Melanopsin--shedding light on the elusive circadian photopigment

Abstract

Circadian photoentrainment is the process by which the brain's internal clock becomes synchronized with the daily external cycle of light and dark. In mammals, this process is mediated exclusively by a novel class of retinal ganglion cells that send axonal projections to the suprachiasmatic nuclei (SCN), the region of the brain that houses the circadian pacemaker. In contrast to their counterparts that mediate image-forming vision, SCN-projecting RGCs are intrinsically sensitive to light, independent of synaptic input from rod and cone photoreceptors. The recent discovery of these photosensitive RGCs has challenged the long-standing dogma of retinal physiology that rod and cone photoreceptors are the only retinal cells that respond directly to light and has explained the perplexing finding that mice lacking rod and cone photoreceptors can still reliably entrain their circadian rhythms to light. These SCN-projecting RGCs selectively express melanopsin, a novel opsin-like protein that has been proposed as a likely candidate for the photopigment in these cells. Research in the past three years has revealed that disruption of the melanopsin gene impairs circadian photo- entrainment, as well as other nonvisual responses to light such as the pupillary light reflex. Until recently, however, there was no direct demonstration that melanopsin formed a functional photopigment capable of catalyzing G-protein activation in a light-dependent manner. Our laboratory has recently succeeded in expressing melanopsin in a heterologous tissue culture system and reconstituting a pigment with the 11-cis-retinal chromophore. In a reconstituted biochemical system, the reconstituted melanopsin was capable of activating transducin, the G-protein of rod photoreceptors, in a light-dependent manner. The absorbance spectrum of this heterologously expressed melanopsin, however, does not match that predicted by previous behavioral and electophysiological studies. Although melanopsin is clearly the leading candidate for the elusive photopigment of the circadian system, further research is needed to resolve the mystery posed by its absorbance spectrum and to fully elucidate its role in circadian photoentrainment.

Figure 1

Melanopsin-containing retinal ganglion cells form a meshwork of cells that are intrinsically sensitive to light. (A) Approximately 1–2% of retinal ganglion cells express melanopsin. Shown is a confocal projection of a rat retinal wholemount immunostained with affinity-purified rabbit polyclonal antibodies raised against the amino-terminal peptide of melanopsin. (B) SCN-projecting RGCs are intrinsically sensitive to light. The light response shown was recorded in whole-cell perforated-patch clamp mode from a rat RGC that was labeled following stereotaxic injection of a fluorescent retrograde tracer into the SCN. All rod-and cone-driven synaptic inputs were blunted by prior exposure of the isolated retina to bright light, which bleaches the photopigments in traditional photoreceptors, rendering them unresponsive to light. The bar at the top indicates the duration of the light stimulus.

Figure 2

Deduced amino acid sequence and predicted secondary structure of mouse melanopsin. Predicted transmembrane regions [NORSp algorithm (Liu and Burkhard, 2002)] are indicated by shaded rectangles.

Figure 3

Spectrum of purified expressed melanopsin reconstituted with 11-cis-retinal. Melanopsin was expressed in COS cells reconstituted with 11-cis-retinal and purified by immunoaffinty chromatography. Purified melanopsin was solubilized in a PBS buffer containing 0.1% dodecyl maltoside. (A) Raw absorbance spectra of dark and illuminated melanopsin pigment. (B) Difference spectrum of purified expressed melanopsin. The spectrum (shown as filled circles) was obtained by subtracting the spectrum obtained following hydroxylamine treatment (50 mM hydroxylamine, pH 7.0) from that of melanopsin in the dark prior to treatment with hydroxylamine. The positive component of the spectrum reveals the dark pigment with a peak at 420 nm, which is susceptible to hydroxylamine attack. The smooth curve is a fit based upon the combination of a rhodopsin template (λ_max of 421 nm) with the spectrum of ll-cis-retinaldehyde oxime.

Figure 4

Melanopsin stimulates GTP-γ-³⁵S uptake by transducin. (A) Melanopsin-mediated GTP-γ-³⁵S binding in bright white light (□) and in the dark (■). Reaction rates were 0.04 pmol GTP-γ-³⁵S bound/s/pmol receptor for the light reaction and 0.02 pmol GTP-γ-³⁵S bound/s/pmol receptor for the dark reaction. In this assay, reaction mixtures contained 2.1 μM transducin, 3 μM GTP-γ-³⁵S, and 9.14 nM melanopsin in a buffer containing 10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM MgCl₂, and 1 mM DTT. Circles represent GTP-γ-³⁵S binding by untransfected COS-1 membranes in bright white light (○), and in the dark (●). Reaction mixtures contained 2.1 μM transducin, 3 μM GTP-γ-³⁵S and untransfected COS-1 membranes in the Tris buffer described above. (B) Melanopsin is activated most efficiently by blue light. Mean rates of melanopsin-catalyzed GTP-γ-³⁵S binding following illumination with UV (381 ±10 nm), blue (443 ±19 nm), green (516 ±30 nm), and red (> 700 nm) light. Illumination was provided by a calibrated light source. The samples were illuminated for 10 s. and the rate of transducin activation was determined. The irradiance (photons/s/cm²) was: UV—1.3 ×10¹³; Blue—1.54 ×10¹³; Green—2.72 ×10¹³; Red—1.9 ×10¹⁵. Wavelengths were selected using bandpass or longpass filters. Intensities were chosen to provide clear discrimination amongst action spectra for candidate pigments that have absorption maxima in the spectral range from the UV to the green. The intensity of red light was 100-fold greater than the other stimuli to rule out any contribution of photoactivation by light outside the intended spectral bands. The GTP-γ-³⁵S binding rate in the dark (4.8 ×10⁻⁴ pmol/s) was subtracted from raw rate values to generate the data shown (Mean ± SD). Reaction mixtures contained 2.1 μM transducin, 3 μM GTP-γ-³⁵S, and 0.3 nM melanopsin.