Two types of photoreceptors, rods and cones, coexist in the vertebrate retina. An in-depth analysis of the retinal circuitry that transmits rod and cone signals has been hampered by the presence of intimate physical and functional connections between rod and cone pathways. By deleting the cyclic nucleotide-gated channel CNG3 we have generated a mouse lacking any cone-mediated photoresponse. In contrast, the rod pathway is completely intact in CNG3-deficient mice. The functional loss of cone function correlates with a progressive degeneration of cone photoreceptors but not of other retinal cell types. CNG3-deficient mice provide an animal model to dissect unequivocally the contribution of rod and cone pathways for normal retinal function.

Cyclic nucleotide-gated (CNG) channels are important mediators in the transduction pathways of rod and cone photoreceptors. Native CNG channels are heterotetramers composed of homologous A and B subunits. In heterologous expression systems, B subunits alone cannot form functional CNG channels, but they confer a number of channel properties when coexpressed with A subunits. To investigate the importance of the CNGB subunits in vivo, we deleted the CNGB1 gene in mice. In the absence of CNGB1, only trace amounts of the CNGA1 subunit were found on the rod outer segment. As a consequence, the vast majority of isolated rod photoreceptors in mice lacking CNGB1 (CNGB1 Ϫ/Ϫ ) failed to respond to light. In electroretinograms (ERGs), CNGB1 Ϫ/Ϫ mice showed no rod-mediated responses. The rods also showed a slow-progressing degeneration caused by apoptotic death and concurred by retinal gliosis. Cones were primarily unaffected and showed normal ERG responses up to 6 months, but they started to degenerate in later stages. At the age of ϳ1 year, CNGB1 Ϫ/Ϫ animals were devoid of both rods and cones. Our results show that CNGB1 is a crucial determinant of native CNG channel targeting. As a result of the lack of rod CNG channels, CNGB1 Ϫ/Ϫ mice develop a retinal degeneration that resembles human retinitis pigmentosa.

The loss of CNGA3 impairs the targeting of cone opsins and the expression of other visual cascade proteins. In addition, CNGA3 appears to be essential for normal postnatal migration of cone somata. After loss of cone outer segment proteins, CNGA3(-/-) cones induce apoptotic cell death.

Cyclic nucleotide-gated (CNG) channels play a key role in olfactory and visual transduction. Native CNG channels are heteromeric complexes consisting of the principal alpha subunits (CNG1-3), which can form functional channels by themselves, and the modulatory beta subunits (CNG4-5). The individual alpha and beta subunits that combine to form the CNG channels in rod photoreceptors (CNG1 + CNG4) and olfactory neurons (CNG2 + CNG4 + CNG5) have been characterized. In contrast, only an alpha subunit (CNG3) has been identified so far in cone photoreceptors. Here we report the molecular cloning of a new CNG channel subunit (CNG6) from mouse retina. The cDNA of CNG6 encodes a peptide of 694 amino acids with a predicted molecular weight of 80 kDa. Among the CNG channel subunits, CNG6 has the highest overall similarity to the CNG4 beta subunit (47% sequence identity). CNG6 transcripts are present in a small subset of retinal photoreceptor cells and also in testis. Heterologous expression of CNG6 in human embryonic kidney 293 cells did not lead to detectable currents. However, when coexpressed with the cone photoreceptor alpha subunit, CNG6 induced a flickering channel gating, weakened the outward rectification in the presence of extracellular Ca(2+), increased the sensitivity for L-cis diltiazem, and enhanced the cAMP efficacy of the channel. Taken together, the data indicate that CNG6 represents a new CNG channel beta subunit that may associate with the CNG3 alpha subunit to form the native cone channel.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr 476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr 476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.