We previously developed a general method to express orthogonal prokaryotic tRNAs in eukaryotic cells by using a type-3 Pol III promoter in mammalian cells[7
] and a posttranscriptionally cleavable Pol III promoter in yeast.[4
] Here we demonstrate that this strategy can be extended to neural stem cells. The type-3 H1 promoter that drives the expression of human H1RNA was successfully used to express both
of E. coli
origin in HCN-A94 cells throughout their neural differentiation program. Other members of the type-3 class of polymerase III promoter, such as U6, 7SK and MRP/7-2, should also work in a similar manner. Among the three strategies used for constructing lentiviral vectors to drive expression of the orthogonal tRNA/synthetase pair and the gene of interest, the second strategy employing the H1-tRNA cassette inserted in the 3′-LTR yielded the most efficient amino acid incorporation at the UAG amber codon site. The key feature of this strategy is that while only a single H1-tRNA cassette is inserted in the lentiviral vector, it generates two genomically incorporated copies of the transcription unit after lentiviral reverse transcription and integration into the host cells. The third strategy employing four tandem repeats of the H1-tRNA cassette resulted in lower amino acid incorporation, possibly because repeated promoter and tRNA sequences within the same lentiviral construct resulted in recombination during the production of transducible lentivirus, leading to deletion of the H1-tRNA cassette. Similar lentiviral genetic instability has been reported when a promoter was repeatedly used to express shRNAs encoded in the same lentiviral vector.[26
] Insertion of a single H1-tRNA cassette in the 3′-LTR circumvents this problem while maintaining increased gene dosage upon integration into the host cell. Notably, amino acid incorporation efficiency by the orthogonal tRNA/synthetase increases with progression of HCN-A94 cell differentiation. It has been reported that CMV, PGK and CAG promoters all drive transgene expression more efficiently at the late stages of mouse embryonic stem cell differentiation.[27
Fluorophores have been chemically attached to ion channels using engineered Cys residues and sulfhydryl reactive fluorophore conjugates. Together with voltage-clamp fluorometry,[28
] which detects fluorescence changes upon application of different voltages to a cell membrane, they have provided valuable information on conformational changes of voltage- or ligand-gated channels.[29
] This pioneering technique has been largely used in Xenopus
oocytes, and only extracellular amino acid residues that are accessible for Cys labeling can be readily modified and then studied using fluorescence spectroscopy. Moreover, inconsistent behaviors of VSDs in Xenopus
oocytes compared to more natural mammalian cells have been noted. For instance, the gating charge movement of CiVSP in mammalian PC12 cells is 10-times faster than in Xenopus
oocytes, possibly due to differences in lipid environments.[24
] Mammalian and neuronal cells are native hosts for mammalian ion channels, receptors and transporters, and the genetic encoding method reported here should enable such proteins to be more quantitatively studied in these cells. In addition, instead of being limited to certain extracellular residues, genetically encoding Uaas with fluorogenic properties allows spatial precision for fluorophore incorporation without the limitation inherent to traditional chemical modification strategies. For instance, the S4 Phe234 position chosen in this study resides on the cytoplasmic face of the lipid bilayer and is not readily accessible for Cys-mediated labeling. Moreover, in comparison to Cys labeling and fluorescent protein tagging, genetically encoded fluorescent Uaas permit introduction of the fluorophore closer to the protein backbone, so that the fluorophore follows domain movements closer.[35
] Therefore, the method reported here should significantly expand our ability to investigate ion channel and other membrane protein behavior on the molecular level with spatial and temporal precision and in their native cellular environments.
A physical-chemical model for how the VSD transfers gating charges on S4 across the low dielectric membrane bilayer is fundamental for understanding the switch-like responses of ion channels to membrane voltage changes. One model suggests that the conserved basic residues on S4 move up to 15–20 Å sweeping across the membrane.[36
] A second model posits that there is very little physical movement of S4 and instead a reorganization of the electric field around the S4 charges through changes in the local water structure and/or membrane bilayer deformation.[30
By genetically incorporating DanAla into the VSD of CiVSP in HCN-A94 cells, we showed that DanAla fluorescence changes optically reported VSD conformational changes in response to membrane depolarization in differentiated neurons. Fluorescence increases and decreases were respectively observed at opposite ends of S4, suggesting that the microenvironments of the two ends of S4 change in opposing manners upon membrane depolarization. The experimentally measured increase in fluorescence observed for DanAla incorporated a position 234 of the VSD of CiVSP represents the first measurement of a fluorescence change of a residue on the intracellular end of S4. While the observed fluorescence increase is consistent with either of the above models, it provides an experimental platform for more spatially and temporally defined fluorescent experiments aimed at resolving how charge moves in response to membrane depolarization.
The fluorescence decrease observed for DanAla incorporated at position 208 is more intriguing. Residue 208 sits on the S3–S4 loop near the extracellular surface of the lipid bilayer. The observed decrease in DanAla fluorescence suggests that this position along S3–S4 is sequestered from solvent possibly buried within the hydrophobic membrane in the closed VSD state and then moves to a more hydrophilic environment upon membrane depolarization and VSD opening. The appearance of a second component on the Q-V
curve for the VSD(Gln208DanAla) mutant lends support for this model. Mutagenic replacement of a hydrophilic Gln with a substantially more hydrophobic DanAla residue should make it more difficult to “pull” the DanAla side chain from a buried hydrophobic layer. This “difficulty” would likely be evidenced by the requirement for a more positive membrane voltage to exert force on the VSD transiting to the open conformation. While preliminary in nature, these latter results are consistent with a gating model involving a large S4 sweep across the membrane. In contrast, small changes in S4 position or localized membrane reconstructuring would be unlikely to effectuate such a pronounced fluorescence change in DanAla that already resides on the extracellular side of the transmembrane region. Nonetheless, a systematic fluorescence mapping of VSD residues at more loop sites and throughout the transmembrane helices is needed to provide a sufficient amount of data to better infer the global conformational changes of VSD in response to voltage changes of the membrane potential.[40
In summary, we developed lentiviral vectors for the long-term genetic incorporation of Uaas into translated proteins in neural stem cells. Uaa incorporation does not change the overall differentiation process of the neural progenitor HCN-A94 cells, and Uaa incorporation efficiency increases as the differentiation program ensues. Moreover, a genetically encoded fluorescent Uaa optically reports the conformational change of a VSD in response to membrane polarization in neurons differentiated from HCN-A94 cells, setting the stage for the systematic functional mapping of global conformation changes of VSDs in response to membrane voltage changes.