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Abstract: Calmodulin (CaM) is known to play an important role in the regulation of TRP channels activity. Although it has been reported that CaM binds to the C-terminus of TRPV1 (TRPV1-CT), no classic CaM-binding motif was found in this region. In this work, we explored this unusual TRPV1 CaM-binding motif in detail and found that five residues from a putative CaM-binding motif are important for TRPV1-CT’s binding to CaM, with arginine R785 being the most essential residue. The homology modelling suggests that a CaM-binding motif of TRPV1-CT forms an alpha helix that docks into the central cavity of CaM. [Copyright &y& Elsevier]

Abstract: Ivermectin (IVM) applied extracellularly increases the sensitivity of P2X4 receptor (P2X4R) to ATP, enhances the maximum current amplitudes, and greatly prolongs the deactivation kinetics. In this manuscript, we focused on identification of receptor-specific residues responsible for IVM effects on channel gating using the wild-type rat homomeric P2X4R, several chimeric P2X2/P2X4 receptors, and single-point P2X4R-specific mutants in the ectodomain and two transmembrane domains. Experiments with chimeric receptors revealed that the Val49–Val61 but not the Val64–Tyr315 ectodomain sequence is important for the effects of IVM on channel deactivation. Receptor-specific mutations placed in the Gly29–Val61 and Asp338–Leu358 regions showed the importance of Trp50, Val60, and Val357 residues in IVM regulation of the rate of channel deactivation, but not on the maximum current amplitude. These results suggest that the transmembrane domains and the nearby ectodomain region contribute to the effects of IVM on channel deactivation. [Copyright &y& Elsevier]

Abstract: To better understand the mechanism of interactions between G-protein-coupled melatonin receptors and their ligands, our previously reported homology model of human MT2 receptor with docked 2-iodomelatonin was further refined and used to select residues within TM3, TM6, and TM7 potentially important for receptor–ligand interactions. Selected residues were mutated and radioligand-binding assay was used to test the binding affinities of hMT2 receptors transiently expressed in HEK293 cells. Our data demonstrate that residues N268 and A275 in TM6 as well as residues V291 and L295 in TM7 are essential for 2-iodomelatonin binding to the hMT2 receptor, while TM3 residues M120, G121, V124, and I125 may participate in binding of other receptor agonists and/or antagonists. Presented data also hint at possible specific interaction between the side-chain of Y188 in second extracellular loop and N-acetyl group of 2-iodomelatonin. [Copyright &y& Elsevier]

Site-directed mutagenesis was applied to modify phenylalanines (Phe475Trp, Phe548Tyr, and both) to generate mutants on the basis of molecular modeling of the ATP-binding domain of Na+/K+-ATPase, in order to characterize the forces that stabilize ATP in its binding pocket. Each of the mutants was examined by Raman difference spectroscopy, i.e., as a difference between the spectrum of the domain with and without bound ATP. It was shown that Phe475 plays a key role in stabilizing ATP-binding by a stacking interaction. Phe548 co-stabilizes ATP on the opposite site of the binding pocket and its type of interaction with ATP-binding differs from that of Phe475. [Copyright &y& Elsevier]

The ATP-binding site of Na+/K+-ATPase is localized on the large cytoplasmic loop of the α-subunit between transmembrane helices H4 and H5. Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser445, Glu446, and Phe475 were proposed to be close to the binding pocket. Replacement of Phe475 with Trp and Glu446 with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser445 with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser445 is close to the binding site, although it does not participate in binding. [Copyright &y& Elsevier]