Gating Mechanism Of Channels Research Articles

Conformational Changes Underlying Glutamate Receptor Gating

The gating mechanism of glutamate receptor ion channels involves conformational changes triggered by the binding of glutamate that open the ion channel, followed within milliseconds by nearly complete desensitization. In a collaboration with the Subramaniam Lab at NCI, cryo-EM structures of full length AMPA (GluA2) receptors, stabilized in the active state by allosteric modulators, reveal concerted conformational changes in the ligand binding domain that produce a vertical contraction accompanied by a corkscrew like rotation that leads to expansion of the linkers leading to the M3 helix bundle; in the desensitized state the ATD dimer pairs adopts multiple conformations with evidence for substantial reorganization of the ligand binding domain tetramer. By contrast, for the GluK2 kainate receptor the ATD dimer assembly remains intact in the desensitized state, while the LBD rearranges into a 4-fold symmetric structure in which the linkers leading to the M3 helix bundle adopt a contracted conformation that is however different from the closed state. Measurements of ATD tetramer association by analytical ultracentrifugation reveal a 1000-fold high higher affinity for GluK2 versus GluA2 that underlies the different behavior of the ATD in the desensitized state, movements of which however are secondary to and play no major role in desensitization.

EPR Studies of Gating Mechanisms in Ion Channels.

Ion channels open and close in response to diverse stimuli, and the molecular events underlying these processes are extensively modulated by ligands of both endogenous and exogenous origin. In the past decade, high-resolution structures of several channel types have been solved, providing unprecedented details of the molecular architecture of these membrane proteins. Intrinsic conformational flexibility of ion channels critically governs their functions. However, the dynamics underlying gating mechanisms and modulations are obscured in the information from crystal structures. While nuclear magnetic resonance spectroscopic methods allow direct measurements of protein dynamics, they are limited by the large size of these membrane protein assemblies in detergent micelles or lipid membranes. Electron paramagnetic resonance (EPR) spectroscopy has emerged as a key biophysical tool to characterize structural dynamics of ion channels and to determine stimulus-driven conformational transition between functional states in a physiological environment. This review will provide an overview of the recent advances in the field of voltage- and ligand-gated channels and highlight some of the challenges and controversies surrounding the structural information available. It will discuss general methods used in site-directed spin labeling and EPR spectroscopy and illustrate how findings from these studies have narrowed the gap between high-resolution structures and gating mechanisms in membranes, and have thereby helped reconcile seemingly disparate models of ion channel function.

Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel.

SummaryActivation of mechanosensitive ion channels by physical force underlies many physiological processes including the sensation of touch, hearing and pain1–5. TRAAK ion channels are neuronally expressed members of the two-pore domain K+ (K2P) channel family and are mechanosensitive6. They are involved in controlling mechanical and temperature nociception in mice7. Mechanosensitivity of TRAAK is mediated directly through the lipid bilayer: it is a membrane tension gated channel8. However, the molecular mechanism of TRAAK channel gating and mechanosensitivity is unknown. Here we present crystal structures of TRAAK in conductive and nonconductive conformations defined by the presence of permeant ions along the conduction pathway. In the nonconductive state, a lipid acyl chain accesses the channel cavity through a 5 Å-wide lateral opening in the membrane inner leaflet and physically blocks ion passage. In the conductive state, rotation of a transmembrane helix (TM4) about a central hinge seals the intramembrane opening, preventing lipid block of the cavity and permitting ion entry. Additional rotation of a membrane interacting TM2-TM3 segment, unique to mechanosensitive K2Ps, against TM4 may further stabilize the conductive conformation. Comparison of the structures reveals a biophysical explanation for TRAAK mechanosensitivity: an expansion in cross sectional area up to 2.7 nm2 in the conductive state is expected to create a membrane tension-dependent energy difference between conformations that promotes force activation. Our results show how tension of the lipid bilayer can be harnessed to control gating and mechanosensitivity of a eukaryotic ion channel.

Structure of a mammalian ryanodine receptor.

Ryanodine receptors (RyRs) mediate rapid release of calcium (Ca2+) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here, we report the closed-state structure of the 2.3 MDa complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle cryo-electron microscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3939 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane (6TM) ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca2+.

STIM1 triggers a gating rearrangement at the extracellular mouth of the ORAI1 channel.

The ER-resident regulatory protein STIM1 triggers store-operated Ca2+ entry by direct interaction with the plasma membrane Ca2+ channel ORAI1. The mechanism of channel gating remains undefined. Here we establish that STIM1 gates the purified recombinant ORAI1 channel in vitro, and use Tb3+ luminescence and, separately, disulfide crosslinking to probe movements of the pore-lining helices. We show that interaction of STIM1 with the cytoplasmic face of the human ORAI1 channel elicits a conformational change near the external entrance to the pore, detectable at the pore Ca2+-binding residue E106 and the adjacent pore-lining residue V102. We demonstrate that a short nonpolar segment of the pore including V102 forms a barrier to ion flux in the closed channel, implicating the STIM1-dependent movement in channel gating. Our data explain the close coupling between ORAI1 channel gating and ion selectivity, and open a new avenue to dissect the gating, modulation, and inactivation of ORAI-family channels.

The Region Adjacent to the C-end of the Inner Gate in Transient Receptor Potential Melastatin 8 (TRPM8) Channels Plays a Central Role in Allosteric Channel Activation

The ability of transient receptor potential (TRP) channels to sense and respond to environmental and endogenous cues is crucial in animal sensory physiology. The molecular mechanism of channel gating is yet elusive. The TRP box, a conserved region in the N-end of the C terminus domain, has been signaled as pivotal for allosteric activation in TRP channels. Here, we have examined the role of the linker region between the TRPM8 inner gate and the TRP box (referred to as the S6-TRP box linker) to identify structural determinants of channel gating. Stepwise substitutions of segments in the S6-TRP box linker of TRPM8 channel with the cognate TRPV1 channel sequences produced functional chimeric channels, and identified Tyr(981) as a central molecular determinant of channel function. Additionally, mutations in the 986-990 region had a profound impact on channel gating by voltage and menthol, as evidenced by the modulation of the conductance-to-voltage (G-V) relationships. Simulation of G-V curves using an allosteric model for channel activation revealed that these mutations altered the allosteric constants that couple stimuli sensing to pore opening. A molecular model of TRPM8, based on the recently reported TRPV1 structural model, showed that Tyr(981) may lie in a hydrophobic pocket at the end of the S6 transmembrane segment and is involved in inter-subunit interactions with residues from neighbor subunits. The 986-990 region holds intrasubunit interactions between the TRP domain and the S4-S5 linker. These findings substantiate a gating mechanism whereby the TRP domain acts as a coupling domain for efficient channel opening. Furthermore, they imply that protein-protein interactions of the TRP domain may be targets for channel modulation and drug intervention.

Interaction of the BKCa channel gating ring with dendrotoxins

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca2+-activated K+ channel (BKCa, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BKCa channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BKCa channel. Calculation of an electrostatic surface map of the BKCa gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BKCa channel activation gating.

Cooperative subunit interactions mediate fast C-type inactivation of hERG1 K+ channels.

At depolarized membrane potentials, the conductance of some voltage-gated K(+) channels is reduced by C-type inactivation. This gating process is voltage independent in Kv1 and involves a conformational change in the selectivity filter that is mediated by cooperative subunit interactions. C-type inactivation in hERG1 K(+) channels is voltage-dependent, much faster in onset and greatly attenuates currents at positive potentials. Here we investigate the potential role of subunit interactions in C-type inactivation of hERG1 channels. Point mutations in hERG1 known to eliminate (G628C/S631C), inhibit (S620T or S631A) or enhance (T618A or M645C) C-type inactivation were introduced into subunits that were combined with wild-type subunits to form concatenated tetrameric channels with defined subunit composition and stoichiometry. Channels were heterologously expressed in Xenopus oocytes and the two-microelectrode voltage clamp was used to measure the kinetics and steady-state properties of inactivation of whole cell currents. The effect of S631A or T618A mutations on inactivation was a graded function of the number of mutant subunits within a concatenated tetramer as predicted by a sequential model of cooperative subunit interactions, whereas M645C subunits increased the rate of inactivation of concatemers, as predicted for subunits that act independently of one another. For mutations located within the inactivation gate proper (S620T or G628C/S631C), the presence of a single subunit in a concatenated hERG1 tetramer disrupted gating to the same extent as that observed for mutant homotetramers. Together, our findings indicate that the final step of C-type inactivation of hERG1 channels involves a concerted, all-or-none cooperative interaction between all four subunits, and that probing the mechanisms of channel gating with concatenated heterotypic channels should be interpreted with care, as conclusions regarding the nature of subunit interactions may depend on the specific mutation used to probe the gating process.

Characterization of the Intrinsic Voltage-Dependent Gating Mechanism of CALHM Ion Channels

Human CALHM1, which plays important roles in cortical neuron excitability and taste perception, belongs to a new family of evolutionarily conserved ion channels that are regulated by voltage and extracellular Ca2+ (Ca2+o) [1,2]. There is a single hCALHM1 homolog in C. elegans, CLHM-1 [3]. Although hCALHM1 and CLHM-1 have little sequence identity (16%), these ion channels exhibit similar biophysical properties when expressed in Xenopus oocytes and are functionally interchangeable when expressed in vivo [3].Since neither hCALHM1 nor CLHM-1 contain known Ca2+-binding sequences or canonical voltage sensing domains, we are seeking to identify residues responsible for the intrinsic voltage-dependent gating mechanism for CALHM channels. To understand the Ca2+o and voltage-dependent gating mechanisms utilized by these ion channels, we determined activation and deactivation kinetics as well as apparent conductance-voltage (G-V) relations for CLHM-1 and hCALHM1 in divalent-free and Ca2+ containing solutions. While both channels were regulated by Ca2+o and voltage, there were significant differences in the kinetics and voltage dependencies in the absence of Ca2+o. These results suggest that the intrinsic gating of these channels is significantly different. To define the channel region(s) required for intrinsic voltage-dependent gating, we have examined the gating kinetics and apparent G-V relationships of CLHM-1 / hCALHM1 chimeric proteins. A chimeric protein consisting of CLHM-1 with the second extracellular loop from hCALHM1 forms a functional ion channel. Like hCALHM1, it is difficult to close this chimeric channel in the absence of Ca2+o at hyperpolarized voltages. This result suggests that the second extracellular loop may play a role in intrinsic voltage-dependent gating of CALHM ion channels. [1] Ma et al., PNAS (2012). [2] Taruno et al., Nature (2013). [3] Tanis et al., J. Neurosci. (2013). Supported by NIH DC012538.

An Overview on Recent Trends in Catalysis

Copyright: © 2014 Shehadi IA. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The vast majority of chemical and biological activities and processes involve catalytic steps. Catalysis (homogeneous or heterogeneous) play an important role for chemicals manufacturing, utilizing of energy resources, energy conversion systems, devices and environmental control system, biological activities and drug discovery and design. However, the most important issues in any catalytic process are the activity and the selectivity of the catalysts by which processes are performed. In order to understand and control how a catalysts function, one must understand the energy profile through which the catalytic processes must go through. The energy content and transfer of any catalytic processes is easily understood by applying the principles of thermodynamics which provides an insight whether the process is attainable or not. Therefore, thermodynamics is used to describe the macroscopic behavior of chemical systems through understanding the properties of atoms and molecules. The area of thermodynamics is fully established and all laws that govern the energy flow and transfer are developed. Alternatively, the routes and the rate of the chemical processes happening are not yet fully comprehended. Hence, the application of chemical kinetics to understand catalytic activity and selectivity is still under investigation. Moreover, tremendous amounts of scientific discoveries are continually being achieved and more are to be revealed in the years to come. Even though a unifying principle has been identified in the area of chemical kinetics and compiled, but none are faultless. Each principle has its own defects and limitations. Understanding the mechanistic behavior of chemical systems in general and catalysis in particular, lead to significant discoveries in current scientific research. These days, scientists can study, monitor and characterize fast and ultra-fast reactions using laser pulses involved in numerous techniques. One of the most prominent areas of catalytic research these days is to understand the electronic and ionic transfer in biological systems. Functional ion channels in particular are the focus of many recent catalytic research works directed to understand gating, closing and filtering mechanism of the channel. In order to understand the basic channel mechanics, researchers have focused upon questions surrounding the functioning, selectivity, and gating mechanisms of ion channels. Another attractive area for research is to study electron transfer in proteins and enzymes in order to understand functions and properties. Such areas have boomed after the discovery of the genomic code of many living organisms which led to the discovery of thousands of proteins and enzymes of unknown functions and structures. However, recent advances in biophysics especially in X-ray crystallography, high-resolution electron microscopy and molecular biology have helped shed light on some of the issues in scientific quests.

Binding Structure & Dynamics for Toxins Modifying the Gating Mechanism of Kv Channels

Voltage gated ion channels allow the flux of specific ions through the cell membrane to be controlled by the membrane potential, which makes them a critical component for electro-chemical signaling in the nervous system. The gating is regulated by the voltage sensor domains (VSD), in which the charged S4 helix moves to induce the conformational transition between open/closed states. This serves as an ideal model system for membrane proteins with multiple conformations, and if the gating can be modulated it would potentially enable a whole range of new drugs that could fine-tune the gating response. This is strikingly manifested in the case of tarantula spiders, which produce a potent venom that specifically target the VSD to causing dysfunctions in the neuronal and cardiac systems.Here, we report on molecular simulations of interactions between toxins and different Kv channels, in particular related to their influence of the gating transition. We have studied the binding to multiple intermediate states and effect of Stromatoxin (ScTx1) on gating transitions in both Kv2.1 and a mutant Shaker channel with enhanced toxin affinity. Results from toxin-protein docking show the toxin binding to S2 and S3 helices. An interaction pair is formed between E277 (Kv2.1 numbering) in the top-most part of S3 and either R4 or K22 on opposite sides of the toxin, resulting in two distinct poses in the binding site. In addition to docking, we have also explored multi-microsecond molecular dynamics simulations of toxins bound in both open and closed states.We propose a specific binding site for the toxin in the VSD of both channels, and predict that differences in residue interactions are responsible for selectively stabilizing S4 in either the open or closed state.

Gating Mechanism of Mechanosensitive Ion Channels Studied by Continuum Mechanics

To complement the existing experimental and computational methods used for studies of membrane protein structure and dynamics a finite element (FE) model for multi length-scale and time-scale investigations of the gating mechanism of mechanosensitive (MS) ion channels has been established. The main advantage of the FE simulation over molecular dynamic simulation is its flexibility in handling complex geometries and boundary conditions. In this study the FE model of MscL, the large conductance bacterial MS channel, has been developed as an initial template providing a continuum structural framework for a mechanistic understanding of the gating mechanism in MS channels, which can also be employed to develop structural dynamic models of other types of MS channels. A typical tensional force needed to activate MS channels was first applied to the FE model of the lipid bilayer and was then varied depending on the mechanical properties and cholesterol content of the membrane bilayer surrounding MscL. In agreement with the experimental results the FE model showed that adding cholesterol to the lipid bilayer caused a significant increase in the tensional force required for MscL activation. Furthermore, the current model helped to define the membrane stress distribution around a ‘hole’ in the membrane bilayer containing the MscL protein as well as to identify stress areas where the MscL protein is attached to the hole boundary by applying an appropriate mesh refinement and defining contact conditions between the channel and the bilayer. Cholesterol, although stiffening the lipid bilayer, increased the stress intensity in the highly stressed areas of the MscL protein under tension and thus facilitated rupturing of the lipid bilayer. Although initially surprising, this result is consistent with the patch clamp experiments, which demonstrated that adding cholesterol markedly reduced the bilayer lytic tension.

Probing gating mechanisms of sodium channels using pore blockers.

Several classes of small molecules and peptides bind at the central pore of voltage-gated sodium channels either from the extracellular or intracellular side of the membrane and block ion conduction through the pore. Biophysical studies that shed light on the chemical nature, accessibility, and kinetics of binding of these naturally occurring and synthetic compounds reveal a wealth of information about how these channels gate. Here, we discuss insights into the structural underpinnings of gating of the channel pore and its coupling to the voltage sensors obtained from pore blockers including site 1 neurotoxins and local anesthetics.

Control of KirBac3.1 Potassium Channel Gating at the Interface between Cytoplasmic Domains

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating.

A Lack of Significant Lipid Interactions in the Open State of MSCS Implies a Jack-In-The-Box Type Channel Gating Mechanism

Bacterial mechanosensitive channels are important for cell survival in changing osmotic environments. For the mechanosensitive channel of small conductance from Escherichia coli (Ec-MscS), seven residues have previously been shown to form important lipid contacts in the closed state of the channel. Based on open state crystal structures and closed state models, these residues interact with lipids in the closed state of the channel. Decreasing the hydrophobicity of these residues reduces bacterial survival upon osmotic downshock. Since the closed state appears to be stabilized by lipid interactions, we hypothesized that similar stabilizing lipid interactions could be identified in the open state. Using a computational model of open state Ec-MscS embedded in a lipid bilayer, eleven residues were determined to be lipid exposed with ten of these residues being unique to the open state of the channel. To identify the role of lipid interactions these residues were mutated to alanine and leucine to alter their interaction with the hydrophobic lipids. The effects of these mutations on channel function were assayed using osmotic downshock lysis assays, growth assays, and patch clamp electrophysiology. Mutations of the ten residues that are exposed to the lipid bilayer only in the open state of the channel effected a wildtype phenotype in all of these assays. The lack of phenotypic changes, for residues that interact with the lipid bilayer solely in the open state, suggests that these interactions are not critical for channel gating. This has led us to propose a Jack-In-The-Box model of gating for MscS, in which intrinsic lipid bilayer pressure holds the channel in the closed state. Upon relief of the intrinsic bilayer pressure by application of opposing extrinsic tension, MscS springs into the open state.

Transient receptor potential channels and regulation of lung endothelial permeability.

This review highlights our current knowledge regarding expression of transient receptor potential (TRP) cation channels in lung endothelium and evidence for their involvement in regulation of lung endothelial permeability. Six mammalian TRP families have been identified and organized on the basis of sequence homology: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin), and TRPA (ankyrin). To date, only TRPC1/4, TRPC6, TRPV4, and TRPM2 have been extensively studied in lung endothelium. Calcium influx through each of these channels has been documented to increase lung endothelial permeability, although their channel-gating mechanisms, downstream signaling mechanisms, and impact on endothelial structure and barrier integrity differ. While other members of the TRPC, TRPV, and TRPM families may be expressed in lung endothelium, we have little or no evidence linking these to regulation of lung endothelial permeability. Further, neither the expression nor functional role(s) of any TRPML, TRPP, and TRPA family members has been studied in lung endothelium. In addition to this assessment organized by TRP channel family, we also discuss TRP channels and lung endothelial permeability from the perspective of lung endothelial heterogeneity, using outcomes of studies focused on TRPC1/4 and TRPV4 channels. The diversity within the TRP channel family and the relative paucity of information regarding roles of a number of these channels in lung endothelium make this field ripe for continued investigation.

A gating mechanism of pentameric ligand-gated ion channels

Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communication in the nervous system and are involved in fundamental processes such as attention, learning, and memory. They are oligomeric protein assemblies that convert a chemical signal into an ion flux through the postsynaptic membrane, but the molecular mechanism of gating ions has remained elusive. Here, we present atomistic molecular dynamics simulations of the prokaryotic channels from Gloeobacter violaceus (GLIC) and Erwinia chrysanthemi (ELIC), whose crystal structures are thought to represent the active and the resting states of pLGICs, respectively, and of the eukaryotic glutamate-gated chloride channel from Caenorhabditis elegans (GluCl), whose open-channel structure was determined complexed with the positive allosteric modulator ivermectin. Structural observables extracted from the trajectories of GLIC and ELIC are used as progress variables to analyze the time evolution of GluCl, which was simulated in the absence of ivermectin starting from the structure with bound ivermectin. The trajectory of GluCl with ivermectin removed shows a sequence of structural events that couple agonist unbinding from the extracellular domain to ion-pore closing in the transmembrane domain. Based on these results, we propose a structural mechanism for the allosteric communication leading to deactivation/activation of the GluCl channel. This model of gating emphasizes the coupling between the quaternary twisting and the opening/closing of the ion pore and is likely to apply to other members of the pLGIC family.

Automated Parallel Recordings of Topologically Identified Single Ion Channels

Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.

Ion selectivity and gating mechanisms of FNT channels

The phospholipid bilayer has evolved to be a protective and selective barrier by which the cell maintains high concentrations of life sustaining organic and inorganic material. As gatekeepers responsible for an immense amount of bidirectional chemical traffic between the cytoplasm and extracellular milieu, ion channels have been studied in detail since their postulated existence nearly three-quarters of a century ago. Over the past fifteen years, we have begun to understand how selective permeability can be achieved for both cationic and anionic ions. Our mechanistic knowledge has expanded recently with studies of a large family of anion channels, the Formate Nitrite Transport (FNT) family. This family has proven amenable to structural studies at a resolution high enough to reveal intimate details of ion selectivity and gating. With five representative members having yielded a total of 15 crystal structures, this family represents one of the richest sources of structural information for anion channels.

Dual Gating Mechanism and Function of P2X7 Receptor Channels

The ATP-gated P2X7 receptor channel (P2X7R) operates as a cytolytic and apoptotic receptor but also controls sustained cellular responses, including cell growth and proliferation. However, it has not been clarified how the same receptor mediates such opposing effects. To address this question, we have combined electrophysiological, imaging, and mathematical studies using wild-type and mutant rat P2X7Rs. Activation of naïve (not previously stimulated) receptors by low agonist concentrations caused monophasic slow desensitizing currents and internalization of receptors without other changes in the cellular morphology, much like other P2XRs. In contrast, saturating agonist concentrations induced high-amplitude biphasic currents, reflecting pore dilation and causing rapid cell swelling and lysis. The existence of these two signaling patterns was accounted for using a revised Markov-state model that included, in addition to naïve and sensitized states, desensitized states. Occupancy of one or two ATP-binding sites of naïve receptors favored a slow transition to desensitized states, whereas occupancy of the third binding site favored a transition to sensitized/dilated states. Consistent with model predictions, nondilating P2X7R mutants always generated desensitizing currents. These results suggest that the level of saturation of the ligand binding sites determines the nature of the P2X7R gating and cellular actions.