Mufeng Li

Ivermectin Interaction with Transmembrane Helices Reveals Widespread Rearrangements during Opening of P2X Receptor Channels

P2X receptors are trimeric cation channels that open in response to binding of extracellular ATP. Each subunit contains a large extracellular ligand binding domain and two flanking transmembrane (TM) helices that form the pore, but the extent of gating motions of the TM helices is unclear. We probed these motions using ivermectin (IVM), a macrocyclic lactone that stabilizes the open state of P2X(4) receptor channels. We find that IVM partitions into lipid membranes and that transfer of the TM regions of P2X(4) receptors is sufficient to convey sensitivity to the lactone, suggesting that IVM interacts most favorably with the open conformation of the two TM helices at the protein-lipid interface. Scanning mutagenesis of the two TMs identifies residues that change environment between closed and open states, and substitutions at a subset of these positions weaken IVM binding. The emerging patterns point to widespread rearrangements of the TM helices during opening of P2X receptor channels.

Gating the pore of P2X receptor channels

Three families of ligand-activated ion channels mediate synaptic communication between excitable cells in mammals. For pentameric channels related to nicotinic acetylcholine receptors and tetrameric channels such as glutamate receptors, the pore-forming and gate regions have been studied extensively. In contrast, little is known about the structure of trimeric P2X receptor channels, a family of channels that are activated by ATP and are important in neuronal signaling, pain transmission and inflammation. To identify the pore-forming and gate regions in P2X receptor channels, we introduced cysteine residues throughout the two transmembrane (TM) segments and studied their accessibility to thiol-reactive compounds and ions. Our results show that TM2 lines the central ion-conduction pore, TM1 is positioned peripheral to TM2 and the flow of ions is minimized in the closed state by a gate formed by the external region of TM2.

Pore-opening mechanism in trimeric P2X receptor channels.

The opening of ion channels in response to ligand binding, voltage or membrane stretch underlies electrical and chemical signalling throughout biology. Two structural classes of pore-opening mechanisms have been established, including bending of pore-lining helices in the case of tetrameric cation channels, or tilting of such helices in mechanosensitive channels. In this paper, we explore how the structure of the pore changes during opening in P2X receptors by measuring the modification of introduced cysteine residues in transmembrane helices by thiol-reactive reagents, and by engineering metal bridges. Our results are consistent with the X-ray structure of the closed state, and demonstrate that expansion of the gate region in the external pore is accompanied by a significant narrowing of the inner pore, indicating that pore-forming helices straighten on ATP binding to open the channel. This unique pore-opening mechanism has fundamental implications for the role of subunit interfaces in the gating mechanism of P2X receptors and points to a role of the internal pore in ion permeation.

Ion access pathway to the transmembrane pore in P2X receptor channels

P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain. The x-ray structure of the P2X4 receptor from zebrafish (zfP2X4) receptor reveals that the extracellular vestibule above the gate opens to the outside through lateral fenestrations, providing a potential pathway for ions to enter and exit the pore. The extracellular region also contains a void at the central axis, providing a second potential pathway. To investigate the energetics of each potential ion permeation pathway, we calculated the electrostatic free energy by solving the Poisson-Boltzmann equation along each of these pathways in the zfP2X4 crystal structure and a homology model of rat P2X2 (rP2X2). We found that the lateral fenestrations are energetically favorable for monovalent cations even in the closed-state structure, whereas the central pathway presents strong electrostatic barriers that would require structural rearrangements to allow for ion accessibility. To probe ion accessibility along these pathways in the rP2X2 receptor, we investigated the modification of introduced Cys residues by methanethiosulfonate (MTS) reagents and constrained structural changes by introducing disulfide bridges. Our results show that MTS reagents can permeate the lateral fenestrations, and that these become larger after ATP binding. Although relatively small MTS reagents can access residues in one of the vestibules within the central pathway, no reactive positions were identified in the upper region of this pathway, and disulfide bridges that constrain movements in that region do not prevent ion conduction. Collectively, these results suggest that ions access the pore using the lateral fenestrations, and that these breathe as the channel opens. The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.

Physical basis of apparent pore dilation of ATP-activated P2X receptor channels

The selectivity of ion channels is fundamental for their roles in electrical and chemical signaling and in ion homeostasis. Although most ion channels exhibit stable ion selectivity, the prevailing view of purinergic P2X receptor channels, transient receptor potential V1 (TRPV1) channels and acid-sensing ion channels (ASICs) is that their ion conduction pores dilate upon prolonged activation. We investigated this mechanism in P2X receptors and found that the hallmark shift in equilibrium potential observed with prolonged channel activation does not result from pore dilation, but from time-dependent alterations in the concentration of intracellular ions. We derived a physical model to calculate ion concentration changes during patch-clamp recordings, which validated our experimental findings and provides a quantitative guideline for effectively controlling ion concentration. Our results have fundamental implications for understanding ion permeation and gating in P2X receptor channels, as well as more broadly for using patch-clamp techniques to study ion channels and neuronal excitability.

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

Significance The opening of P2X receptor channels by extracellular ATP underlies purinergic signaling in many tissues. Here we use computational and functional approaches to study helix interactions within the transmembrane domain of P2X receptors. Our results suggest that the intersubunit crevices observed in the X-ray structure of detergent-solubilized ATP-bound receptors are nonnative but confirm helix interactions within individual subunits observed in both apo and ATP-bound receptors and identify a hot spot within a narrow internal region where the gating and permeation properties can be readily tuned. P2X receptor channels open in response to the binding of extracellular ATP, a property that is essential for purinergic sensory signaling. Apo and ATP-bound X-ray structures of the detergent-solubilized zebrafish P2X4 receptor provide a blueprint for receptor mechanisms but unexpectedly showed large crevices between subunits within the transmembrane (TM) domain of the ATP-bound structure. Here we investigate both intersubunit and intrasubunit interactions between TM helices of P2X receptors in membranes using both computational and functional approaches. Our results suggest that intersubunit crevices found in the TM domain of the ATP-bound crystal structure are not present in membrane-embedded receptors but substantiate helix interactions within individual subunits and identify a hot spot at the internal end of the pore where both the gating and permeation properties of P2X receptors can be tuned. We propose a model for the structure of the open state that has stabilizing intersubunit interactions and that is compatible with available structural constraints from functional channels in membrane environments.

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

Significance ATP is an important extracellular signal that activates P2X receptor channels. Although a large fraction of ATP is bound to divalent cations in vivo, the forms of ATP that activate P2X receptors are unknown. Here we show how the activity of homomeric P2X receptors is tuned by Mg2+ in some subtypes by preventing activation by free ATP, and in others by binding to a distinct regulatory site. We also find that both regulatory mechanisms are disengaged in heteromeric channels to form a sensitive ATP signaling pathway. These fundamental properties of P2X receptors will be valuable for investigating their physiological functions. The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg2+. Here we investigated the active forms of ATP and found that the action of MgATP2− and ATP4− differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP2− promotes opening with very low efficacy. In contrast, both free ATP and MgATP2− robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg2+ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP2− and weak regulation by Mg2+. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP2− and regulation by Mg2+, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.

Expression Level Dependence of the Gating and Permeation Properties of P2X Receptor Channels

P2X receptors are cation channels that are activated by extracellular ATP, and that are widely expressed in virtually every tissue. Seven subtypes of P2X receptors have been identified in mammals, and they play important roles in sensory signaling and inflammation. The expression levels of P2X receptors have been reported to change in response to various stimuli. For instance, the expression level of P2X4 receptors increased strikingly in microglia of spinal cord after nerve injury, and these receptors were suggested to play important roles for tactile allodynia1. Interestingly, it has also been reported that both gating and permeation properties of some P2X receptors change in a channel-density dependent manner2,3. Here we transfected P2X2 receptor channels into HEK293 cells with either high or low expression vectors, and then studied the properties of these channels at varying expression levels. Our results suggest that the selectivity, rectification and ATP sensitivity of P2X receptor channels exhibit expression level dependence, as if distinct open states predominate at different expression levels. These changes do not appear to result from artefactual changes in intracellular ion concentrations or voltage errors, and we are currently studying the underlying mechanism.References1. Tsuda, M. et al. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424, 778-83 (2003).2. Fujiwara, Y. & Kubo, Y. Density-dependent changes of the pore properties of the P2X2 receptor channel. J Physiol 558, 31-43 (2004).3. Clyne, J.D., Brown, T.C. & Hume, R.I. Expression level dependent changes in the properties of P2X2 receptors. Neuropharmacology 44, 403-12 (2003).

Ion Accumulation and Depletion in Patch Clamp Experiments

The flow of ions through channels and transporters depends sensitively on the concentration of the ionic species and voltage at the membrane. In the widely used patch-clamp technique, voltage and current are measured using electrodes in the pipette and bath that are distant from the membrane. Although the importance of voltage differences between the membrane and electrodes is widely appreciated, concentration gradients arising from the flow of ions are often neglected. In this study, we modeled changes in voltage and ion concentrations during patch-clamp experiments using the Nernst-Plank equation and we derived simple formulas for estimating the timescale and extent of ion accumulation and depletion. For excised patch experiments, ions crossing the membrane can directly diffuse into or out of the patch pipette and ion concentrations stabilize on the millisecond timescale after a change in membrane current. In contrast, in whole-cell experiments the cytosol acts as a reservoir and ion concentrations change on the timescale of seconds. In either configuration, ion accumulation or depletion at steady-state depends primarily on the electrode access resistances and currents carried by each ionic species. As a practical illustration, simulations were performed for bi-ionic protocols previously used to characterize the dynamic ionic selectivity of P2X and TRPV channels. Importantly, even when the net current was small and the membrane voltage effectively clamped, ion accumulation and depletion could cause significant, time-dependent changes in current resembling reported examples of “pore dilation”. Thus, limitations for clamping ion concentrations should be considered when performing and interpreting patch-clamp experiments.

Rapid Activation of Distinct Conducting States in P2X Receptor Channels

P2X receptors are trimeric cation channels that are activated by extracellular ATP. These purinergic receptors have been reported to undergo pore dilation following activation by high concentrations of ATP, a phenomenon thought to mediate apoptosis in the immune system. Here we demonstrate that the widely reported slow time-dependent increase in the relative permeability of NMDG to Na, thought to reflect pore dilation, results from a gradual inhibition of small-cation-selective channels following depletion of intracellular alkali ions. Moreover, we find that P2X receptors enter both small-cation-selective open states and large-cation-permeable open states within milliseconds of ATP application. Taken together, our results demonstrate that P2X receptors can rapidly enter a large-cation-permeable open state without requiring either high ATP concentrations or high channel density, indicating that the ability of ATP to permeabilize cells can occur under physiologically realistic conditions.