The Glutamic Acid-Rich Protein Is a Gating Inhibitor of Cyclic Nucleotide-Gated Channels

Abstract

The cyclic nucleotide-gated (CNG) cation channel of rod photoreceptors is a heterotetramer consisting of homologous CNGA1 and CNGB1a subunits. While CNGA1 is indispensable for channel activation, the specific role of CNGB1a in this process has remained elusive. Here, we show that the N-terminal glutamic acid-rich protein (GARP) domain of CNGB1a and soluble GARP2, which corresponds to the proximal portion of the GARP domain, act as autoinhibitory domains that decrease the opening probability of the CNG channel. In the presence of mutations that structurally impair the cyclic nucleotide-binding domain (CNBD) of CNGB1a, the GARP domain completely abolishes channel activity. In agreement with an inhibitory function of GARP, the activity of mutant CNG channels could be fully restored by deletion of the GARP domain. We identified two sequences within the GARP domain that confer most of the inhibitory effect and demonstrate that the profound inhibition imposed by the GARP domain is caused by direct and autonomous protein-protein interaction with the CNG channel complex. In wild-type rod CNG channels, this inhibitory effect can be relieved by binding of cGMP to the CNBD of CNGB1a. In conclusion, we propose that the N terminus of CNGB1a and soluble GARPs act as molecular gate keepers that control the activation of heteromeric rod CNG channels. Our results suggest that the GARP domain has evolved in rod photoreceptors to reduce current noise resulting from openings of CNG channels in the absence of cGMP.

Varying the GARP2-to-RDS Ratio Leads to Defects in Rim Formation and Rod and Cone Function

“…RDS haploinsufficiency is known to cause severe structural and functional defects alone, 27,43 and recent work has suggested that GARP2 may play a role in regulating rod sensitivity by altering PDE6 activity and cGMP turnover. [9][10][11]44 In addition, our observation that CNGB1 levels were decreased in the presence of excess GARP suggests that CNG channel function or trafficking may be slightly altered in GARP2-Tg animals. Because our CNG antibodies recognize both CNGB1 and GARP, it is not possible to assess CNGB1 alone by immunofluorescence (although the GARP2-TG protein can be visualized by using the c-Myc antibody).…”

Up-regulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 (HCN3) by Specific Interaction with K+ Channel Tetramerization Domain-containing Protein 3 (KCTD3)

“…Surface Biotinylation-Cell surface proteins were labeled with 0.8 mM non-cell-permeable sulfo-NHS-SS-biotin (Pierce) and precipitated using streptavidin-agarose beads (Pierce) as described (44). Quantitative Western blots were performed on both total and biotinylated surface proteins using NIH ImageJ software.…”

Alternative Splicing Governs Cone Cyclic Nucleotide-gated (CNG) Channel Sensitivity to Regulation by Phosphoinositides

“…Soluble GARP proteins and the GARP region of CNGB1a are thought to interact with proteins in the rim of outer segment disc membranes, including phosphodiesterase (26) and peripherin (27). Furthermore, soluble GARP2 and the GARP region intrinsic to CNGB1a can act as gating inhibitors of the rod CNG channel (28). For human CNGA3, four protein isoforms can arise from alternative splicing because of different exon combinations within the 5Ј region of the mRNA encoding the N-terminal cytoplasmic domain of the subunit (24,29).…”