Light-induced movement of the transmembrane helix B in channelrhodopsin-2.

In the past decade, its versatile usage has established channelrhodopsin-2 (ChR2) as the most prominent optogenetic tool. The precise spatio-temporal control of the activity of a neuron by light allows noninvasive in vitro or in vivo investigations of neural circuits. The key is the heterologous expression of ChR2, a member of the type-I rhodopsins, that enters a photocycle upon light activation starting with the isomerization around the C13–C14 bond of its chromophore retinal. The intermediates of the photocycle are linked to a closed and an open state of an ion pore. Cation flux during the open state depolarizes the cell membrane thereby triggering neuronal action potentials. A coupling of isomerization with structural alterations is wellknown for bacteriorhodopsin (bR) and sensory rhodopsin II (SRII) that eventually leads to movement of the cytoplasmic part of transmembrane helices (TMHs) F and G. Conformational changes are also anticipated for ChR2 propagating from the chromophore to the putative cation pore. According to a homology model of ChR2 and the closed state structure of a chimera (C1C2) of ChR1 (TMHs A to E) and ChR2 (TMHs F and G), the pore comprises residues from TMHs A, B, C, and G, including a ChR characteristic patch of glutamate residues in TMH B. In this study, we followed the relative (re-)arrangements of TMHs B and F by EPR spectroscopy (double electron electron resonance, DEER) on spin-labeled mutants retaining wild-type (WT) character in ion conductance and photocycle kinetics. We could monitor a conformational change at TMH B upon light activation. Because of the unique channel properties of ChR2 and the major structural deviations in TMH A and B compared to other rhodopsins, the movement of TMH B might be a key element for channel opening. A monomeric form of ChR2 is unknown and a cysteinefree mutant shows very low light-induced currents in electrophysiological experiments and insufficient expression in yeast cells. ChR2 cysteine mutant screening led to two constructs. The first mutant has six cysteines replaced and three remaining: C34SC36SC87SC179LC183LC259L (Mut3C). The second mutant has seven cysteines replaced and only two remaining: C34SC36SC87SC179LC183LC208AC259L (Mut2C) (Figure 1A). Spin labeling of Mut3C with native cysteines C79, C128, and C208 using (1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl) methanethiosulfonate spin label (MTSSL) allows the study of interspin distances between TMHs B and TMHs F. Mut2C with native cysteines C79 and C128 reduces the analysis to interspin distances between two TMHs B sites, C79R1 and C79R1’, in the ChR2 dimer (R1 denotes the spin-labeled side chain). We found C128 to be inaccessible for MTSSL. Light-induced currents recorded from oocytes expressing WT ChR2, Mut3C, and Mut2C (Figure 1B) show typical inward rectification. All currents densities are normalized to the WT value at 60 mV (Figure 1C). Mut3C shows a 14% reduced amplitude. A further decrease to 25% of the WT ChR2 value is found for Mut2C. The effect of the C208A mutation is also observed in a triple point mutation (C34SC36SC208A). Here, the C208A mutation shows a 28 or 12 % reduction compared to WT ChR2 or C34SC36S ChR2 (Figure 1C), respectively. We cannot discriminate between a lower expression level and a functional defect in the C208A mutant. Amino acids other than alanine reduce the current densities even more. The sensitivity of position 208 is unclear as there is no obvious interaction site for the cysteine side chain in the chimera structure. The mutation of C79 to alanine (C34SC36SC79A) leads to a reduced current amplitude of about 52% of the WT ChR2 value (Figure 1 C). As for C208, other mutations at position 79 (to Leu, Thr, or Ser) provoke strong current density reductions. Replacing C79 and C208 together (C34SC36SC79AC208A) creates a variant with very low current amplitude (< 10 %) comparable to that of the cysteine-less ChR2 mutant. C79 is located at the N-terminus of TMH B close to the intracellular loop A-B. This loop is not resolved in the C1C2 structure. Hence, the flexibility of the loop together with the positional sensitivity of C79 could indicate a structurally relevant factor that is needed for the open state. This view is in line with a constriction site formed by the neighboring Y70 in the closed dark state structure. Compared to WT ChR2 no significant change in the mono[*] T. Sattig, Prof. Dr. E. Bamberg, Dr. C. Bamann Abteilung f r Biophysikalische Chemie Max-Planck-Institut f r Biophysik Max-von-Laue Str. 3, 60438 Frankfurt a. M. (Germany) E-mail: christian.bamann@biophys.mpg. de C. Rickert, Prof. Dr. H. -J. Steinhoff Fachbereich Physik, Universit t Osnabr ck Barbarastr. 7, 49076 Osnabr ck (Germany) E-mail: hsteinho@uni-osnabrueck.de [] These authors contributed equally to this work.