H uman e ther-à-go-go- r elated g ene (Kv11.1, or hERG) is a potassium channel that conducts the delayed rectifier potassium current (I Kr ) during the repolarization phase of cardiac action potentials. hERG channels have a larger pore than other K + channels and can trap many unintended drugs, often resulting in acquired LQTS (aLQTS). R -roscovitine is a cyclin-dependent kinase (CDK) inhibitor that induces apoptosis in colorectal, breast, prostate, multiple myeloma, other cancer cell lines, and tumor xenografts, in micromolar concentrations. It is well tolerated in phase II clinical trials. R -roscovitine inhibits open hERG channels but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus oocytes expressing wild-type (WT) or hERG pore mutant channels (T623A, S624A, Y652A, F656A) demonstrated that compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 μM R -roscovitine was ~ 48%, 29%, and 73% weaker, respectively. In contrast, S624A hERG was inhibited more potently than WT hERG, with a ~ 34% stronger inhibition. These findings were further supported by the IC 50 values, which were increased for T623A, Y652A and F656A (by ~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data suggest that while T623, Y652, and F656 are critical for R -roscovitine-mediated inhibition, S624 may not be. Docking studies further support our findings. Thus, R- roscovitine’s relatively unique features, coupled with its tolerance in clinical trials, could guide future drug screens.

14Human ether-à-go-go-related gene (Kv11.1, or hERG) is a potassium channel that 15 conducts the delayed rectifier potassium current (I Kr ) during the repolarization phase of cardiac 16 action potentials. hERG channels have a larger pore than other K + channels and can trap many 17 unintended drugs, often resulting in acquired LQTS (aLQTS). R-roscovitine, a cyclin-dependent 18 kinase (CDK) inhibitor that also inhibits L-type calcium channels, inhibits open hERG channels 19 but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus 20 oocytes expressing wild-type (WT) or mutant (T623A, S624A, Y652A, F656A) hERG channels 21 demonstrated that, compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 µM R-22 roscovitine was ~ 48 %, 29 %, and 73 % weaker, respectively. In contrast, S624A hERG was 23 inhibited more potently than WT hERG, with an ~ 34 % stronger inhibition. These findings were 24 further supported by the IC 50 values, which were increased for T623A, Y652A and F656A (by 25~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data 26 suggest that while T623, Y652, and F656 are critical for R-roscovitine-mediated inhibition, S624 27 may not be. This relatively unique feature, coupled with R-roscovitine's tolerance in clinical 28 trials, could guide future drug screens. We discuss our findings and how they lend support for the 29 recent Comprehensive In Vitro Proarrhythmia Assay (CiPA) guidelines on the re-evaluation of 30 potentially useful drugs that had failed testing due to unintended interactions with hERG. 31 32 Introduction 33 Human ether-à-go-go-related gene, or hERG [Kv11.1], is a voltage-gated potassium 34 channel critical for nerve and cardiac function [1,2]. In the heart, hERG channels initially open 3 35 during the depolarization phase of the cardiac action potential (cAP) but immediately inactivate. 36 Upon cAP repolarization, hERG channels de-inactivate and reopen, which allows the ensuing 37 large K + efflux to speed cAP repolarization [1], limit cardiac excitability, and maintain normal 38 QT intervals [3]. Consequently, mutations in hERG are one of the leading causes of congenital 39 long QT syndrome (cLQTS), with a neonatal incidence rate of up to 1 in 2,500 [4]; abnormal 40 cardiac phenotypes are usually triggered during exercise, arousal, or rest [5].41 hERG channels are tetrameric proteins, with each monomer consisting of six 42 transmembrane alpha helices (S1-S6) and cytoplasmic amino and carboxy termini [6]. Similar to 43 other voltage-gated channels, S1-S4 is considered the primary voltage-sensing region, with S4 44 containing positively-charged residues that move slowly outward to induce the characteristically 45 slow activation kinetics of hERG [7,8]. The four S5 and S6 helices and their intervening 46 sections, including the P-loops and P-helices, form the pore and selectivity filter of the channel 47 [9,10]. The pore is thought to have a region between the pore helix and the S6 segments that may 48 provide p...

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).

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...