrode voltage-clamp recordings from Xenopus oocytes revealed that current amplitudes of N-glycosylation mutants were reduced by 80% as compared to wildtype TRESK. To investigate membrane targeting, GFP-tagged TRESK subunits were expressed in Xenopus oocytes and fluorescence intensity at the cell surface was measured by confocal microscopy. Signals of the N-glycosylation mutants were reduced by >50%, indicating that their lower current amplitudes substantially result from inadequate surface expression of the channel.

Molecular Basis for Zinc Transporter 1 Action as an Endogenous Inhibitor of L-type Calcium Channels

The L-type calcium channel (LTCC) has a variety of physiological roles that are critical for the proper function of many cell types and organs. Recently, a member of the zinc-regulating family of proteins, ZnT-1, was recognized as an endogenous inhibitor of the LTCC, but its mechanism of action has not been elucidated. In the present study, using two-electrode voltage clamp recordings in Xenopus oocytes, we demonstrate that ZnT-1-mediated inhibition of the LTCC critically depends on the presence of the LTCC regulatory beta-subunit. Moreover, the ZnT-1-induced inhibition of the LTCC current is also abolished by excess levels of the beta-subunit. An interaction between ZnT-1 and the beta-subunit, as demonstrated by co-immunoprecipitation and by fluorescence resonance energy transfer, is consistent with this result. Using surface biotinylation and total internal reflection fluorescence microscopy in HEK293 cells, we show a ZnT-1-dependent decrease in the surface expression of the pore-forming alpha(1)-subunit of the LTCC. Similarly, a decrease in the surface expression of the alpha(1)-subunit is observed following up-regulation of the expression of endogenous ZnT-1 in rapidly paced cultured cardiomyocytes. We conclude that ZnT-1-mediated inhibition of the LTCC is mediated through a functional interaction of ZnT-1 with the LTCC beta-subunit and that it involves a decrease in the trafficking of the LTCC alpha(1)-subunit to the surface membrane.

Rearrangements in the Relative Orientation of Cytoplasmic Domains Induced by a Membrane-anchored Protein Mediate Modulations in Kv Channel Gating

Interdomain interactions between intracellular N and C termini have been described for various K(+) channels, including the voltage-gated Kv2.1, and suggested to affect channel gating. However, no channel regulatory protein directly affecting N/C interactions has been demonstrated. Most Kv2.1 channel interactions with regulatory factors occur at its C terminus. The vesicular SNARE that is also present at a high concentration in the neuronal plasma membrane, VAMP2, is the only protein documented to affect Kv2.1 gating by binding to its N terminus. As its binding target has been mapped near a site implicated in Kv2.1 N/C interactions, we hypothesized that VAMP2 binding to the N terminus requires concomitant conformational changes in the C terminus, which wraps around the N terminus from the outside, to give VAMP2 access. Here, we first determined that the Kv2.1 N terminus, although crucial, is not sufficient to convey functional interaction with VAMP2, and that, concomitant to its binding to the "docking loop" at the Kv2.1 N terminus, VAMP2 binds to the proximal part of the Kv2.1 C terminus, C1a. Next, using computational biology approaches (ab initio modeling, docking, and molecular dynamics simulations) supported by molecular biology, biochemical, electrophysiological, and fluorescence resonance energy transfer analyses, we mapped the interaction sites on both VAMP2 and Kv2.1 and found that this interaction is accompanied by rearrangements in the relative orientation of Kv2.1 cytoplasmic domains. We propose that VAMP2 modulates Kv2.1 inactivation by interfering with the interaction between the docking loop and C1a, a mechanism for gating regulation that may pertain also to other Kv channels.

Magnitude of a Conformational Change in the Glycine Receptor β1-β2 Loop Is Correlated with Agonist Efficacy

The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the alpha1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > beta-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52.

Abstract: 126 ABDOMINAL OBESITY AND METABOLIC SYNDROME: IMPLICATIONS FOR ASSESSMENT AND MANAGEMENT OF GLOBAL CARDIOMETABOLIC RISK

Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupled receptors (GPCRs) to an ion channel. Binding of ligands on the GPCR triggers conformational changes of the receptor that are mechanically transmitted to the ion channel gates, generating an electrical signal easily detectable with conventional electrophysiological techniques. ICCRs are heterologously expressed in Xenopus oocytes and offers several advantages such as: (i) real-time recordings on single cells, (ii) standard laboratory environment and inexpensive media for Xenopus oocytes maintenance, (iii) absence of protein purification steps, (iv) sensitivity to agonists and antagonists in concentration-dependent manner, (v) compatibility with a Gi/o protein activation assay based on Kir3.x channels, and (vi) ability to detect receptor activation independently of intracellular effectors. This last characteristic of ICCRs led to the development of a functional assay for G protein-“uncoupled” receptors such as GPCRs optimized for crystallization by alteration of their third intracellular (i3) loop. One of the most widely used approaches consists in replacing the i3 loop with the T4 phage lysozyme (T4L) domain that obstructs the access of G proteins to their binding site. We recently demonstrated that the ICCR technology can functionally characterize GPCRs(T4L). Two-electrode voltage-clamp (TEVC) recordings revealed that apparent affinities and sensitivities to ligands are not affected by T4L insertion, while ICCRs(T4L) displayed a partial agonist phenotype upon binding of full agonists, suggesting that ICCRs could detect intermediate-active states. This chapter aims to provide exhaustive details from molecular biology steps to electrophysiological recordings for the design and the characterization of ICCRs and ICCRs(T4L).

Inhibition of rat recombinant GluN1/GluN2A and GluN1/GluN2B NMDA receptors by ethanol at concentrations based on the US/UK drink-drive limit

Many studies examine the actions of ethanol on N-methyl-D-aspartate (NMDA) receptors using concentrations that are highly toxic (≥100 mM). This study re-assesses the actions of ethanol at concentrations based around the US/UK ‘drink-drive’ limit (17 mM). Using two-electrode voltage-clamp recordings we examined the actions of ethanol on recombinant GluN1/GluN2A and GluN1/GluN2B NMDA receptors expressed in Xenopus laevis oocytes. We also investigated its actions on NMDA receptors containing GluN2A subunits with truncated or deleted carboxy terminal domains. Ethanol inhibition was voltage-independent and for GluN1/GluN2A NMDA receptors mean inhibition (20 mM at −60 mV) was 9.5±0.8% (n=33) while corresponding values for GluN1/GluN2B NMDA receptors were 6.5±0.8% (n=21). EC50 values for glutamate at GluN1/GluN2A and glutamate and glycine at GluN1/GluN2B NMDA receptors were unaffected by the presence of ethanol. We did however observe a small increase in glycine potency, in the presence of ethanol, at GluN1/GluN2A NMDA receptors. Neither voltage-dependent Mg2+ block nor memantine block was affected by ethanol. Reduced ethanol inhibition was observed however at NMDA receptors containing GluN2A subunits with mutated carboxy terminal domains. We conclude that the levels of inhibition seen with ethanol concentrations near to the US/UK drink-driving limit are very modest and even at higher (intoxicating) concentrations do not alter characteristic NMDA receptor properties.

Structural Insights into KChIP4a Modulation of Kv4.3 Inactivation

Dynamic inactivation in Kv4 A-type K(+) current plays a critical role in regulating neuronal excitability by shaping action potential waveform and duration. Multifunctional auxiliary KChIP1-4 subunits, which share a high homology in their C-terminal core regions, exhibit distinctive modulation of inactivation and surface expression of pore-forming Kv4 subunits. However, the structural differences that underlie the functional diversity of Kv channel-interacting proteins (KChIPs) remain undetermined. Here we have described the crystal structure of KChIP4a at 3.0A resolution, which shows distinct N-terminal alpha-helices that differentiate it from other KChIPs. Biochemical experiments showed that competitive binding of the Kv4.3 N-terminal peptide to the hydrophobic groove of the core of KChIP4a causes the release of the KChIP4a N terminus that suppresses the inactivation of Kv4.3 channels. Electrophysiology experiments confirmed that the first N-terminal alpha-helix peptide (residues 1-34) of KChIP4a, either by itself or fused to N-terminal truncated Kv4.3, can confer slow inactivation. We propose that N-terminal binding of Kv4.3 to the core of KChIP4a mobilizes the KChIP4a N terminus, which serves as the slow inactivation gate.

Inhibition of Neuronal Nicotinic Acetylcholine Receptor Subtypes by α-Conotoxin GID and Analogues

alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha4beta2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha4beta2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha7, alpha3beta2, and alpha4beta2 subtypes. Although the proline residue was the most critical residue for activity at the alpha3beta2 subtype, Asp(3), Arg(12), and Asn(14) are also critical at the alpha7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha4beta2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha4beta2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.