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Insight into the functional of N irl-3 and DrdgB function has been gained from the genetic studies of Hyde and co-workers. Specifically, they demonstrated that the expression of the cDNA of Nir2, Nir3 or a zebrafish N irl homologue in rdgB

null mutant flies rescued the mutant phenotype; however, the extent of the rescue differed with each isoform (Chang et al., 1997; Elagin et a l , 2000; Lu et a l, 1999). These data suggest some conservation of biochemical activity between DrdgB and N irl-3 with respect to function in the phototransduction pathway of Drosophila. The expression of Nir2 cDNA in rdgB mutant flies was sufficient for complete rescue of the retinal degeneration and electrophysiological defects associated with the with the

rdgB m utant phenotype (Chang et al., 1997). In contrast, N irl and Nir3 cDNA expression only delayed DrJgB^-depenent retinal degeneration. While Nir3 was able to partially rescue the defective ERG response (Lu et al., 1999), N irl offered no detectable rescue of the electrophysiological defects associated with the mutant phenotype (Elagin et al., 2000). The differences between N irl-3 in suppressing the

DrdgB^ mutant phenotype further suggested that there are functional differences between these mammalian homologues and that Nir2 is functionally more similar to DrdgB than N irl and Nir3.

The observation that N irl, which lacks a Ptdlns-T P domain, is able to partially rescue the mutant phenotype is consistent with the hypothesis that the carboxy-terminal residues of DrdgB are functionally relevant. Furthermore, these data suggest that Nir3 possess an activity that promotes photoreceptor viability but is unable to participate in the PLC-mediated signalling cascade (Elagin et al., 2000). In contrast to N irl, the expression in rdgB^ flies of a DrdgB cDNA, in which the Ptdlns-TP domain was deleted, offered no detectable rescue of the mutant phenotype (Elagin et al., 2000). Since both proteins appeared to possess similar structural motifs and were stability expressed at high levels on several independent transgenic lines, Hyde and co-workers proposed that an intrinsic functional difference exists between N irl and the non-Ptdlns-TP domains of DrdgB (Elagin et al., 2000).

The observation that all three N ir proteins exhibit some activity during invertebrate phototransduction, together with the observation that N irl-3 map to three distinct chrom osom e regions known to contain m ultiple retinopathy loci (Section 3.1), raises the possibility that these proteins may play a role in vertebrate phototransduction. Evolution has adapted seem ingly divergent paradigm s for invertebrate and vertebrate visual processing (Section 1.8.1), suggesting that N irl-3

do not play a direct role in the major vertebrate phototransduction pathway. While a direct role for PLC isoforms during vertebrate phototransduction has yet to be established, PLCB, PLCy and PLC5 isoforms have been detected through out the retina (reviewed by Giusto et al., 2000). Furthermore, Ghalayini and co workers have demonstrated that PLC activity under light conditions is significantly higher than the activity found under dark conditions (Ghalayini et ah, 1998). However, this study did not reveal which PLC isozyme was responsible for the increase inactivity. A vertebrate retina-specific homologue of the Drosophila PLC encoded by the

NorpA gene, is expressed throughout the retina (Alvarez et a l, 1995; Ferreira et a l,

1993; Lee et a l, 1993). Specifically, this PLCB4 isoform has been detected within the photoreceptor cell layer, bipolar cells, horizontal cells and the ganglion cells (Peng et a l , 1997). Although the specific function of mammalian retinal PLC isoforms remain to be clearly defined, experiments in which murine PLCB4 was knocked out demonstrated altered visual processing and suggested that PLCB4 plays a modulatory role in the rod-rod-bipolar signalling cascade, rather that acting as a primary signalling interm ediary as it does during D ro sophila phototransduction (Jiang et a l, 1996).

In addition to PLC isoforms, immunolocalisation studies in various vertebrate retinas, have indicated the presence of eight PKC subspecies with unique cellular distributions within this tissue (reviewed by Giusto et a l , 2000). PKC-catalysed phosphorylation of rhodopsin, transducin, arrestin and PDE has been reported (Giusto et a l , 2000). W hile, this phosphorylation causes a decrease in the light response, the functional significance of phosphorylation by PKC remains to be examined (Giusto et a l, 2000). These data suggest the existence of novel aspects of the vertebrate phototransduction cascade. The roles of N irl-3 and PLC- and PKC- isoforms within vertebrate phototransduction, particularly during light recovery and adaptation, await further investigation.

Recent data by W omack and colleagues have provided further evidence for a role of phosphoinositide signalling during vertebrate phototransduction (Womack et a l, 2000). W omack and co-workers investigated the effects of PtdIns(4,5)P2 and anti-Ptdlns(4,5) ? 2 antibodies on guanidine nucleotide-stim ulated PDE activity in a

biochemical assay using membranes from bovine rod outer segments. Using this assay, it was demonstrated that PtdIns(4,5)P2-enhanced PDE activity, suggesting a regulatory role for PtdIns(4,5)P2 on PDE activity (Womack et a l, 2000). In the same study it was shown, prim arily using electrophysiological recordings, that

PtdIns(4,5)P2 has a strong inhibitory effect on bovine rod cyclic nucleotide gated (CNG) channels when a and 6 subunits were expressed in X e n o p u s oocytes (W omack et al., 2000). In agreement with other studies, similar effects on rod CNG channels were also observed by the application of MgATP to the cytoplasmic face of the inside-out patch. Furthermore, this inhibition was reversed by the application of anti-PtdIns(4,5)?2 antibodies (Womack et al., 2000). Previous studies have indicated that the inhibitory effect of ATP on CNG channels expressed in oocyte membranes is due to tyrosine phosphorylation (Molokanova et al., 1999; M olokanova et al., 1997). W omack and colleagues have thus proposed that the inhibition by Ptdlns (4,5)^2 and ATP are interdependent (W omack et al., 2000). This notion is consistent with the observation that the stim ulatory effects o f cAM P dependent protein kinase phosphorylation and Ptdlns(4,5)P2 binding on G protein-sensitive inwardly rectifying potassium (ROMK) channels is synergistic (Liou et al., 1999).

The findings of W omack and colleagues are consistent with previous reports demonstrating that Ptdlns(4,5)?2 can modulate the function of several ion channels and transporters, independent of lns(l,4,5)Pg, Ca^^, DAG and PKC (Womack et al.,

2000). The physiological significance and the mechanism by which Ptdlns(4,5)?2 promotes the activation of PDE by transducin, and modulates CNG channel activity is unknown, as is the physiological significance.

1.9.3 A role for rdgB proteins during capacitative Ca^"^ entry in non-excitable