Rhodopsin and Signal Transduction

Some time ago, Mentzer (previously unpublished) envisioned eye drops with ligand effectors binding to protein disruptions in the rhodopsin chain, as an immediate indicator of mTBI. Clinical pupillary measurements could be enhanced with a colorimetric quantification based on changes related to mTBI. Correlates with eye tracking devices for mTBI assessment would benefit as well.

Rhodopsin is a light-activated G protein-coupled receptor (GPCR) in rod cells of the eye—specifically located in flat discs in the outer portion of the rod cells. The rhodopsin GPCR is covalently bound to 11-cis-retinal, which is a visual pigment that responds to the “visible” portion of the electromagnetic spectrum, and is completely surrounded by the seven membrane spanning regions of the GPCR. In the case of rhodopsin the coupled trimeric G protein is transducin (Gt), with the Ga class subunit Gat (Lodish et al.. 2008).

The 11-cis-retinal lysine side chain couples to opsin in the rhodopsin, which becomes activated through absorption of a photon of light, concurrent with the 11-cis-retinal isomerization to the all-trans-retinal moiety, now coupled to the activated opsin, in what we call meta-rhodopsin II. Note that the 11-cis-retinal couples to the opsin’s lysine side chain, and all-trans retinal is restored to 11-cis-retinal.

This meta-stable intermediate activates the Gt protein’s Gat subunit. After several seconds, the trans moiety dissociates from opsin and converts back to the cis moiety for the subsequent binding to inactivated opsin. Analogous to the conformational change associated with ligand binding in other GPCRs, there is a conformational change in the opsin upon photon activation. Light absorption causes nonselective ion channels (for Na+, Ca2+, K+) in the rod membrane to close, polarizing the resting potential of the membrane to a higher inside negativity, which is transmitted to the brain and perceived as light (Lodish et al., 2008).

Light absorption induced closing of the nonselective ion channels in the plasma membrane of the rod cells involves the cGMP second messenger, which keeps the channels open in the absence of light. Light-activated opsin, bound to Gat, mediates GDP>GTP; and activates cGMP phosphodiesterase (PDE), which converts cGMP to GMP. The drop in cytosolic cGMP causes the ion channels to close, leading to the electrical polarization transient across the rod membrane that is detected in the brain as light. So, the PDE is the effector protein, and the cGMP is the secondary messenger (Lodish et al., 2008).

Having outlined the variant of the GPCR represented by rhodopsin and conversion of optical photons to creation of visual images in the brain, we are equipped to properly assess the work of Smith (2010). Smith reviews rhodopsin as a special case of the GPCR, analyzes signal transduction from a 3D structural perspective, provides a structural explanation for the Ga class genetically conserved amino acids and structural motifs, and more specifically, conserved residues in visual receptors, leading to convergence with the more general models for the Ga class of GPCRs. Smith focuses especially on the activation mechanism in rhodopsin, remarkable in many respects—including a photoreaction quantum yield of 0.67 (better than our best engineered photovoltaic solar cells) and a photoisomerization process that occurs in 200 femtoseconds).

The most loosely packed of the beta-sheet folds in the seven transmembrane helices (H5 and H6) are shown to undergo the largest reconfiguration (displacement) upon activation of the 11-cis-retinal. Hydrogen bonded networks are of great importance in the maintenance of a high dissociation constant of the pro- tonated Schiff’s base linking the 11-cis-retinal to the receptor protein; as well as importance in stabilization of the extracellular loops; and finally to their rearrangement upon isomerization/activation. Indeed, I would note the quantum yield for 11-cis-retinal in solution is only 0.3, and accompanied by multiple isomeriza- tions. Hydrogen bonding in vivo certainly contributes to stabilization and reconfigurations of the four molecular switch “microdomains” observed in rhodopsin (Smith, 2010).

The absorption band shifts observed during the thermal relaxation of the photoreaction intermediates, further defined as a “series of distinct, spectrally defined intermediates,” provides numerous intriguing insights into overall signal transduction—namely, the relation of wavelength shift to protonation of the Schiff’s base, wavelength/conformation relationships and controls, temporal characterization of reaction intermediates, motion of H5 and H6 within the protein binding pocket, and ultimately, the ability to ascertain the function of conserved residues to validate the generalized GPCR models proposed for retinal. This leads to improved understanding of disease states with respect to mutations of these conserved residues— along with avenues for therapeutic drug targets. It may also lead to insights regarding the GTP>GDP exchange mechanism, which is not fully understood in terms of signal transduction mechanism (Smith, 2010).

Smith illustrates, in a series of motif, domain, conformation -driven reaction sequences, conserved functionality identification, and water-mediated hydrogen bonding stabilizations; how rhodopsin evolved as a photoreceptor with remarkable dynamic range and sensitivity. He points the way for further investigation into the nature of the generalized Ga class of trimeric G proteins in GPCRs as the basis for improved understanding of the unique specifics of the rhodopsin receptor. Rather than signal transduction via movement of an electrical or chemical potential through a reaction mechanism circuit, signal transduction in rhodopsin is seen as a sequence of consecutive “information” transfers involving modulation of binding potentials, metastable intermediates, conformational signals and effectors, and direct control activation via light photons rather than ligand binding.

Further to the specifics of the second messenger phototransduction cascade in rhodopsin photopigment, some additional detail of what happens in the activation process in the disk membrane goes like this:

~hv » ll-cis to all-trans retinal isomer » transducin activation » phosphodiesterase (PDE) activation » hydrolysis of cGMP, reducing cGMP concentration available to bind to channels » closing of channels in outer membrane » producing differential cation transients across the membrane » producing transient hyperpolarization transients.

And deactivation proceeds like this:

rhodopsin kinase » phosphorylation of active rhodopsin » permitting arrestin to bind to rhodopsin » blocking transducin activation by activated rhodopsin » ending the phototransduction cascade

Via the retinoid cycle, after photoisomerization, retinal is restored back to the all-trans form like this:

all trans retinal » converts to all trans retinol » which is transported by interphotoreceptor retinoid binding protein (IRBP) chaperone into pigment epithelium » where it gets transformed to 11-cis retinal » and then chaperoned again by IRBP to the outer membrane segment » and combined with opsin.

Additional signaling pathways include the light adaptation process. This results in greater gray-scale sensitivity over a wider dynamic range, analogous to photomultipliers rather than the limited performance of, for instance, a conduction mode pn junction diode—analogizing to the solid-state detector world, in which we’ve not achieved comparable success.

Further to the concept of nuclear signaling—we can separate the mammalian code—of approximately 25,000 genes, translating to more than 200 different cell types—into two groups: germ cells and somatic cells. Germ cells have the ability to divide and produce all the other cell types, and somatic cells represent the specific engines of life. The epigenome is modulated by activation of eight major classes of cell surface receptors, translating signaling molecules into cellular transcription response.

After the cell cycle progresses to the point where somatic differentiation results in cells progressing beyond the germ and stem cell phase, the transcription factors are influenced by two categories of signals: First is the set of external signals we’ve been discussing, to include cytokines, growth factors, hormones, etc., along with stress type responses (Lodish et al., 2008); and second is a set of intrinsic factors to include transcription itself, replication of DNA, and chromosome modifications and segregation. Polycomb and trithorax (PcG and trxG) are two regulators of differentiation and cell specification in the eukaryotes (Allis et ah, 2009).

Histone modifications result in repression or alteration of proper functioning of the genome (chromatin deregulation) and, along with our extrinsic factor regulation, may collectively supply the approach to a personalized therapy for a wide range of maladies. In fact, the epigenomic code, when established, may provide the basis for determination of which kinase cascades are malfunctional and require “adjustments”.

Transprocess signaling between the extrinsic and intrinsic factors discussed provides an additional regulatory signaling mechanism. Therefore—just as we’ve looked at how protein domain functionality assists with cascades of kinase reactions—the epigenetic condition determines which portions of the genetic sequence are available to be transcribed.

Recent progress in the field of optogenetics (Farmer, 2020; Zhang et al., 2011) provides the means to activate or deactivate transmembrane rhodopsins to manipulate neuronal circuits at the cellular level. This provides better understanding of neuronal circuits and brain synapses.

REFERENCES

Allis, C.D., T. Jenuwein, D. Reinberg, and M. Caparros, eds. 2009. Epigenetics. Cold Spring Harbor Press, New York. pp. 43-50.

Farmer, D. 2020. Opsins travel to the brain’s hidden places. A rising number of light- sensitive proteins and optogenetics are enabling precise imaging of brain cells, as well as the potential to adapt functioning in neuronal networks. BioPhotonics. Pittsfield, MA: Lauren Publishing. https://www.photonics.com/Articles/ Opsins_Travel_to_the_Brains_Hidden_Places/a65549 Lodish, H., A. Berk, C.A. Kaiser, et al. 2008. Molecular cell biology, 6th edition. New York: W.H. Freeman and Company.

Smith, S. 2010. Structure and activation of the visual pigment rhodopsin. Annu Rev Biophys. 39:309-328.

Zhang, F., J. Vierock, O. Yizhar, et al. 2011. The microbial opsin family of optogenetic tools. Cell. 147(7): 1446-1457.

 
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