Debayan Sarkar

Background and Purpose: P2X receptors are trimeric ligand‐gated ion channels that open a cation‐selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains.Experimental Approach: Here, we examine the impact of P2X2 receptor inter‐subunit interface missense variants identified in the human population or by structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches.Key Results: We demonstrate that predicted extracellular domain inter‐subunit interfaces display a higher‐than‐expected density of missense variations and that the majority of mutations that disrupt putative inter‐subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity.Conclusion and Implications: We provide the first structural mapping of the mutational distribution across the human population in a ligand‐gated ion channel and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other ligand‐gated ion channels, P2X2 receptors have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

Background and Purpose: P2X receptors (P2XRs) are trimeric ligand-gated ion channels (LGICs) that open a cation-selective pore in response to ATP binding to their large extracellular domain (ECD). The seven known P2XR subtypes can assemble as homo- or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2XRs is well established, but little is known about the spectrum and prevalence of human genetic variations and the functional implications in specific domains. Experimental Approach: Here we examine the impact of P2X2 receptor (P2X2R) inter-subunit interface missense variants identified in the human population or through structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches. Key results: We demonstrate that predicted ECD inter-subunit interfaces display a higher-than-expected density of missense variations and that the majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity. Conclusions and Implications: We provide the first structural mapping of the mutational burden across the human population in a LGIC and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other LGICs, P2X2Rs have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation. Bullet point summary: ‘What is already known’: P2X2 receptors are ATP-activated ion channels implicated in hearing and nociceptice pathways ‘What this study adds’: A structural mapping of missense variants observed in the human population We identify the intersubunit-interface as a variant hotspot and decipher functional impact of mutations ‘Clinical significance’: The development of both inhibitors and activators of P2X2 receptor function may be required

Significance: Since fever is frequently a response to infection, immune responses likely occur commonly at fever temperatures. Are fever temperatures detected by immune cells, what molecular pathways are involved in this sensing, and what are the functional consequences? Our data show that CD4 T lymphocytes detect moderate fever temperature. A TRPV channel-mediated pathway involving Notch activation leads to a Th2 skewing of the effector T cell response. However, the antigen presenting dendritic cells that stimulate CD4 T cells, in turn, detect fever temperature and overproduce interleukin-12 to counter this Th2 skewing. The evolutionary genesis of these pathways and the functional consequences of this complex regulatory loop in specific infectious circumstances are, thus, likely to be of great interest.

Understanding the topology adopted by individual G-quadruplex (GQ)-forming sequences in vivo and targeting a specific GQ motif among others in the genome will have a profound impact on GQ-directed therapeutic strategies. However, this remains a major challenge as most of the tools poorly distinguish different GQ conformations and are not suitable for both cell-free and in-cell analysis. Here, we describe an innovative probe design to investigate GQ conformations and recognition in both cell-free and native cellular environments by using a conformation-sensitive dual-app nucleoside analogue probe. The nucleoside probe, derived by conjugating fluorobenzofuran at the 5-position of 2′-deoxyuridine, is composed of a microenvironment-sensitive fluorophore and an in-cell NMR compatible ¹⁹F label. This noninvasive nucleoside, incorporated into the human telomeric DNA oligonucleotide repeat, serves as a common probe to distinguish different GQ topologies and quantify topology-specific binding of ligands by fluorescence and NMR techniques. Importantly, unique signatures displayed by the ¹⁹F-labeled nucleoside for different GQs enabled a systematic study in Xenopus laevis oocytes to provide new structural insights into the GQ topologies adopted by human telomeric overhang in cells, which so far has remained unclear. Studies using synthetic cell models, immunostaining on fixed cells, and crystallization conditions suggest that parallel GQ is the preferred conformation of telomeric DNA repeat. However, our findings using the dual-app probe clearly indicate that multiple structures including hybrid-type parallel-antiparallel and parallel GQs are formed in the cellular environment. Taken together, our findings open new experimental strategies to investigate topology, recognition, and therapeutic potential of individual GQ-forming motifs in a biologically relevant context.

The roles of surrounding membrane lipids in the functions of transmembrane and peripheral membrane proteins are largely unknown. Herein, we utilize the recently reported structures of the TRPV1 ion channel protein bound to its potent protein agonist, the double-knot toxin (DkTx), as a model system to investigate the roles of toxin–lipid interfaces in TRPV1 activation by characterizing a series of DkTx variants electrophysiologically. Together with membrane partitioning experiments, these studies reveal that toxin–lipid interfaces play an overwhelmingly dominant role in channel activation as compared to lipid-devoid toxin–channel interfaces. Additionally, we find that whereas the membrane interfaces formed by one of the knots of the toxin endow it with its extremely low channel-dissociation rate, those formed by other knot contribute primarily to its potency. These studies establish that protein–lipid interfaces play nuanced yet profound roles in the function of protein–protein complexes within membranes.