Achromatopsia-associated Mutation in the Human Cone Photoreceptor Cyclic Nucleotide-gated Channel CNGB3 Subunit Alters the Ligand Sensitivity and Pore Properties of Heteromeric Channels

Changhong Peng,Elizabeth D Rich,Michael D Varnum

doi:10.1074/jbc.m305102200

Changhong Peng, Elizabeth D Rich + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m305102200

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Abstract

Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to form by assembly of two different subunit types, CNGA3 and CNGB3. Recently, mutations in the gene encoding the CNGB3 subunit have been linked to achromatopsia in humans. Here we describe the functional consequences of two achromatopsia-associated mutations in human CNGB3 (hCNGB3). Co-expression in Xenopus oocytes of human CNGA3 (hCNGA3) subunits with hCNGB3 subunits containing an achromatopsia-associated mutation in the S6 transmembrane domain (S435F) generated functional heteromeric channels that exhibited an increase in apparent affinity for both cAMP and cGMP compared with wild type heteromeric channels. In contrast, co-expression of a presumptive null mutation of hCNGB3 (T383f.s.Delta C) with hCNGA3 produced channels with properties indistinguishable from homomeric hCNGA3 channels. The effect of hCNGB3 S435F subunits on cell-surface expression of green fluorescent protein-tagged hCNGA3 subunits and of non-tagged hCNGA3 subunits on surface expression of green fluorescent protein-hCNGB3 S435F subunits were similar to those observed for wild type hCNGB3 subunits, suggesting that the mutation does not grossly disturb subunit assembly or plasma membrane targeting. The S435F mutation was also found to produce changes in the pore properties of the channel, including decreased single channel conductance and decreased sensitivity to block by l-cis-diltiazem. Overall, these results suggest that the functional properties of cone CNG channels may be altered in patients with the S435F mutation, providing evidence supporting the pathogenicity of this mutation in humans. Thus, achromatopsia may arise from a disturbance of cone CNG channel gating and permeation or from the absence of functional CNGB3 subunits.

Highlights

The effect of human CNGB3 (hCNGB3) S435F subunits on cell-surface ex- of CNGA3 subunits alone can generate functional homomeric pression of green fluorescent protein-tagged human CNGA3 (hCNGA3) sub- channels [3,4,5], but CNGB3 subunits cannot [6, 7]
Co-expression in Xenopus oocytes of human CNGA3 subunits with hCNGB3 subunits containing an achromatopsia-associated mutation in the S6 transmembrane domain (S435F) generated functional heteromeric channels that exhibited an increase in apparent affinity for both cAMP and cGMP compared with wild type heteromeric channels
Functional Expression of Mutant Human CNGB3 Subunits—To investigate the functional consequences of two achromatopsia-associated mutations in the human cone photoreceptor cyclic nucleotide-gated (CNG) channel ␤ subunit gene (CNGB3) [8, 9], we introduced these mutations into cDNA encoding the human CNGB3 subunit and co-expressed mutant or wild type CNGB3 with human cone CNG channel ␣ subunits [4] in Xenopus oocytes

Summary

EXPERIMENTAL PROCEDURES

Molecular Biology—The human retinal cone CNG channel ␤ subunit clone, hCNGB3, was isolated from human retinal cDNA as previously described [7]. The coding sequence of this clone differs from the complete published sequence for CNGB3 of Kohl et al [9] (AF272900) at two positions (nucleotide 1789g3a and nucleotide 1834a3g), representing either sequence polymorphisms or Taq polymerase errors; neither nucleotide change alters the amino acid sequence It was subcloned into pGEMHE [16] for heterologous expression in Xenopus oocytes. Oocytes expressing GFP-tagged CNG channel subunits 64 –72 h after injection of mRNA were placed in borosilicate coverglass chambers such that the equator was approximately perpendicular to the plane of imaging. Biochemistry—To assess the overall abundance of cone CNG channel subunits expressed in Xenopus oocytes, we used Western blot analysis of proteins from oocytes expressing GFP-tagged hCNGA3 or hCNGB3. The approximate molecular weights of the GFP-tagged subunits were calculated by interpolation using the linear relationship between the log of molecular weight for protein standards (Invitrogen), as reported by the manufacturer for these buffer conditions, and the migration distance of the proteins

RESULTS

TABLE I Permeability and conductance ratios

DISCUSSION