Ion transport activity and optogenetics capability of light-driven Na+-pump KR2

PLoS One. 2021 Sep 10;16(9):e0256728. doi: 10.1371/journal.pone.0256728. eCollection 2021.

Abstract

KR2 from marine bacteria Krokinobacter eikastus is a light-driven Na+ pumping rhodopsin family (NaRs) member that actively transports Na+ and/or H+ depending on the ionic state. We here report electrophysiological studies on KR2 to address ion-transport properties under various electrochemical potentials of Δ[Na+], ΔpH, membrane voltage and light quality, because the contributions of these on the pumping activity were less understood so far. After transient expression of KR2 in mammalian cultured cells (ND7/23 cells), photocurrents were measured by whole-cell patch clamp under various intracellular Na+ and pH conditions. When KR2 was continuously illuminated with LED light, two distinct time constants were obtained depending on the Na+ concentration. KR2 exhibited slow ion transport (τoff of 28 ms) below 1.1 mM NaCl and rapid transport (τoff of 11 ms) above 11 mM NaCl. This indicates distinct transporting kinetics of H+ and Na+. Photocurrent amplitude (current density) depends on the intracellular Na+ concentration, as is expected for a Na+ pump. The M-intermediate in the photocycle of KR2 could be transferred into the dark state without net ion transport by blue light illumination on top of green light. The M intermediate was stabilized by higher membrane voltage. Furthermore, we assessed the optogenetic silencing effect of rat cortical neurons after expressing KR2. Light power dependency revealed that action potential was profoundly inhibited by 1.5 mW/mm2 green light illumination, confirming the ability to apply KR2 as an optogenetics silencer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Flavobacteriaceae / metabolism*
  • Ions / metabolism*
  • Light*
  • Neurons / cytology
  • Neurons / metabolism*
  • Rats
  • Sodium-Potassium-Exchanging ATPase / metabolism*

Substances

  • Ions
  • Sodium-Potassium-Exchanging ATPase

Supplementary concepts

  • Dokdonia eikasta

Associated data

  • figshare/10.6084/m9.figshare.16417236

Grants and funding

This work was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology (18H03986 to H.K and 18K06109 to S.P.T.), JST CREST grant (JPMJCR1753 to H.K.) and JST PRESTO grant (JPMJPR1688 to S.P.T). S.H. is a Research Fellow of the Japan Society for the Promotion of Science (JSPS Research Fellow). https://www.jst.go.jp/kisoken/presto/en/index.htmlhttps://www.jst.go.jp/kisoken/crest/en/index.htmlhttps://www.jsps.go.jp/english/index.html.