Ion Flux Assays Research Articles

Fluorescence-Based HTS Assays for Ion Channel Modulation in Drug Discovery Pipelines.

Ion channels represent a druggable family of transmembrane pore-forming proteins with important (patho)physiological functions. While electrophysiological measurement (manual patch clamp) remains the only direct method for detection of ion currents, it is a labor-intensive technique. Although automated patch clamp instruments have become available to date, their high costs limit their use to large pharma companies or commercial screening facilities. Therefore, fluorescence-based assays are particularly important for initial screening of compound libraries. Despite their numerous disadvantages, they are highly amenable to high-throughput screening and in many cases, no sophisticated instrumentation or materials are required. These features predispose them for implementation in early phases of drug discovery pipelines (hit identification), even in an academic environment. This review summarizes the advantages and pitfalls of individual methodological approaches for identification of ion channel modulators employing fluorescent probes (i.e., membrane potential and ion flux assays) with emphasis on practical aspects of their adaptation to high-throughput format.

Evidence for evolution and selection of drought-resistant genes based on high-throughput resequencing in weedy rice.

Weedy rice (Oryza sativa f. spontanea) is a relative of cultivated rice that propagates in paddy fields and has strong drought resistance. In this study, we used 501 rice accessions to reveal the selection mechanism of drought resistance in weedy rice through a combination of selection analysis, genome-wide association studies, gene knockout and overexpression analysis, and Ca2+ and K+ ion flux assays. The results showed that the weedy rice species investigated have gene introgression with cultivated rice, which is consistent with the hypothesis that weedy rice originated from de-domestication of cultivated rice. Regions related to tolerance have particularly diversified during de-domestication and three drought-tolerance genes were identified. Of these, Os01g0800500 was also identified using an assay of the degree of leaf withering under drought, and it was named as PAPH1, encoding a PAP family protein. The drought-resistance capacity of PAPH1-knockout lines was much lower than that of the wild type, while that of overexpression lines was much higher. Concentrations of Ca2+ and K+ were lower in the knockout lines and higher in the overexpression lines compared with those of the wild type, suggesting that PAPH1 plays important roles in coping with drought stress. Our study therefore provides new insights into the genetic mechanisms underlying adaptive tolerance to drought in wild rice and highlights potential new resistance genes for future breeding programs in cultivated rice.

Proton-modulated interactions of ions with transport sites of prokaryotic and eukaryotic NCX prototypes

The cytosolic pH decline from 7.2 to 6.9 results in 90% inactivation of mammalian Na+/Ca2+ exchangers (NCXs) due to protons interactions with regulatory and transport domains (“proton block”). Remarkably, the pH titration curves of mammalian and prokaryotic NCXs significantly differ, even after excluding the allosteric effects through regulatory domains. This is fascinating since “only” three (out of twelve) ion-coordinating residues (T50S, E213D, and D240N) differ between the archaeal NCX_Mj and mammalian NCXs although they contain either three or two carboxylates, respectively. To resolve the underlying mechanisms of pH-dependent regulation, the ion-coordinating residues of NCX_Mj were mutated to imitate the ion ligation arrays of mammalian NCXs; the mutational effects were tested on the ion binding/transport by using ion-flux assays and two-dimensional infrared (2D IR) spectroscopy. Our analyses revealed that two deprotonated carboxylates ligate 3Na+ or 1Ca2+ in NCX prototypes with three or two carboxylates. The Na+/Ca2+ exchange rates of NCX_Mj reach saturation at pH 5.0, whereas the Na+/Ca2+ exchange rates of the cardiac NCX1.1 gradually increase even at alkaline pHs. The T50S replacement in NCX_Mj "recapitulates" the pH titration curves of mammalian NCX by instigating an alkaline shift. Proteolytic shaving of regulatory CBD domains activates NCX1.1, although the normalized pH-titration curves are comparable in trypsin treated and untreated NCX1.1. Thus, the T50S-dependent alkaline shift sets a dynamic range for "proton block" function at physiological pH, whereas the CBDs (and other regulatory modes) modulate incremental changes in the transport rates rather than affect the shape of pH dependent curves.

TaPUB15, a U‐Box E3 ubiquitin ligase gene from wheat, enhances salt tolerance in rice

AbstractPlant U‐box (PUB) E3 ubiquitin ligases play essential roles in response to environmental stresses and hormone signaling, but little is known about them in wheat (Triticum aestivum L.). We characterized a U‐box E3 ubiquitin ligase gene, TaPUB15, which is conserved in wheat cultivars. TaPUB15 was expressed in multiple tissues, and the expression was much higher in roots than in other tissues. Transcription of TaPUB15 was induced by salt, abscisic acid, 4°C, and polyethylene glycol (PEG). Overexpression of TaPUB15‐D led to improve salt tolerance in transgenic rice, along with more roots in transgenic rice. Ion flux assays showed that transgenic rice plants had improved salt tolerance and maintained low Na+/K+ ratios under salinity stress. Gene expression assays indicated that several salt stress‐related genes were significantly upregulated in transgenic rice relative to the wild type. These results from transgenic rice were also verified in transgenic Arabidopsis. Our data showed that TaPUB15 plays crucial roles in enhancing salt tolerance in crop plants and served a valuable gene resource for promoting the breeding of salt‐tolerant wheat in the future.

Purification and biochemical characterisation of a putative sodium channel agonist secreted from the South African Knobbly sea anemone Bunodosoma capense

Voltage gated ion channels have become a subject of investigation as possible pharmaceutical targets. Research has linked the activity of ion channels directly to anti-inflammatory pathways, energy homeostasis, cancer proliferation and painful diabetic neuropathy. Sea anemones secrete a diverse array of bioactive compounds including potassium and sodium channel toxins. A putative novel sodium channel agonist (molecular mass of 4619.7 Da) with a predicted sequence: CLCNSDGPSV RGNTLSGILW LAGCPSGWHN CKKHKPTIGW CCK was isolated from Bunodosoma capense using a modified stimulation technique to induce the secretion of the neurotoxin rich mucus confirmed by an Artemia nauplii bio-assay. The peptide purification combined size-exclusion and reverse-phase high performance liquid chromatography. A thallium-based ion flux assay confirmed the presence of a sodium channel agonist/inhibitor and purity was determined using a modified tricine SDS-PAGE system. The peptide isolated indicated the presence of multiple disulfide bonds in a tight β-defensin cystine conformation. An IC50 value of 26 nM was determined for total channel inhibition on MCF-7 cells. The unique putative sodium channel agonist initiating with a cystine bond indicates a divergent evolution to those previously isolated from Bunodosoma species.

Structure-dynamic and functional relationships in a Li+-transporting sodium‑calcium exchanger mutant

The cell membrane (NCX) and mitochondrial (NCLX) Na+/Ca2+ exchangers control Ca2+ homeostasis. Eleven (out of twelve) ion-coordinating residues are highly conserved among eukaryotic and prokaryotic NCXs, whereas in NCLX, nine (out of twelve) ion-coordinating residues are different. Consequently, NCXs exhibit high selectivity for Na+ and Ca2+, whereas NCLX can exchange Ca2+ with either Na+ or Li+. However, the underlying molecular mechanisms and physiological relevance remain unresolved. Here, we analyzed the NCX_Mj-derived mutant NCLX_Mj (with nine substituted residues) imitating the ion selectivity of NCLX. Site-directed fluorescent labeling and ion flux assays revealed the nearly symmetric accessibility of ions to the extracellular and cytosolic vestibules in NCLX_Mj (Kint = 0.8–1.4), whereas the extracellular vestibule is predominantly accessible to ions (Kint = 0.1–0.2) in NCX_Mj. HDX-MS (hydrogen-deuterium exchange mass-spectrometry) identified symmetrically rigidified core helix segments in NCLX_Mj, whereas the matching structural elements are asymmetrically rigidified in NCX_Mj. The HDX-MS analyses of ion-induced conformational changes and the mutational effects on ion fluxes revealed that the “Ca2+-site” (SCa) of NCLX_Mj binds Na+, Li+, or Ca2+, whereas one or more additional Na+/Li+ sites of NCLX_Mj are incompatible with the Na+ sites (Sext and Sint) of NCX_Mj. Thus, the replacement of ion-coordinating residues in NCLX_Mj alters not only the ion selectivity of NCLX_Mj, but also the capacity and affinity for Na+/Li+ (but not for Ca2+) binding, bidirectional ion-accessibility, the response of the ion-exchange to membrane potential changes, and more. These structure-controlled functional features could be relevant for differential contributions of NCX and NCLX to Ca2+ homeostasis in distinct sub-cellular compartments.

Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies.

Quantitative investigations into functional properties of purified ion channel proteins using standard electrophysiological methods are challenging, in particular for the determination of average ion channel behavior following rapid changes in experimental conditions (e.g., ligand concentration). Here, we describe a method for determining the functional activity of liposome-reconstituted K+ channels using a stopped-flow fluorometric ion flux assay. Channel activity is quantified by measuring the rate of fluorescence decrease of a liposome-encapsulated fluorophore, specifically quenched by thallium ions entering the liposomes via open channels. This method is well suited for studying the lipid bilayer dependence of channel activity, the activation and desensitization kinetics of ligand-dependent K+ channels, and channel modulation by channel agonists, blockers, or other antagonists.

High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor

High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

Synthesis and biological evaluation of novel hybrids of highly potent and selective α4β2-Nicotinic acetylcholine receptor (nAChR) partial agonists

We previously reported the cyclopropylpyridine and isoxazolylpyridine ether scaffolds to be versatile building blocks for creating potent α4β2 nicotinic acetylcholine receptor (nAChR) partial agonists with excellent selectivity over the α3β4 subtype. In our continued efforts to develop therapeutic nicotinic ligands, seven novel hybrid compounds were rationally designed, synthesized, and evaluated in [3H]epibatidine binding competition studies. Incorporation of a cyclopropane- or isoxazole-containing side chain onto the 5-position of 1-(pyridin-3-yl)-1,4-diazepane or 2-(pyridin-3-yl)-2,5-diazabicyclo[2.2.1]heptane led to highly potent and selective α4β2* nAChR partial agonists with Ki values of 0.5–51.4 nM for α4β2 and negligible affinities for α3β4 and α7. Moreover, compounds 21, 25, and 30 maintained the functional profiles (EC50 and IC50 values of 15–50 nM) of the parent azetidine-containing compounds 3 and 4 in the 86Rb+ ion flux assays. In vivo efficacy of the most promising compound 21 was confirmed in the mouse SmartCube® platform and classical forced swim tests, supporting the potential use of α4β2 partial agonists for treatment of depression.

Development of an Online Cell-Based Bioactivity Screening Method by Coupling Liquid Chromatography to Flow Cytometry with Parallel Mass Spectrometry.

This study describes a new platform for the fast and efficient functional screening for bioactive compounds in complex natural mixtures using a cell-based assay. The platform combines reversed-phase liquid chromatography (LC) with online flow cytometry (FC) and mass spectrometry (MS). As a model (an example or proof-of-concept study) we have used a functional calcium-flux assay in human neuroblastoma SH-SY5Y cells stably overexpressing the α-7 nicotinic acetylcholine receptor (α7-nAChR), a potential therapeutic target for central nervous system (CNS) related diseases. We have designed the coupled LC-FC system employing the neuroblastoma cells followed by analytical and pharmacological evaluation of the hyphenated setup in agonist and mixed antagonist-agonist assay modes. Using standard receptor ligands we have validated pharmacological responses and standardized good assay quality parameters. The applicability of the screening system was evaluated by analysis of various types of natural samples, such as a tobacco plant extract (in agonist assay mode) and snake venoms (in mixed antagonist-agonist assay mode). The bioactivity responses were correlated directly to the respective accurate masses of the compounds. Using simultaneous functional agonist and antagonist responses nicotine and known neurotoxins were detected from tobacco extract and snake venoms, respectively. Thus, the developed analytical screening technique represents a new tool for rapid measurement of functional cell-based responses and parallel separation and identification of compounds in complex mixtures targeting the α7-nAChR. It is anticipated that other fast-response cell-based assays (e.g., other ion flux assays) can be incorporated in this analytical setup.

Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors.

Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS), which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K) channels and downregulating nicotinic acetylcholine receptors (nAChRs) in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function.

Design and construction of conformational biosensors to monitor ion channel activation: A prototype FlAsH/BRET-approach to Kir3 channels

Ion channels play a vital role in numerous physiological functions and drugs that target them are actively pursued for development of novel therapeutic agents. Here we report a means for monitoring in real time the conformational changes undergone by channel proteins upon exposure to pharmacological stimuli. The approach relies on tracking structural rearrangements by monitoring changes in bioluminescence energy transfer (BRET). To provide proof of principle we have worked with Kir3 neuronal channels producing 10 different constructs which were combined into 17 donor–acceptor BRET pairs. Among these combinations, pairs bearing the donor Nano-Luc (NLuc) at the C-terminal end of Kir3.2 subunits and the FlAsH acceptor at the N-terminal end (NT) or the interfacial helix (N70) of Kir3.1 subunits were identified as potential tools. These pairs displayed significant changes in energy transfer upon activation with direct channel ligands or via stimulation of G protein-coupled receptors. Conformational changes associated with channel activation followed similar kinetics as channel currents. Dose response curves generated by different agonists in FlAsH–BRET assays displayed similar rank order of potency as those obtained with conventional BRET readouts of G protein activation and ion flux assays. Conformational biosensors as the ones reported herein should prove a valuable complement to other methodologies currently used in channel drug discovery.

The Arabidopsis Ca2 +-dependent protein kinase CPK27 is required for plant response to salt-stress

Ca2+-dependent protein kinases (CDPKs) play vital roles in plant adaptations to environmental challenges. The precise regulatory mechanism of CDPKs in mediating salt stress still remains unclear, although several CDPK members have been identified to be involved in salt stress accumulation in various plants, such as Arabidopsis thaliana and Oryza sativa. Here, we investigated the function of an Arabidopsis CDPK, CPK27, in salt stress-signaling. CPK27 is a membrane-localized protein kinase; its expression was induced by NaCl. cpk27-1, a T-DNA insertion mutant of CPK27, was much more sensitive to salt stress than wild-type plants in terms of seed germination and post-germination seedling growth. In ion-flux assay, cpk27-1 mutants exhibited a lower capacity than wild-type plants to extrude Na+ and import H+ after a long-term salt treatment (110mM NaCl for 10days). Moreover, the content of Na+ was higher and K+ was lower in cpk27-1 mutants than in wild-type plants under salt stress. In addition, the level of salt-elicited H2O2 production was higher in cpk27-1 mutants than in wild-type plants Col after a short-term NaCl shock and long-term salt treatment. Collectively, our results suggest that CPK27 is required for plant adaptation to salt stress.

Axial hydrogen at C7 position and bumpy tetracyclic core markedly reduce sterol's affinity to amphotericin B in membrane.

The interaction of amphotericin B (AmB) with fungal ergosterol (Erg) is stronger than its interaction with mammalian cholesterol (Cho), and this property of AmB as an antifungal drug is thought to be responsible for its selective toxicity toward fungi. However, the mechanism by which AmB recognizes the structural differences between sterols, particularly minor difference in the sterol alicyclic portion, is largely unknown. Thus, to investigate the mode of interaction between AmB and the sterol core, we assessed the affinity of AmB to various sterols with different alicyclic structures. Ion flux assays and UV spectral measurements clearly revealed the importance of the Δ7-double bond of the sterol B-ring for interaction with the drug. AmB showed lower affinity for triene sterols, which have double bonds at the Δ5, Δ7, and Δ9 positions. Intermolecular distance measurements by (13)C{(19)F} rotational echo double resonance (REDOR) revealed that the AmB macrolide ring is in closer contact with the steroid core of Erg than it is with the Cho core in the membrane. Conformational analysis suggested that an axial hydrogen atom at C7 of Δ5-sterol (2, 6) and the protruded A-ring of Δ5,7,9-sterol (4, 8) sterically hampered face-to-face contact between the van der Waals surface of the sterol core and the macrolide of AmB. These results further suggest that the α-face of sterol alicycle interacts with the flat macrolide structure of AmB.

Prevention and treatment of colon cancer by peroral administration of HAMLET (human α-lactalbumin made lethal to tumour cells)

BackgroundMost colon cancers start with dysregulated Wnt/β-catenin signalling and remain a major therapeutic challenge. Examining whether HAMLET (human α-lactalbumin made lethal to tumour cells) may be used for colon cancer...

Cadmium impairs ion homeostasis by altering K+ and Ca2+ channel activities in rice root hair cells

Cadmium (Cd2+) interferes with the uptake, transport and utilization of several macro- and micronutrients, which accounts, at least in part, for Cd2+ toxicity in plants. However, the mechanisms underlying Cd2+ interference of ionic homeostasis is not understood. Using biophysical techniques including membrane potential measurements, scanning ion-selective electrode technique for non-invasive ion flux assays and patch clamp, we monitored the effect of Cd2+ on calcium (Ca2+) and potassium (K+) transport in root hair cells of rice. Our results showed that K+ and Ca2+ contents in both roots and shoots were significantly reduced when treated with exogenous Cd2+. Further studies revealed that three cellular processes may be affected by Cd2+, leading to changes in ionic homeostasis. First, Cd2+ -induced depolarization of the membrane potential was observed in root hair cells, attenuating the driving force for cation uptake. Second, the inward conductance of Ca2+ and K+ was partially blocked by Cd2+, decreasing uptake of K+ and Ca2+ . Third, the outward K+ conductance was Cd2+ -inducible, decreasing the net content of K+ in roots. These results provide direct evidence that Cd2+ impairs uptake of Ca2+ and K+, thereby disturbing ion homeostasis in plants.

Syndrome de Sweet révélant une micropolyangéite : première description

The tight junction of epithelial cells excludes macromolecules but allows permeation of ions. However, it is not clear whether this ion-conducting property is mediated by aqueous pores or by ion channels. To investigate the permeability properties of the tight junction, we have developed paracellular ion flux assays for four major extracellular ions, Na+, Cl−, Ca2+, and Mg2+. We found that the tight junction shares biophysical properties with conventional ion channels, including size and charge selectivity, dependency of permeability on ion concentration, competition between permeant molecules, anomalous mole-fraction effects, and sensitivity to pH. Our results support the hypothesis that discrete ion channels are present at the tight junction. Unlike conventional ion channels, which mediate ion transport across lipid bilayers, the tight junction channels must orient parallel to the plane of the plasma membranes to support paracellular ion movements. This new class of paracellular-tight junction channels (PTJC) facilitates the transport of ions between separate extracellular compartments.

Studying Potassium Conductance in Isolated Mouse Heart Using Thallium Flux Assay

Inward rectifier potassium channels IK1 underlie major resting ionic conductance in the heart. A wealth of research on IK1 has been conducted on isolated cells using voltage-clamp techniques but studying IK1 in intact hearts is limited to ion flux assays. Additionally, in the past, flux studies were primarily performed in beating hearts thus eliminating the possibility of measuring any resting conductance. We have implemented a novel approach based on recently discovered thallium-dependent fluorescence of a low-affinity calcium-sensititive dye (BTC). Langendorff-perfused isolated mouse hearts were (i) loaded with membrane permeant BTC and (ii) stopped using verapamil and lidocaine, in order to achieve stable hyperpolarized membrane potential across the heart (thus silencing voltage-dependent potassium conductances). (iii) 1 mM thallium was then applied and (iv) fluorescence of BTC captured using a camera. In all hearts the time course of the relative increase in fluorescence, ΔF, was characterized by (1) well-defined fast (tau∼50÷150 sec) and (2) highly variable slow components. The total initial rate of ΔF was significantly increased in transgenic hearts with upregulated IK1 (up to 2-fold vs control). The initial rates of both components of ΔF were strongly IK1 dependent. Both, transgenic down regulation of IK1 and blockade of IK1 with Ba2+ ions led to a ∼10-20% decrease in the rate of ΔF (not statistically significant) suggesting other major pathways for thallium influx (e.g. Na+/K+ ATPase). Confocal microscopy of isolated ventricular myocytes revealed significant accumulation of BTC in the mitochondria consistent with the hypothesis that the slow component of ΔF reflects mitochondrial uptake of thallium. Results of this study show the possibility of studying sarcolemmal and mitochondrial K+ conductances including IK1 and other pathways (KATP, Na+/K+ ATPase, Na+/K+/CL- symporter) in intact isolated hearts with high temporal resolution.

Discerning Data Analysis Methods to Clarify Agonistic/Antagonistic Actions on the Ion Flux Assay of Ligand-Gated Ionotropic Glutamate Receptor on Engineered Post-Synapse Model Cells

Cell-based experiments provide the efficacy of chemicals through the biological function. The authors have described post-synapse model cell-based assay based on qualified analysis for neural drug discoveries. However, in general, cell-based assays often include data fluctuation. This may be a barrier preventing the performance for various practical purposes. In this study, we tried discerning data analysis for clarify the chemical action to the ionotoropic glutamate receptor (GluR), whereby an ion-flux assay of post-synapse model cells is performed and are analyzed based on a chemometrics approach. The dynamic behavior of the GluR of post-synapse model cell was assayed with multivariate data analysis methods namely hierarchical cluster analysis (HCA) and principal component analysis (PCA). By using HCA, we can identify and remove the non-responding samples. By using PCA, the effect of chemicals on the dynamic behavior of ion flux through GluR can be recognized clearly; as either agonist or antagonist. As shown in the results, the GluR-based assay by post-synapse model cell with data analysis methods provide a sodium influx profile which is derived by an agonists or antagonists application. By employing the data analysis methods, PCA and HCA, it is possible to develop a smart cellular biosensing system for qualified analysis.

Kinetics of desensitization and recovery from desensitization for human alpha4beta2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells.

Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human alpha4beta2-nicotinic acetylcholine receptors (nAChR) heterologously expressed in the SH-EP1 human epithelial cell line. Whole-cell patch clamp recordings were performed to evaluate alpha4beta2-nAChR currents. Application of 0.1 micromol/L nicotine or 1 mmol/L acetylcholine (ACh) for 1 s or longer induced two phases, with time constants of approximately 70 and approximately 700 ms, for the onset of alpha4beta2-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of alpha4beta2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs(1) from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human alpha4beta2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings(2) using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse. These findings indicate the existence of alpha4beta2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.