Phanindra Velisetty

Desensitization Mechanism in Prokaryotic Ligand-gated Ion Channel

Background: GLIC, a prokaryotic homologue of pentameric ligand-gated ion channel (LGIC), is activated by protons, and crystal structures suggest a putative open conformation. Results: GLIC function characterized in proteoliposomes reveals rapid activation and slow desensitization. Desensitization is modulated by voltage, blockers, and membrane cholesterol. Conclusion: GLIC desensitization shows many hallmark features of the mechanism in LGIC. Significance: Understanding GLIC desensitization is an important step toward structure-function characterization of LGIC. Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.

Conformational Transitions Underlying Pore Opening and Desensitization in Membrane-embedded Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Background: GLIC, a prokaryotic homologue of pentameric ligand-gated ion channels (LGIC), is activated by protons, and crystal structures suggest a putative open conformation. Results: EPR-spectroscopic studies in the pore-lining segment reveals major conformational changes during activation and desensitization. Conclusion: Gating mechanism in GLIC involves distinct activation and desensitization gates. Significance: These studies provide insights into the role of structural dynamics in the functioning of LGIC. Direct structural insight into the mechanisms underlying activation and desensitization remain unavailable for the pentameric ligand-gated channel family. Here, we report the structural rearrangements underlying gating transitions in membrane-embedded GLIC, a prokaryotic homologue, using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. We particularly probed the conformation of pore-lining second transmembrane segment (M2) under conditions that favor the closed and the ligand-bound desensitized states. The spin label mobility, intersubunit spin-spin proximity, and the solvent-accessibility parameters in the two states clearly delineate the underlying protein motions within M2. Our results show that during activation the extracellular hydrophobic region undergoes major changes involving an outward translational movement, away from the pore axis, leading to an increase in the pore diameter, whereas the lower end of M2 remains relatively immobile. Most notably, during desensitization, the intervening polar residues in the middle of M2 move closer to form a solvent-occluded barrier and thereby reveal the location of a distinct desensitization gate. In comparison with the crystal structure of GLIC, the structural dynamics of the channel in a membrane environment suggest a more loosely packed conformation with water-accessible intrasubunit vestibules penetrating from the extracellular end all the way to the middle of M2 in the closed state. These regions have been implicated to play a major role in alcohol and drug modulation. Overall, these findings represent a key step toward understanding the fundamentals of gating mechanisms in this class of channels.

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Background: Allosteric mechanisms in ligand-gated ion-channels (pLGIC) that couple neurotransmitter binding to channel opening are poorly understood. GLIC is an important prokaryotic surrogate. Results: EPR studies at the junctional interface of GLIC reveal structural changes during desensitization. Conclusion: The closed conformation is characterized by extensive intrasubunit interactions at the junctional interface that weaken during desensitization. Significance: These studies elucidate the role of structural dynamics in pLGIC function. Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.

A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

A chimeric ligand–gated channel is activated by the ligands that activate both parent channels, which gate it through two distinct mechanisms.