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AbstractRecent advances in mass spectrometry coupled with chemical crosslinking (CX-MS) can be applied for the structural interrogation of macromolecular complexes to identify statedependent distance constraints and provides a powerful complementary technique to other structural methods. In this study, we develop a CX-MS approach to identify the sites of crosslinking from a single targeted location within the human glycine receptor (GlyR) in a single apo/resting state. The GlyR belongs to the family of pentameric ligand-gated ion channel receptors that function in fast neuronal transmission. A single cysteine residue was re-introduced into Cys null GlyR construct at position 41 within the extracellular domain of an overexpressed human a1 homomeric GlyR. After purification and reconstitution into vesicles, a methanethiosulfonate benzophenone heterobifunctional crosslinker was attached via formation of a disulfide bond, and the resting receptor is subsequently photocrosslinked. Monomeric and oligomeric bands from SDS-PAGE gels were then trypsinized and analyzed by tandem MS in bottom-up studies. Dozens of intra- and inter-subunit sites of crosslinking were differentiated and identified from single gel bands (pmols of purified GlyR), showing the utility of this approach to identify a diverse array of distance constraints of GlyR in its resting state. These studies highlight the potential of CX-MS as an experimental approach to identify state-dependent crosslinks of full length integral membrane protein assemblies in a native-like lipid environment.

the entire LBD or the whole protein. Hence, b-TF1 is viewed as a suppressor of InsP 3 binding. Additional studies suggest that b-TF1 not only helps stabilize LBD but also couples its conformational changes to the gate of the ion pore. The crystal structures of b-TF1 alone and of b-TF2 plus ARF bound with InsP 3 have been solved separately. The latter structure reveals how InsP 3 is coordinated by various side-chains in the binding sites at the ARF-b-TF2 interface, and mutation of these side-chains weakens InsP 3 binding. Despite this progress, the fundamental question of how InsP 3 biases the gating conformation of LBD remains. To address this question, we have determined the crystal structure of the entire LBD of rat InsP 3 R1 in both InsP 3-bound and-unbound conformations, revealing a triangular architecture. Comparison of the InsP 3bound and-unbound conformations strongly implies that b-TF1 and ARF move as a rigid unit with respect to b-TF2. While LBD without InsP 3-bound may spontaneously transition between gating states, binding of InsP 3 between b-TF2 and ARF locks it in a state that would strongly bias the gating equilibrium toward the open state of the ion pore.

Emerging evidences show involvement of chloride channels present in cardiac cells, in the adaptive remodeling of the heart during myocardial hypertrophy and heart failure. Previously it was demonstrated that blocking cardiac chloride channels with IAA-94, an inhibitor for chloride intracellular channel proteins (CLICs), prevented the protective effects of ischemic preconditioning and cyclosporine A-mediated cardioprotection but the molecular identity and specific role of CLICs in cardiac tissue is not known. Therefore, we attempted to establish molecular identity of CLICs and understand mechanism of IAA-94-mediated cardio deleterious effect. We demonstrate that IAA-94 increases myocardial infarction after ischemic/reperfusion injury in in vivo rat model also. Amongst the six paralogs of CLICs present in mammals, qPCR showed that CLIC1 (63.2þ3.8%), CLIC4 (89.6þ10.4%), and CLIC5 (66.7þ14.0%) are the most abundant CLICs in the heart. We recorded IAA-94 sensitive channel conductance of 110þ10 pS in isolated mitoplasts as reported for CLICs. Further, organelle-immunochemistry showed that CLIC5 (69.1512.2%), CLIC4 (48.956.9%), but not CLIC1 (29.5511.2%) localized to the ultrapure mitochondria. DmCLIC, an ortholog of mammalian CLIC present in D. melanogaster also localizes to cardiac mitochondria. We further demonstrate that rate of reactive oxygen species (ROS) production is reduced upon IAA-94 (100 mM) addition in both cardiac mitochondria (82.553.0%) and H9C2 cells (20.154.6%). ROS modulation was specific to IAA-94 and was not observed with other chloride channel inhibitors. IAA-94 significantly reduced the calcium retention capacity of the mitochondria (90.056.0%), hence reducing a threshold for opening mitochondrial permeability transition pore, but did not affect its membrane potential. Our study highlights the presence of CLIC4 and CLIC5 in cardiac mitochondria, where they modulate mitochondrial function, therefore playing a potential role in cardioprotection.