The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.
Measuring the rate of dinitrophenylation of a specific lysine residue (called a) that is allosterically linked to the transfer site, it could be demonstrated that the anion transport protein may exist in two different conformational states, designated cis and trans. In the cis conformation a is easily accessible for reaction with dinitrofluorobenzene; in the trans conformation, a is less accessible. In the presence of the substrate anion Cl, the equilibrium between the cis and trans conformation is towards the cis conformation. Reversibly acting inhibitors of anion transport arrest the transport system, either predominantly in the cis or in the trans conformation. Phlorizin and certain positively charged derivatives of furosemide produce arrest in cis conformation, internal 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonate (APMB) and Ca++ in trans conformation. Within this frame of reference, the different susceptibilities of the transfer site to internal and external 4,4' diacetamido-2,2'-stilbene disulfonate (DAS) are interpreted on the assumption that the conformation of the transfer site changes during the transition of the transport protein from the cis to the trans conformation, so that in the trans conformation a reaction with DAS is no longer possible.
A vector was constructed containing a cDNA for mouse band 3 obtained from Demuth et al. (1986, EMBO J., 5, 1205‐1214), a synthetic linker (containing 5′‐non‐translated region, start codon and a coding region for the first 12 N‐terminal amino acids), and RNA polymerase promoters suitable for in vitro transcription of cRNA. After injection of the cRNA into the cytoplasm of Xenopus oocytes and incubation for 16 h, expression of mouse band 3 was demonstrated by immunoprecipitation, immunohistochemical methods and influx or efflux measurements with 36Cl‐. Antisense cRNA inhibits the expression. Lysines 558 and 561 were replaced by asparagines using oligonucleotide‐directed mutagenesis. Like the original band 3, the mutant shows stilbene disulfonate‐inhibitable anion exchange. However, in contrast to the original band 3, inhibition by 4,4′‐diisothiocyano dihydrostilbene‐2,2′‐disulfonate (H2DIDS) is no longer irreversible. This indicates that thiourea bond formation between H2DIDS and band 3 involves one of the two modified lysine residues. It also shows that the two lysine residues are not essential for the execution of the anion transport function of band 3. The results described suggest that the cDNA clone of Demuth et al. (1986) encodes a protein with properties that are representative for the properties of the bulk of the band 3 protein in the plasma membrane of the red cell of the mouse.
4,4′‐Diisothiocyanatodihydrostilbene‐2,2′‐disulfonate and 4,4′‐dibenzoylstilbene‐2,2′‐disulfonate potently inhibit the erythrocyte anion transporter. These inhibitors act by binding, with a 1:1 stoichiometry, to the band 3 transport protein. We have studied, by sedimentation equilibrium analysis in an analytical ultracentrifuge, the effect of the two closely related stilbenedisulfonates on the state of association of band 3 in the nonionic detergent nonaethyleneglycol lauryl ether. It was found that covalent binding of 4,4′‐diisothiocyanatodihydrostilbene‐2,2′‐disulfonate to band 3 did not significantly disturb the monomer/dimer/tetramer association equilibrium shown by the unliganded protein. An entirely different result was obtained after addition of 4,4′‐dibenzoylstilbene‐2,2′‐disulfonate to the protein, at both low and high chloride concentrations. The amount of band 3 dimer in the samples increased with increasing inhibitor concentration cI, and for cI≥15 μM virtually all of the protein was present as dimer. After removal of the inhibitor (by gel filtration or dialysis), the original monomer/dimer/tetramer distribution of the band 3 protein was restored.
Our data show that the (noncovalent) binding of 4,4′‐dibenzoylstilbene‐2,2′‐disulfonate drastically changes the coupling between band 3 protomers. In addition, a reversible change in the state of association of band 3 induced by ligand binding is demonstrated.
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