Background: TRPM7 channels are key regulators of cell growth and proliferation. Results: A natural compound from a Hawaiian soft coral blocks TRPM7 currents and inhibits proliferation. Conclusion: Waixenicin A represents the first potent and relatively specific inhibitor of TRPM7 ion channels. Significance: Waixenicin A or structural analogs may have cancer-specific therapeutic potential.

Summary Synaptotagmin 1 (syt1) is a synaptic vesicle membrane protein that functions as the Ca2+-sensor in neuronal exocytosis. Here, site-directed spin labeling was used to generate models for the solution and membrane bound structures of a soluble fragment of syt1 containing its two C2 domains, C2A and C2B. In solution, distance restraints between the two C2 domains of syt1 were measured using double electron-electron resonance (DEER) and used in a simulated annealing routine to generate models for the structure of the tandem C2A-C2B fragment. The data indicate that the two C2 domains are flexibly linked and do not interact with each other in solution, with or without Ca2+. However, the favored orientation is one where the Ca2+-binding loops are oriented in opposite directions. A similar approach was taken for membrane associated C2A–C2B, combining both distances and bilayer depth restraints with simulated annealing. The restraints can only be satisfied if the Ca2+ and membrane binding surfaces of the domains are oriented in opposite directions so that C2A and C2B are docked to opposing bilayers. The result suggests that syt1 functions to bridge across the vesicle and plasma membrane surfaces in a Ca2+-dependent manner.

Synaptotagmin 1 (syt1) is a synaptic vesicle-anchored membrane protein that acts as the calcium sensor for the synchronous component of neuronal exocytosis. Using site-directed spin labeling, the position and membrane interactions of a fragment of syt1 containing its two C2 domains (syt1C2AB) were determined in bilayers containing phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol, 4,5-bisphosphate (PIP2). Addition of 1 mole% PIP2 to a lipid mixture of PC and PS results in a deeper membrane penetration of the C2A domain and alters the orientation of the C2B domain so that the polybasic face of C2B comes into close proximity to the bilayer interface. The C2B domain is found to contact the membrane interface in two regions, the Ca2+-binding loops and a region opposite the Ca2+-binding loops. This suggests that syt1C2AB is configured to bridge two bilayers and is consistent with a model generated previously for syt1C2AB bound to membranes of PC and PS. Point-to-plane depth restraints, obtained by progressive power saturation, and interdomain distance restraints, obtained by double electron-electron resonance, were obtained in the presence of PIP2 and used in a simulated annealing routine to dock syt1C2AB to two membrane interfaces. The results yield a different average structure than is found in the absence of PIP2 and indicate that bilayer-bilayer spacing is decreased in the presence of PIP2. The results indicate that PIP2, which is necessary for bilayer fusion, alters C2 domain orientation, enhances syt1-membrane electrostatic interactions and acts to drive vesicle and cytoplasmic membrane surfaces closer together.

SummaryThe Ca 2+ -independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling (SDSL) and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine (PS) and phosphatidylcholine (PC) in a Ca 2+ -independent manner with a lipid partition coefficient of approximately 3.0 × 10 2 M −1 . A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to PC/PS bilayers in the absence of Ca 2+ . Although the Ca 2+ -independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. SDSL was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca 2+ . In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer, but is localized within the aqueous double-layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca 2+ , the membrane affinity of C2B is increased approximately 20 fold, and the domain rotates so that the Ca 2+ binding loops of C2B insert into the bilayer. This Ca 2+ -triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B, and control interactions of syt1 with other components of the fusion machinery.