Voltage-gated ion channels feature voltage sensor domains (VSDs) that exist in three distinct conformations during activation: resting, intermediate, and activated. Experimental determination of the structure of a potassium channel VSD in the intermediate state has previously proven elusive. Here, we report and validate the experimental three-dimensional structure of the human KCNQ1 voltage-gated potassium channel VSD in the intermediate state. We also used mutagenesis and electrophysiology in Xenopus laevis oocytes to functionally map the determinants of S4 helix motion during voltage-dependent transition from the intermediate to the activated state. Finally, the physiological relevance of the intermediate state KCNQ1 conductance is demonstrated using voltage-clamp fluorometry. This work illuminates the structure of the VSD intermediate state and demonstrates that intermediate state conductivity contributes to the unusual versatility of KCNQ1, which can function either as the slow delayed rectifier current (IKs) of the cardiac action potential or as a constitutively active epithelial leak current.
The structures determined in this work have been deposited into the Protein Databank (PDB ID 6MIE).NMR data assignments and structural restraints have been deposited in the BioMagResBank (BMRB ID 30517).All electrophysiology and voltage-clamp fluorometry data generated or analysed during this study are included in the manuscript, supporting files, and source data file.
NMR structure of the KCNQ1 voltage-sensing domainProtein Data Base, 6MIE.
Solution NMR structure of the KCNQ1 voltage-sensing domainBioMagResBank, 30517.
- Alfred L George
- Jens Meiler
- Charles R Sanders
- Jianmin Cui
- Jianmin Cui
- Keenan C Taylor
- Panpan Hou
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Oocytes from Xenopus laevis (frogs) were employed in this work (at Washington University) and the frogs were cared for in accordance with the protocol approved by the Washington University Animal Studies Committee (Protocol # 20190030).
- Merritt Maduke, Stanford University School of Medicine, United States
© 2020, Taylor et al.
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 transcriptional regulator SsrB acts as a switch between virulent and biofilm lifestyles of non-typhoidal Salmonella enterica serovar Typhimurium. During infection, phosphorylated SsrB activates genes on Salmonella Pathogenicity Island-2 (SPI-2) essential for survival and replication within the macrophage. Low pH inside the vacuole is a key inducer of expression and SsrB activation. Previous studies demonstrated an increase in SsrB protein levels and DNA-binding affinity at low pH; the molecular basis was unknown (Liew et al., 2019). This study elucidates its underlying mechanism and in vivo significance. Employing single-molecule and transcriptional assays, we report that the SsrB DNA binding domain alone (SsrBc) is insufficient to induce acid pH-sensitivity. Instead, His12, a conserved residue in the receiver domain, confers pH sensitivity to SsrB allosterically. Acid-dependent DNA binding was highly cooperative, suggesting a new configuration of SsrB oligomers at SPI-2-dependent promoters. His12 also plays a role in SsrB phosphorylation; substituting His12 reduced phosphorylation at neutral pH and abolished pH-dependent differences. Failure to flip the switch in SsrB renders Salmonella avirulent and represents a potential means of controlling virulence.
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