10.3  Current Research

Fig. 10.3  UV light or multiphoton excitation of caged amino acid neurotransmitters can be converted into biologically active amino acids

provide a means of controlling the release – both spatially and temporally – of agonists for kinetic studies of receptor binding or channel opening. The technique of rapid light-induced release of signaling molecules [34] descends from the flash photolysis experiments of Norrish and Porter [24]. Calloway and Katz pioneered a photochemical approach for high-spatial-resolution mapping of functional circuitry in living mammalian brain slices [4]. Photostimulation was achieved by bathing brain slices in a molecularly caged form of the neurotransmitter glutamate [L-glutamic acid alpha- (4,5-dimethoxy-2-nitrobenzyl) ester], which was then converted to the active form by brief pulses (<1 ms) of ultraviolet irradiation. Using this technique, the locations of neurons making functional synaptic connections to a single neuron were revealed by photostimulation of highly restricted areas of the slice (50–100 mm in diameter) while maintaining a whole-cell recording of the neuron of interest.

10.3.2  Pore Blocker and Photoisomerization

Building on the work of Lester et al. [17], Banghart et al. [2] used structure-based design to develop a chemical gate that confers light sensitivity to an ion channel for remote control of neuronal firing using a pore blocker and photoisomerizable azobenzene structure. Figure 10.4 illustrates the basic idea. Bistable positioning of the pore blocker was achieved with light of two different wavelengths. Absorption of a 500 nm photon triggered a cis-trans isomerization. The ~1.7 nm length of the trans isomer moved the blocker to the pore of the ion channel. Conversely, absorption of a near UV 380 nm photon triggered a trans-cis isomerization, The ~1.0 nm length of the cis isomer caused retraction of the pore blocker. The light-activated gate was covalently linked to the ion channel and the ion channel was integral to the neuronal cell membrane. The control over individual neurons was spatially accurate and did not rely on diffusible ligands. Also, the gate could be reversibly photo switched, allowing recurrent control of neural activity. Inside-out patches from an oocyte were treated with 100 mM of the triad maleimide + azo linkage chromophores – triethanolamine for 30 min. The patch showed a large Shaker current in 380 nm light (cis) and almost complete block in 500 nm light (trans). Current block in the dark followed a biexponential time course with t1 = 0.49 min and t2 = 4.79 min.

Fig. 10.4  Application of azobenzene chromophores to the gating of ionic currents through modified Shaker channels. MAL is maleimide for cystine tethering. QA is a quaternary ammonium group to block the channel [from ref. [2], Nature Publishing Co., © 2004, used with permission]

Volgraf et al. [32] applied the azobenzene technique to a ligand-gated ion channel, the ionotropic glutamate receptor (iGluR). Using structure-based design, they modified the ligand-binding domain to develop a light-activated channel. An agonist was covalently tethered to the protein through an azobenzene moiety, which functioned as the optical switch. The agonist was reversibly presented to the binding site upon photoisomerization, initiating domain closure and channel gating. Photoswitching occurred on a millisecond timescale, with channel conductances that reflect the photostationary state of the azobenzene at a given wavelength.

10.3.3  The Channelrhodopsins

Nagel et al. have shown that Channelrhodopsins 1 and 2 (ChR1 and ChR2) are involved in generation of photocurrents of the green alga Chlamydomonas reinhardtii. ChR1 is a light-gated proton channel [22]. ChR2, on the other hand, is a directly lightswitched cation-selective ion channel [23]. It opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. Nagle et al. have demonstrated that ChR2 may be used to depolarize cells by illumination [23]. Boyden et al. [3] and Li et al. [19] achieved temporally precise, noninvasive control in well-defined neuronal populations by adapting the naturally occurring algal protein

Fig. 10.7  Schematic illustration of PSI adjacent to a voltage-gated ion channel. One or more PSI reaction centers may be able to trigger the ion channels. Depending on orientation, PSI can depolarize or hyperpolarize the membrane

in close proximity to voltage-gated ion channels, either in the membrane or externally at the lipid–water interface, may be capable of generating a local electrical disturbance of sufficient magnitude to create an excitatory postsynaptic potential and generate an action potential. Kuritz et al. [14] have shown that PSI-proteoliposomes incubated with retinoblastoma cells imparted optical activity to the cells as measured by the light-induced slow movement of calcium ions into the cells. Pennisi et al. have performed experimental and theoretical studies on the incorporation of PSI reaction centers in human cells and lipid vesicles [25–27]. In particular, new methods of delivery and detection of PSI in the membrane of human cells have been developed (Fig. 10.8) [27]. Purified fractions of PSI were reconstituted in proteoliposomes that were used as vehicles for the membrane incorporation. A fluorescent impermeable dye was entrapped in the vesicles to qualitatively analyze the nature of the vesicle–cell interaction. After incorporation, the localization and orientation of the complexes in the membrane was studied using immunofluorescence microscopy. The results showed complexes oriented as in native membranes, which were randomly distributed in clusters over the entire surface of the cell. Additionally, analysis of cell viability showed that the incorporation process does not damage the cell membrane. Taken together, the results of this work suggest that the mammalian cellular membrane is a reasonable environment for the incorporation of PSI complexes, which opens the possibility of using these molecular photovoltaic structures for optical control of cell activity.

The direct use of chlorophyll itself as a visual pigment in animals has been observed in nature and attempted in the laboratory. Chlorophyll-like pigments have been found to be associated with the rhodopsin of deep sea fish [40, 41]. These pigments are believed to be used for dim light vision, as this would enable using the red part of the incident light spectrum, which has tenfold less loss due to scattering. Based on these observations, direct utilization of chlorophyll-derived pigments for

Fig. 10.8  Simultaneous evaluation of pyranine uptake and immunodetection of PSI in adipose tissue-derived stem cells. The images were obtained using differential interference contrasting (DIC; first row), and in the fluorescein isothiocyanate (FITC) and Cy5 fluorescent dye channels (second and third row, respectively). In this experiment, pyranine fluorescence is detected with the FITC channel and the secondary antibody fluorescence with the Cy5 channel. The images corresponding to the Cy5 channel clearly show that PSI complexes are associated with the membrane of the cells from the experimental sample in contrast with the controls. The fourth row has the merged images from the fluorescence channels, where it is possible to see that there is no overlap between the FITC channel (indicative of cytoplasmic localization) and the Cy5 channel (indicative of membrane localization). Individual cells that are separated from the rest and can be more clearly visualized are indicated with arrows in the DIC images. Scale bar = 30 mm. From [27], with permission. Biomedical Engineering Society © 2008

enhancement­ of vision in mammals has been attempted [42]. Dark-adapted mice were injected with the water-soluble chlorophyll derivative chlorin-e6, resulting in its accumulation in the outer segment of the retina. Comparison of the response of chlorin-e6-injected and control live mice to red light indicated that they responded to red light. The electrophysiological response to red light of the melanopsin-expressing retinal ganglion cells was doubled in intensity compared to controls.