Selective Knockout of Murine Glutamic Acid-rich Protein 2 (GARP2) Significantly Alters Cellular Dark Noise in Rod Photoreceptors

Abstract

GARP2, a glutamic-acid-rich protein found exclusively in rod photoreceptors, has been suggested to function as a structural protein, a modulator of the cGMP phosphodiesterase enzyme (PDE6), and a gating inhibitor of the rod cGMP-gated cation channel. GARP2 is a splice variant of the Cngb1 gene, which in the rods encodes the {beta}-subunit of the cyclic nucleotide-gated cation channel. Mutations in Cngb1 cause retinitis pigmentosa (RP45), and {beta}-subunit knockout mice are being studied as models of this disease. In this study, using ZFN-mediated gene editing, we have selectively eliminated GARP2 expression, while not affecting expression of the cyclic nucleotide gated cation channel {beta} -subunit, to determine its essential functions in mouse rods. The absence of GARP2 caused no consistent perturbations of retinal structure. Transiently, rod outer segment length was regionally greater than WT and infrequently misaligned, appearing parallel to the retinal pigment epithelium. Electroretinography of the knockout mice did reveal consistent functional alterations over time, seen as a reduction in the ERG response amplitudes in older mice, albeit with no significant alterations in sensitivity to light. Interestingly, single-cell patch-clamp recordings showed a significant reduction in rod photoreceptor dark noise consistent with a previously proposed role for GARP2 in binding to PDE6 and affecting its basal activity. Our results suggest a role for the GARP2-PDE6 interaction in stabilizing the PDE6 enzyme and controlling the turnover rate of cGMP in darkness, adjusting the level of dark noise and implicating an influence on the signal and noise properties of rod photoreceptors. Key PointsO_LIGlutamic acid-rich protein 2 (GARP2), an alternatively spliced variant of the Cngb1 gene, is exclusively expressed in rod photoreceptors, but the in vivo role of GARP2 remains unestablished. C_LIO_LIWe used precision gene editing technology to selectively knockout soluble GARP2 expression in rods, to determine its essential functions in structure and function of the retina. C_LIO_LIWe show that GARP2 has a minor role in maintaining the structural integrity of the rod outer segments as a function of age. C_LIO_LIOur study indicates that GARP2 has an important role in regulating rod photoreceptor dark noise, likely through stabilization of PDE6 basal activity to maintain the appropriate cGMP turnover rate. C_LIO_LIThis regulation is critical to facilitate the single-photon sensitivity and function of the rod photoreceptors. C_LI