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David L Farrens - One of the best experts on this subject based on the ideXlab platform.

  • evidence that the rhodopsin kinase grk1 n terminus and the transducin gα c terminus interact with the same hydrophobic patch on rhodopsin tm5
    Biochemistry, 2016
    Co-Authors: Amber Jones M Brunette, Abhinav Sinha, Larry L David, David L Farrens
    Abstract:

    Phosphorylation of G protein-coupled receptors (GPCRs) terminates their ability to couple with and activate G proteins by increasing their affinity for arrestins. Unfortunately, detailed information regarding how GPCRs interact with the kinases responsible for their phosphorylation is still limited. Here, we purified fully functional GPCR kinase 1 (GRK1) using a rapid method and used it to gain insights into how this important kinase interacts with the GPCR rhodopsin. Specifically, we find that GRK1 uses the same site on rhodopsin as the transducin (Gt) Gtα C-terminal tail and the arrestin “finger loop”, a cleft formed in the cytoplasmic face of the receptor upon activation. Our studies also show GRK1 requires two conserved residues located in this cleft (L226 and V230) that have been shown to be required for Gt activation due to their direct interactions with hydrophobic residues on the Gα C-terminal tail. Our data and modeling studies are consistent with the idea that all three proteins (Gt, GRK1, and v...

  • Evidence that the Rhodopsin Kinase (GRK1) N‑Terminus and the Transducin Gα C‑Terminus Interact with the Same “Hydrophobic Patch” on Rhodopsin TM5
    2016
    Co-Authors: Amber M. Jones Brunette, Abhinav Sinha, Larry David, David L Farrens
    Abstract:

    Phosphorylation of G protein-coupled receptors (GPCRs) terminates their ability to couple with and activate G proteins by increasing their affinity for arrestins. Unfortunately, detailed information regarding how GPCRs interact with the kinases responsible for their phosphorylation is still limited. Here, we purified fully functional GPCR kinase 1 (GRK1) using a rapid method and used it to gain insights into how this important kinase interacts with the GPCR rhodopsin. Specifically, we find that GRK1 uses the same site on rhodopsin as the transducin (Gt) Gtα C-terminal tail and the arrestin “finger loop”, a cleft formed in the cytoplasmic face of the receptor upon activation. Our studies also show GRK1 requires two conserved residues located in this cleft (L226 and V230) that have been shown to be required for Gt activation due to their direct interactions with hydrophobic residues on the Gα C-terminal tail. Our data and modeling studies are consistent with the idea that all three proteins (Gt, GRK1, and visual arrestin) bind, at least in part, in the same site on rhodopsin and interact with the receptor through a similar hydrophobic contact-driven mechanism

Murray Epstein - One of the best experts on this subject based on the ideXlab platform.

Manfred Lindau - One of the best experts on this subject based on the ideXlab platform.

  • Role of the synaptobrevin C terminus in fusion pore formation
    Proceedings of the National Academy of Sciences, 2010
    Co-Authors: Annita N. Ngatchou, Kassandra Kisler, Qinghua Fang, Alexander M. Walter, Jakob B. Sørensen, Manfred Lindau
    Abstract:

    Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets.

  • Role of the Synaptobrevin C-Terminus in Fusion Pore Formation
    Biophysical Journal, 2010
    Co-Authors: Annita N. Ngatchou, Dieter Bruns, Ying Zhao, Kassandra Kisler, Qinghua Fang, Alexander M. Walter, Jakob B. Sørensen, Manfred Lindau
    Abstract:

    Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII) also called VAMP2, syntaxin and SNAP-25 generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to fusion pore formation remains elusive.To determine a possible role of the sybII TM domain in fusion pore formation, sybII constructs in which one or two polar or non-polar residues were added at the C-Terminus of the protein were expressed in double knock-out (DKO) embryonic mouse chromaffin cells deficient in sybII and cellubrevin1. Exocytosis was stimulated by flash photolysis of caged-calcium (NP-EGTA), and the capability of the mutated constructs to support exocytosis was monitored by whole-cell patch clamp capacitance measurements while the associated transmitter release was monitored by carbon fiber amperometry. We found that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C-Terminus depending on their energy of transfer from water to the membrane interface2, following a Boltzmann distribution. These results suggest that C-terminal zipping of the SNARE complex pulls the C-Terminus of sybII deeper into the vesicle membrane, with this movement disrupting the vesicular membrane continuity and leading to fusion pore formation. In contrast to current models, fusion thus begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed endoplasmic leaflets.1 M. Borisovska, Y. Zhao, Y. Tsytsyura et al., Embo J 24 (12), 2114 (2005).2 W. C. Wimley and S. H. White, Nat Struct Biol 3 (10), 842 (1996).Supported by NIH grants R01NS38200, R01GM85808, T32GM8267, the Nanobiotechnology Center (NSF) and DFG grants SFB523-B16 and SFB530-C10.

Thomas V Mcdonald - One of the best experts on this subject based on the ideXlab platform.

  • functional interactions between kcne1 c terminus and the kcnq1 channel
    PLOS ONE, 2009
    Co-Authors: Jerri Chen, Renjian Zheng, Yonathan F Melman, Thomas V Mcdonald
    Abstract:

    The KCNE1 gene product (minK protein) associates with the cardiac KvLQT1 potassium channel (encoded by KCNQ1) to create the cardiac slowly activating delayed rectifier, IKs. Mutations throughout both genes are linked to the hereditary cardiac arrhythmias in the Long QT Syndrome (LQTS). KCNE1 exerts its specific regulation of KCNQ1 activation via interactions between membrane-spanning segments of the two proteins. Less detailed attention has been focused on the role of the KCNE1 C-Terminus in regulating channel behavior. We analyzed the effects of an LQT5 point mutation (D76N) and the truncation of the entire C-Terminus (Δ70) on channel regulation, assembly and interaction. Both mutations significantly shifted voltage dependence of activation in the depolarizing direction and decreased IKs current density. They also accelerated rates of channel deactivation but notably, did not affect activation kinetics. Truncation of the C-Terminus reduced the apparent affinity of KCNE1 for KCNQ1, resulting in impaired channel formation and presentation of KCNQ1/KCNE1 complexes to the surface. Complete saturation of KCNQ1 channels with KCNE1-Δ70 could be achieved by relative over-expression of the KCNE subunit. Rate-dependent facilitation of K+ conductance, a key property of IKs that enables action potential shortening at higher heart rates, was defective for both KCNE1 C-terminal mutations, and may contribute to the clinical phenotype of arrhythmias triggered by heart rate elevations during exercise in LQTS mutations. These results support several roles for KCNE1 C-Terminus interaction with KCNQ1: regulation of channel assembly, open-state destabilization, and kinetics of channel deactivation.

  • Functional Interactions between KCNE1 C-Terminus and the KCNQ1 Channel
    2008
    Co-Authors: Jerri Chen, Renjian Zheng, Yonathan F Melman, Thomas V Mcdonald
    Abstract:

    The KCNE1 gene product (minK protein) associates with the cardiac KvLQT1 potassium channel (encoded by KCNQ1) to create the cardiac slowly activating delayed rectifier, IKs. Mutations throughout both genes are linked to the hereditary cardiac arrhythmias in the Long QT Syndrome (LQTS). KCNE1 exerts its specific regulation of KCNQ1 activation via interactions between membrane-spanning segments of the two proteins. Less detailed attention has been focused on the role of the KCNE1 C-Terminus in regulating channel behavior. We analyzed the effects of an LQT5 point mutation (D76N) and the truncation of the entire C-Terminus (D70) on channel regulation, assembly and interaction. Both mutations significantly shifted voltage dependence of activation in the depolarizing direction and decreased IKs current density. They also accelerated rates of channel deactivation but notably, did not affect activation kinetics. Truncation of the C-Terminus reduced the apparent affinity of KCNE1 for KCNQ1, resulting in impaired channel formation and presentation of KCNQ1/ KCNE1 complexes to the surface. Complete saturation of KCNQ1 channels with KCNE1-D70 could be achieved by relative over-expression of the KCNE subunit. Rate-dependent facilitation of K+ conductance, a key property of IKs that enables action potential shortening at higher heart rates, was defective for both KCNE1 C-terminal mutations, and may contribute to the clinical phenotype of arrhythmias triggered by heart rate elevations during exercise in LQTS mutations. These results support several roles for KCNE1 C-Terminus interaction with KCNQ1: regulation of channel assembly, open-stat

David L Vesely - One of the best experts on this subject based on the ideXlab platform.