Abstract
Purpose::
GCAP1 and GCAP2 are guanylate cyclase (GC) activating proteins mediating Ca2+ sensitivity of GCs. We wish to determine the expression pattern of GCAP2 in mouse retina and the function of GCAP2 in phototransduction, using GCAP2 and GCAP2-EGFP transgenic mice on wild-type and GCAP1/GCAP2 double knockout background.
Methods::
The 14kb mouse GCAP2 gene containing a 5 kb promoter region and the endogenous polyadenylation signal was subcloned from a BAC clone into pACYC177 vector by ET recombination. The EGFP cDNA sequence was inserted into the construct to generate the GCAP2-GFP fusion protein. The expression levels of GCAP2 and GCAP2-GFP were determined by Western blotting. The expression patterns of GCAP2 and GCAP2-GFP were determined by immunocytochemistry. The function of GCAP2 was determined by paired flash electroretinogram (ERG) with dark-adapted GCAP2 transgenic mice on GCAPs double knockout background.
Results::
GCAP2-GFP is expressed exclusively in rod photoreceptor cells and is not found in the inner retina in adult mice. However, GCAP2-GFP is transiently expressed in the inner retina at around postnatal day 3. Scotopic flash ERG results do not show noticeable difference in the amplitudes of rod a-wave and b-wave among WT, GCAPs double knockout, and GCAP2 transgenic mice. Paired flash ERG results from a high GCAP2 expression line suggest that GCAP2 transgene can rescue the delay of rod recovery and the decrease of rod a-wave amplitude in GCAP1/GCAP2 double knockout mice. Moreover, rod a-wave amplitudes show a correlation with expression levels of GCAP2.
Conclusions::
In the adult mouse retina, GCAP2 is only expressed in rods. Based on preliminary paired flash ERG data, GCAP2 alone can rescue the delay of rod recovery in GCAPs double knockout mouse retina.
Keywords: photoreceptors • signal transduction • calcium