Polycystic kidney disease (PKD) family proteins associate with transient receptor potential (TRP) channel family proteins to form functionally important complexes. PKD proteins differ from known ion channel-forming proteins and are generally thought to act as membrane receptors. Here we find that PKD1L3, a PKD protein, functions as a channel-forming subunit in an acid-sensing heteromeric complex formed by PKD1L3 and TRPP3, a TRP channel protein. Single amino acid mutations in the putative pore region of both proteins alter the channel's ion selectivity. The PKD1L3/TRPP3 complex in the plasma membrane of live cells contains one PKD1L3 and three TRPP3. A TRPP3 C-terminal coiled-coil domain forms a trimer in solution and in crystal and plays a crucial role in the assembly and surface expression of the PKD1L3/TRPP3 complex. These results demonstrate that PKD subunits constitute a new class of channel-forming proteins, enriching our understanding of the function of PKD proteins and PKD/TRPP complexes.

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and different types of learning and memory. Recently, large-scale genetic analysis revealed de-novo missense mutations in their pore-forming a 1 -subunit (CACNA1D gene) in 6 patients associated with a neurodevelopmental syndrome including varying degrees of sporadic autism spectrum disorder (ASD, G407R), intellectual disability (A749G), neurological manifestations (including seizures, V401L) and endocrine symptoms (G403D, I750M). A typical hallmark of these mutations are severe gating changes compatible with a gain-of-channel-function. Here we investigated if similar gating changes are observed in a de-novo CACNA1D mutation (IIS4-S5 linker, Ca v 1.3 a 1mut ) which could explain symptoms in a patient diagnosed with a severe developmental disorder of unknown cause. Methods: Mutant (Ca v 1.3 a 1mut ) and wild-type Ca v 1.3 a 1 were co-expressed together with b 3 and a 2 d-1 subunits in tsA-201 cells and calcium currents (15mM) were measured using the whole cell patch-clamp technique. Results: Very similar to the previously characterized mutation V401L (IS6), A749G and I750M (IIS6), Ca v 1.3 a 1mut dramatically shifted the voltagedependence of Ca v 1.3 steady-state activation and inactivation to more negative voltages ($20 mV) without slowing of inactivation. A complete biophysical analysis revealed that these changes are compatible with a mutational gainof-function phenotype. Conclusion: By demonstrating the typical gating changes previously shown by us for CACNA1D de-novo missense mutations we propose that Ca v 1.3 a 1mut also explains the symptoms in this patient with a severe developmental disorder. Patients carrying such mutations may benefit from treatment with already available L-type Ca 2þ -channel blockers, such as nimodipine. Such CACNA1D missense mutations are likely underreported in large-scale genetic analyses. Support: Austrian Science Fund (FWF F4402, W1101).

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Voltage-gated Ca 2þ channels (VGCC) directly control muscle contraction and neurotransmitter release, and slower processes such as cell differentiation, migration, and death. They are potently inhibited by RGK GTP-ases (Rem1, Rem2, Rad and Gem/Kir), which decrease Ca 2þ channel membrane expression, as well as directly inhibit membrane-resident channels. The mechanisms of membrane-resident channel inhibition are difficult to study because RGKoverexpression causes complete or near complete channel inhibition. Using titrated levels of RGK expression in Xenopus oocytes that inhibit WT P/Q-type calcium channels by 50%, we show that inhibition depends on channel inactivation. Interestingly, fast-inactivating channels, including Familial Hemiplegic Migraine mutants, are more potently inhibited than WT channels, while slow-inactivating channels, such as those expressed with the b 2a auxiliary subunit, are spared. We found similar results in L-type channels, and, remarkably, an insensitivity of Timothy Syndrome mutants to RGK inhibition. Further results suggest that RGKs slow channel recovery from inactivation and identify RGKs as potential modulating factors in channelopathies. Finally, our results confirm a previously proposed immobilization of calcium channel voltage sensors by Rad, which we were now able to observe in electrophysiological experiments with titrated levels of Rad. Thus, RGK-mediated inhibition is a much subtler affair than previously thought, and physiological conditions can control the levels of inhibition. Clathrin-mediated endocytosis is an essential cellular functionof all eukaryotes. It relies on a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies that mediate vesicle formation. How so many proteins can be organized to produce endocytic vesicles with high precision and efficiency is not understood. To address this gap, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. This allowed us tovisualize where individual proteins are localized within the machinery throughout the endocytic process. By combining superresolution imaging, live-cell microscopy and Brownian dynamics simulations, we aim to identify the architectural features that allow the endocytic machinery to create vesicles with high efficiency and robustness. We found that actin filament nucleation is pre-patterned by a nucleation nanotemplate, which directly links molecular organization to the mechanics of endocytosis, and might represent a general design principle for directional force generation in other membrane remodeling processes such as during cell migration and division. 1545-PlatSelf-Organization and Force Production by the Branched Actin Cytoskeleton during Mammalian Clathrin-Mediated Endocytosis During clathrin-mediated endocytosis (CME), the cell's plasma membrane is deformed from a flat sheet into a round vesicle to internalize transmembrane proteins and e...