significant sequence similarity to the Trp protein that is also expressed in photoreceptors. This gene, called Trp-like (trpl), encodes a protein that displays 39% amino acid identity with Trp.

trp mutants were isolated over 25 yr ago (42) and shown to be defective in maintenance of the light response (thus the name transient-receptor-potential). We now know that internal calcium is required to maintain a receptor potential and that trp mutants have lost the major light-activated calcium entry pathway (28,31,34). On the basis of these findings, Hardie and Minke (23) have suggested that the Trp phenotype results from a failure in the refilling of the internal stores, trp mutant photoreceptors are also inactivated after a strong light stimulus. This is likely due to the emptying of the stores and a subsequent decrease in the efficiency of the excitation process ( 43,44).

Analysis of the Trp sequence showed regions of weak similarity with neuronal voltage-gated Ca2+ channels (45,46), consistent with the notion that trp encodes a plasma membrane channel with high calcium permeability. Interestingly, a number of studies suggest that Trp may be related to the elusive vertebrate Icrac ion channel (calcium-release-activated-channel) (11,23), and thus Trp homologs may be critically important in the regulation of intracellular calcium. In efforts to determine whether Trpl also encodes a component of the light-activated conductance, we set up to isolate mutations in this gene. A difficulty in setting up a screen for mutations in trpl is the lack of a reliable, predictable phenotype that defines its loss of function and the possibility that trp and trpl may serve redundant functions. Because of these concerns, we used a screening strategy that was based on the loss of Trpl antigen on immunoblots (20,47). The advantage of this screen is that it does not rely on a hypothetical physiological or behavioral defect but only on the presence or absence of Trpl protein. After screening several thousand chromosomes, we isolated a knock-out mutation in trpl. These mutants are now being subjected to detailed genetic, biochemical, and physiological characterization. Interestingly, Trp and Trpl are not subunits of the same channel (B. Niemeyer and C.S.Z., in preparation). Recently, vertebrate homologs of Trp and Trpl have been cloned. The analysis of these channels and the trp and trpl mutants is likely to provide important insight into the biology of this novel class of ion channels and their role in calcium homeostasis.

Future Challenges

Phototransduction has proven to be an ideal model system for the study of G protein-coupled signaling cascades. Basic cellular phenomena like signal amplification and integration, response deactivation, and adaptation have been first addressed in this signaling pathway. The study of this process also resulted in the molecular cloning of the first seven transmembrane domain receptor (48), the first cyclic nucleotide-gated ion channel (49), and the crystal structure of the first Gα protein subunit (50). Furthermore, the genetic dissection of this pathway in humans and flies has provided fundamental insight into the molecular and cellular basis of inherited retinal disorders (51,52). However, despite these great advances, many important questions still remain. For example, what are the determinants of the kinetics of activation? What are the detailed molecular mechanisms of light and dark adaptation? How do the different signaling molecules interact with each other and regulate their output? How is response deactivation controlled? What are the intracellular messengers in invertebrate phototransduction? How is signal cross-talk prevented?

A complete understanding of the phototransduction process will have to wait until all the gene products that have a role in this process are identified and studied by the physiological effect of their loss or dysfunction. It is here where the study of phototransduction in Drosophila offers unprecedented versatility. The study of this signaling cascade in the fruit fly Drosophila melanogaster makes it possible to use powerful molecular genetic techniques to identify novel transduction molecules and then to examine the function of these molecules in vivo, in their normal cellular and organismal environment. Recent advances in mouse knockout technology also offer an exciting opportunity for a genetic dissection of this process in vertebrates. The combination of these two systems of study may provide the answer to the biggest challenge for the future: how is the entire response of a photoreceptor cell orchestrated in vivo?

I deeply thank past and present members of my laboratory for their contributions. This research was funded by grants from the National Eye Institute, the Pew Foundation, the McKnight foundation, and the March of Dimes. C.S.Z. is an investigator of the Howard Hughes Medical Institute.

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