Limitation of Light Driven Proton Pumps at High Electrochemical Load

Abstract

All known light-driven ion pumps are microbial type rhodopsins that transport protons, sodium or chloride across the plasma membrane. Bacteriorhodopsin (BR) from Halobacterium salinarum is the most widely studied representative that has been investigated mainly by spectroscopic and structural methods. Recently light-driven pumps have been employed in optogenetics for silencing of neuronal activity and for voltage sensing (Arch3) and thus received new attention. Surprisingly, the knowledge about the electrophysiological properties of ion pumps in living cells is still poor. The voltage dependence and the limitation of the pumping activity at high electrochemical load, i.e. negative voltage and low pH, are unknown for most light-driven transporters. We will present experiments on rhodopsins from the alga Chlorella vulgaris (CvRh) and the proteorhodopsin-like pump from cyanobacterium Gloeobacter violaceus (GR). Both exhibit in comparison to BR much larger photocurrents in two-microelectrode voltage-clamp measurements (TEVC) on Xenopus leavis oocytes. We identified the reprotonating E132 in GR (D96 in BR) as the position that mediates high photocycle kinetics but is also responsible for the leakiness of proteorhodopsins at high load. Moreover, the proton shuttle R83 in CvRh (R82 in BR) was discovered as a critical residue in the proton release complex whereas the residues E193 and E203 (194 and 204 in BR) are of minor relevance. Replacement of R83 by Q drastically reduced the power of the pump and caused outward directed pumping at neutral pH but inward directed passive currents at high load. We identified the proton donor Glu (or Asp) that reprotonates the retinal Schiff base and the proton shuttle Arg that transports the proton from the Retinal Schiff base counter ion (D85 in BR) to the extracellular side as the key determinants for the directivity and the power of the pumps.