ecture to Sensory Genomics Class, 10/21/09, Richard Payne ntroduction to eye types ntroduction to ciliate and microvillar photoreceptor types hototransduction in Drosophila photoreceptors involves Ca2+ influx fro xtracellular space his Ca influx occurs through the high Ca permeability of the trp cha hototransduction in Limulus ventral photoreceptors involves release o alcium from internal stores, not influx. hy the difference – are different families of trp channels being use ork shown performed in my lab from Kyrill Ukhanov (post doc), oujun Wang (grad student), Jay Demas, Oluwaseyi Motajo undergrad students).
Transcript
Lecture to Sensory Genomics Class, 10/21/09, Richard Payne
Introduction to eye types
Introduction to ciliate and microvillar photoreceptor types
Phototransduction in Drosophila photoreceptors involves Ca2+ influx from theExtracellular space
This Ca influx occurs through the high Ca permeability of the trp channel.
Phototransduction in Limulus ventral photoreceptors involves release of calcium from internal stores, not influx.
Why the difference – are different families of trp channels being used?
Work shown performed in my lab from Kyrill Ukhanov (post doc), Youjun Wang (grad student), Jay Demas, Oluwaseyi Motajo (undergrad students).
Not every animal has eyes designed like ours. Do they have different photoreceptor types?
Yes, there are different photoreceptor types, but not rigidly linked to eye design
Invertebrates have both simple (single lens) and compound eyes – do they have the same photoreceptor types as ours?No – both the compund fly eye and the squid simple eye shown here have“Microvillar “rather than our “Ciliate” photoreceptors.
What are “ciliate” and “microvillar” photoreceptors?
10.1 Anatomy of the human eye.
Section – see over
10.4 Structure of the retina.
The vertebrate rod photoreceptor is a “ciliate” photoreceptor
cilium
It hyperpolarizes upon illuminationDue to closure of cGMP-activatedNa+ - channels. Because the cGMP –activated channels also let through Ca2+ ions, the [Ca2+] in the cell falls upon illumination.
The reduction in cGMP is achievedVia a biochemical cascade that is linked via a G-protein to the visualpigment, Rhodopsin .
• Compound and other eyes of many invertebrate species (including at least one chordate, Amphioxus) use a different kind of photoreceptor:
The “microvillar” or “rhabdomeric” photoreceptor
By William Harman, Steven Barrett, Cameron Wright, and Michael Wilcox
Search Advanced search Journal home > Archive > insight > Full text > Figure 2
FIGURE 2. Photoreceptor structure.
From the following article:Visual transduction in Drosophila
Roger C. Hardie and Padinjat RaghuNature 413, 186-193(13 September 2001)doi:10.1038/35093002BACK TO ARTICLE
In Drosophila, as in most invertebrate photoreceptors, the photoreceptive membrane is organized into tightly packed, tubular microvilli, each 1–2 m long and 60 nm in diameter, together forming a 100- m-long rhabdomere. At the base of the microvilli a system of submicrovillar cisternae (SMC) have often been presumed to represent smooth endoplasmic reticulum Ca2+ stores endowed with Ins(1,4,5)P3 receptors. However, the SMC may have a more important role in phosphoinositide turnover. Vertebrate rod outer segments (ROS) contain stacks of membranous discs ( 1,000) and are connected to the cell body by a narrow cilium. In both cases the overall structure serves to maximize
The light-induced depolarization of Drosophila photoreceptors results from the opening of Na+ channels, in the microvillar membrane, allowing Na+ ions into the cell. Opposite to rods
Na+
Hardie 1996;
In Drosophila photoreceptors, unlike in rods, there is a light-induced increase in intracellular [Ca2+].
The Ca2+ comes through the light-sensitive channels along with Na+
Hardie, 1996
The current model of Drosophila phototransduction isconfined to a single microvillus, containing 10-30 channels.
After Hardie et al 2002
No role for IP3R or ER calcium stores in excitation– confirmed by genetic knockout of the IP3R
So how does Ca2+ enter the Drosophila photoreceptor?
Drosophila photoreceptors two channel proteins in their microvilli that together constitute the “light-sensitive conductance”
Trp and trpl
Trp;trpl double mutants are blind – no ability to generate a light-induced inward current. So these are the “light-activated” channels
trp has a much higher Ca2+ selectivity than trpl
A third putative channel protein, trpgamma, is also expressed in the photoreceptors – role unknown.
Trp, trpl and trpgamma are all members of the trpC gene family
trp mutants lack the TRP channel protein and have a transient light-activated current and receptor potential. The trp phenotype can be mimicked by blocking trp channels in a wild-type (WT) fly with extracellular lanthanum ions.
In trp mutants, the remaining transient response is carried by the trpl channels.
The light-induced current flowing into WT flies shows a positive , calcium-sensitive “reversal potential” due to a large calcium permeability. Responses of trp mutants show a more negative reversal potential, with less calcium sensitivity, consistent with the elimination of the calcium-permeable trp channel.
An Aspartate at position 621 is largely responsible for the Ca2+ selectivity of trp
Figure 10. TRPC pore sequences and putative structure of the dTRP pore
The current model of Drosophila phototransduction isconfined to a single microvillus
After Hardie et al 2002
No role for IP3R or ER calcium stores in excitation– confirmed by genetic knockout of the IP3R
Is this the model phototransduction scheme for all microvillar photoreceptors?
More-or -less, yes
But the differences are significant
The Limulus
Microvillar photoreceptors are also found in simple eyes without lenses, such as the “ventral eye” in the horseshoe crab, Limulus
(After Calman & Chamberlain, 1982)
Microscopic structure of the photoreceptor
The ventral photoreceptor
1 m
10
m
Ventral nerve
Amplifier
Light
0.1 S
10 m
V
Voltage
Electrode
0.1 SLight
10 nA
Current
Measuring the light induced electrical responsesof Limulus ventral eye photoreceptors shows that, as in
Drosophila, they depolarize in response to lightdue to an influx of Na+ through ion channels
in the plasma membrane
Ventral nerve
Laser
Ca signalPhotomultiplier
(PMT)
PressureInjection of Ca-sensitive fluorescent dye
Electrode
Beamsplitter
Fluorescence Calibration
Intense Light
1 S
Ca concentrations within the Limulus photoreceptors rise upon illumination, as
in Drosophila
Measuring fluorescent Ca signals with confocal microscopy
Removal of extracellular calcium shows that the source of the light-induced calcium increase is different in Drosophila and Limulus photoreceptors
Hardie 1996;
Hardie, 1996
Instead of Ca2+ influx, Ca2+ release from intracellular stores by inositol (1,4,5) trisphosphate occurs in in Limulus ventral photoreceptors
This Ca2+ release stimulates the electrical response
PLC
Microvillus
IP3
DAG
h
Rh
Gq
IP3R
IP3R
Extracellular spaceCytoplasm
Ca
Ca
Ca
Ca?
TRPC ?
Na
Model of the photo-transduction cascade in Limulus ventral photoreceptors – Ca2+is released by IP3 from intracellular stores, and does not flow through the light-sensitive
channels
Compare with the current model of Drosophila phototransductionCa2+ enters through the light-sensitive channels
After Hardie et al 2002
No role for IP3R or ER calcium stores in excitation– confirmed by genetic knockout of the IP3R
But what about Limulus ventral photoreceptors other invertebrate photoreceptors that exhibit little or no light-induced Ca2+ influx?
Do they:
a)Express a trpl ortholog only?
b) Express a trp ortholog, but with a mutated pore region (no D621)
c) Express a trpC channel that is different from either trp or trpl?
If (c), then does this use of a different channel have any correlation with, or impact on the evolution of different eye designs in invertebrate species.
Phylogenetic questions:What trp channel orthologs are present in invertebrate genomes other than Drosophila , what is their phylogeny and how conserved are their putative pore regions?
For example, a trpC - like gene transcript can be cloned from ventral eye tissue, but it only has only 36% identity with Drosophila trp and has a dissimilar pore region (Bandyophadyay and Payne 2004). What is the phylogenetic relationship between this and the Drosophila channel?
Functional questions:How many other microvillar photoreceptors show little or no light-induced Ca2+ influx?What is the role of calcium influx vs. calcium release in phototransduction? What constraints and advantages accompany each mechanism?
