title: Transfer of Kv3.1 voltage sensor features to the isolated Ci-VSP voltage-sensing domain.
abstract: Membrane proteins that respond to changes in transmembrane voltage are critical in regulating the function of living cells. The voltage-sensing domains (VSDs) of voltage-gated ion channels are extensively studied to elucidate voltage-sensing mechanisms, and yet many aspects of their structure-function relationship remain elusive. Here, we transplanted homologous amino acid motifs from the tetrameric voltage-activated potassium channel Kv3.1 to the monomeric VSD of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) to explore which portions of Kv3.1 subunits depend on the tetrameric structure of Kv channels and which properties of Kv3.1 can be transferred to the monomeric Ci-VSP scaffold. By attaching fluorescent proteins to these chimeric VSDs, we obtained an optical readout to establish membrane trafficking and kinetics of voltage-dependent structural rearrangements. We found that motifs extending from 10 to roughly 100 amino acids can be readily transplanted from Kv3.1 into Ci-VSP to form engineered VSDs that efficiently incorporate into the plasma membrane and sense voltage. Some of the functional features of these engineered VSDs are reminiscent of Kv3.1 channels, indicating that these properties do not require interactions between Kv subunits or between the voltage sensing and the pore domains of Kv channels.
authors.affiliation: RIKEN Brain Science Institute, Saitama, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Mishina
authors.forename: Hiroki
authors.initials: H
authors.lastname: Mutoh
authors.forename: Thomas
authors.initials: T
authors.lastname: Knöpfel
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002938:Ciona intestinalis / Q000201:enzymology*; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016062:Porosity; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051670:Shaw Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D051670:Shaw Potassium Channels; C509671:voltage-sensor-containing phosphatase, Ciona intestinalis; D010744:Phosphoric Monoester Hydrolases