Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape
| Autor: | Jason D. Galpin, Elizabeth E. L. Lee, Grace D. Galles, Daniel T. Infield, Francisco Bezanilla, Christopher A. Ahern |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Arginine Physiology Xenopus Kinetics Static Electricity Membrane Potentials 03 medical and health sciences chemistry.chemical_compound Mice Protein Domains Static electricity Citrulline Animals Shaker Research Articles Membrane potential Communication Citrullination Potassium channel 030104 developmental biology chemistry Amino Acid Substitution Biophysics Shaker Superfamily of Potassium Channels Ion Channel Gating |
| Zdroj: | The Journal of General Physiology |
| ISSN: | 1540-7748 0022-1295 |
| Popis: | Activation of voltage-gated channels results from the outward movement of arginine residues on the S4 segments. Infield et al. use in vivo nonsense suppression to replace Shaker's S4 arginine residues with citrulline and reveal that a positive charge is required on R4 for voltage-dependent deactivation. Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance–voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo. |
| Databáze: | OpenAIRE |
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