Stimulated muscarinic acetylcholine receptors (M2Rs) release Gβγ subunits, which slow heart rate by activating a G protein-gated K+ channel (GIRK). Stimulated β2 adrenergic receptors (β2ARs) also release Gβγ subunits, but GIRK is not activated. This study addresses the mechanism underlying this specificity of GIRK activation by M2Rs. K+ currents and bioluminescence resonance energy transfer between labelled G proteins and GIRK show that M2Rs catalyze Gβγ subunit release at higher rates than β2ARs, generating higher Gβγ concentrations that activate GIRK and regulate other targets of Gβγ. The higher rate of Gβγ release is attributable to a faster G protein coupled receptor - G protein trimer association rate in M2R compared to β2AR. Thus, a rate difference in a single kinetic step accounts for specificity.
All data generated or analyzed during this study are included in the manuscript and supporting files.
- Roderick MacKinnon
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: C57BL/6J (Jackson Labs) male and female adult mice (at least 10 weeks old) were used. Animals were kept in cages with a 12 : 12 h light/dark cycle and unrestricted access to food and water. All experimental procedures were carried out according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of The Rockefeller University (Protocol #16864).
- Kenton Jon Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
© 2018, Touhara & MacKinnon
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery mediates the membrane fission step that completes cytokinetic abscission and separates dividing cells. Filaments composed of ESCRT-III subunits constrict membranes of the intercellular bridge midbody to the abscission point. These filaments also bind and recruit cofactors whose activities help execute abscission and/or delay abscission timing in response to mitotic errors via the NoCut/Abscission checkpoint. We previously showed that the ESCRT-III subunit IST1 binds the cysteine protease CAPN7 (Calpain-7) and that CAPN7 is required for both efficient abscission and NoCut checkpoint maintenance (Wenzel et al., 2022). Here, we report biochemical and crystallographic studies showing that the tandem MIT domains of CAPN7 bind simultaneously to two distinct IST1 MIT interaction motifs. Structure-guided point mutations in either CAPN7 MIT domain disrupted IST1 binding in vitro and in cells, and depletion/rescue experiments showed that the CAPN7-IST1 interaction is required for: 1) CAPN7 recruitment to midbodies, 2) efficient abscission, and 3) NoCut checkpoint arrest. CAPN7 proteolytic activity is also required for abscission and checkpoint maintenance. Hence, IST1 recruits CAPN7 to midbodies, where its proteolytic activity is required to regulate and complete abscission.
The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium Vibrio cholerae. Its environmental persistence provoking recurring sudden outbreaks is enabled by V. cholerae's rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies e.g. bile resistance and virulence regulation. The presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. Our data proposes an interdependent functionality between ToxR transcriptional activity and ToxS sensory function. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unravelling the intricate mechanisms governing ToxRS's environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrio's survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.