The heteromeric cyclic nucleotide-gated channel adopts a 3A:1B stoichiometry

Abstract

Cyclic nucleotide-gated (CNG) channels are crucial for visual and olfactory transductions. These channels are tetramers and in their native forms are composed of A and B subunits, with a stoichiometry thought to be 2A:2B (refs 6, 7). Here we report the identification of a leucine-zipper-homology domain named CLZ (for carboxy-terminal leucine zipper). This domain is present in the distal C terminus of CNG channel A subunits but is absent from B subunits, and mediates an inter-subunit interaction. With cross-linking, non-denaturing gel electrophoresis and analytical centrifugation, this CLZ domain was found to mediate a trimeric interaction. In addition, a mutant cone CNG channel A subunit with its CLZ domain replaced by a generic trimeric leucine zipper produced channels that behaved much like the wild type, but less so if replaced by a dimeric or tetrameric leucine zipper. This A-subunit-only, trimeric interaction suggests that heteromeric CNG channels actually adopt a 3A:1B stoichiometry. Biochemical analysis of the purified bovine rod CNG channel confirmed this conclusion. This revised stoichiometry provides a new foundation for understanding the structure and function of the CNG channel family.

Figure 1

Homotypic interaction of the C terminus of CNG channel A subunit. a, Co-immunoprecipitation of epitope-tagged C termini of CNGA3. The constructs were amino-terminally tagged with HA (MGYPYDVPDYADLNGGGGGST) or Myc (MEQKLISEEDLNGGGGGST). The constructs were: A3-C, start of cytoplasmic C terminus (Asn 398) to end of human CNGA3; ShB-C, Asn 482 to end of Shaker B potassium channel (negative control). Inputs before immunoprecipitation (IP) are shown at the bottom and artificially aligned to save space. b, Binding in vitro. The His-tagged protein was detected with an antibody (69522; Novagen) against a T7 epitope right after the His tag. The inputs of GST (control) and the GST-tagged protein, detected with an anti-GST antibody (27-4577-01; Amersham), before binding in vitro are also shown and are artificially aligned (bottom). c, Metabolic labelling. 293T cells transfected with Myc-tagged A3-C were metabolically labelled, then immunoprecipitated with the anti-Myc antibody. The pulled-down proteins and inputs before the immunoprecipitation are shown (see Methods). d, Co-immunoprecipitation of epitope-tagged C-termini of CNGA1 and CNGA2. The experiment was as described in a. The constructs were: A1-C, Asn 393 to end of bovine CNGA1; A2-C, Asn 372 to end of rat CNGA2. Numbers alongside the gels are relative molecular mass (M_r) values in kD.

Figure 2

Identification of the CLZ domain. a, Cytoplasmic C terminus of CNG channel A subunit, showing the cyclic-nucleotide-binding domain (CNB) and the CLZ domain. Sequence alignments of the CLZ domains of vertebrate and invertebrate CNG channel A subunits are shown underneath. dmCNC and Tax-4 are those of Drosophila and C. elegans, respectively. Conserved hydrophobic residues are in bold type. The 22-residue leucine zipper first identified by sequence analysis is underlined. Asterisks label hydrophobic residues found to be essential for the CLZ interaction of hCNGA3 in the co-immunoprecipitation assay. Prefixes: h, human; r, rat. b, Homotypic interactions of various C-terminal constructs of hCNGA3 and mouse (m)CNGB3. In all lanes, the indicated front construct is HA-tagged and after construct is Myc-tagged. The inputs are arbitrarily aligned. The constructs are: A3C-ΔZ, A3-C with Leu 626 to Leu 647 deleted; A3C-28, Lys 581 to end of hCNGA3; A3C-32, A3C-28 with Leu 626 to Leu 647 deleted; A3C-71, A3C-28 with Leu 648 to Arg 661 deleted; B3-C, Gln 432 to end of mCNGB3; B3C-CLZ, B3-C with Ala 655 to Phe 658 replaced by Lys 624 to Gly 672 of hCNGA3 plus two extra residues (Val-Glu) for facilitating insertion by restriction enzymes. a.a., amino acids. Numbers alongside the gels are M_r values in K.

Figure 3

The CLZ domain forms a trimer. a, Gel-filtration experiment with N-terminally HA-tagged A3C-28 (see Fig. 2b). See Methods for details. The M_r values of the peaks were estimated from their Stokes radii based on a standard curve (not shown). b, Cross-linking experiment on N-terminally HA-tagged A3C-28 or A3C-32 (CLZ-disrupted A3C-28, see Fig. 2b) with the cross-linker DSP. The faint >100K band in the gels seems to be non-specific. DTT, dithiothreitol. c, Cross-linking experiments on HA-tagged A1C-28 (Lys 574 to end of human CNGA1) and A2C-28 (Lys 555 to end of rat CNGA2). d, Native-gel electrophoresis. All constructs were N-terminally HA-tagged and detected with an anti-HA antibody. A3C-87 is A3C-28 with mutation L633 A, which disrupted the CLZ-mediated interaction without changing the charge of the construct. The position of A3C-87 indicates the monomer position of A3C-28. The two A3C-28 mutants, A3C-88 (K596E and R603E) and A3C-89 (D613K and E615K), had different mobilities in native-gel electrophoresis. When these two constructs are co-expressed, three bands are expected for a dimeric interaction (P₂, PQ and Q₂) and four bands for a trimer (P₃, P₂Q, PQ₂ and Q₃). Four bands were seen (indicated by arrowheads) when A3C-88 and A3C-89 were co-expressed. In the bottom panel, a fixed amount of A3C-88 was co-transfected with increasing amounts of A3C-89. Four bands can be clearly seen (arrowheads). e, Equilibrium analytical centrifugation. A 48-residue peptide corresponding to the CLZ domain (Lys 624 to Gly 671 of hCNGA3) was commercially synthesized and purified to more than 95% purity (Synpep, Dublin, CA). This peptide was acetylated at the N terminus and amidated at the C terminus, with a calculated M_r of 5,582. The peptide was subjected to analysis by equilibrium analytical centrifugation. A total of nine data sets were collected (0.5, 1.0 and 2.0 mg ml⁻¹, each spun at 40,000, 45,000 and 52,000 r.p.m.). A representative data set (2.0 mg ml⁻¹ centrifuged at 40,000 r.p.m.; open grey squares) and its fitting curves are shown in the upper panel, with residuals (difference between data and fittings) in the lower panel. The data are best fitted by an ideal monomer–trimer model (red) with a M_r of 5,468 (confidence interval 5,375–5,559) and an association constant of 1.43 × 10¹⁰ M⁻² (confidence interval (3.68–71.7) × 10⁹ M⁻²). A similar fit with a monomer–dimer model gave an unrealistic M_r in excess of 7,000. With M_r fixed at 5,582 (the calculated value), neither the monomer–dimer (black) nor the monomer–tetramer models (green) fitted well.

Figure 4

Expression and functional properties of hCNGA3 and its CLZ-substituted mutants. Leu 626 to Val 669 of hCNGA3 was replaced, respectively, by well-characterized dimeric (LEDKVEELLSKNYHLENEVARLKKLVGERI), trimeric (IEDKIEEILSKQYHIENEIARIKKLIGERI) and tetrameric (IEDKLEEILSKLYHIENELARIKKLLGERI) leucine zippers. All transfections and subsequent recordings were performed side by side. a, Saturated cGMP-activated currents of homomeric channels formed by hCNGA3 and the three CLZ-substituted mutants, respectively. The values (means ± s.e.m.) are 207 ± 60 pA (hCNGA3, n = 12), 171 ± 63 pA (dimeric, n = 12), 253 ± 66 pA (trimeric, n = 12) and 159 ± 41 pA (tetrameric, n = 13). b, K_1/2 values of the cGMP dose–response relations for the wild-type and mutant channels. The values (means ± s.e.m.) are 16.9 ± 0.6 μM (hCNGA3, n = 8), 11.0 ± 0.2 μM (dimeric, n = 9), 15.3 ± 0.6 μM (trimeric, n = 9) and 20.7 ± 0.8 μM (tetrameric, n = 9). c, Saturated cAMP/cGMP current ratios of the wild-type and mutant channels. The values (means ± s.e.m.) are 0.122 ± 0.012 (hCNGA3, n = 9), 0.195 ± 0.013 (dimeric, n = 9), 0.106 ± 0.010 (trimeric, n = 9) and 0.076 ± 0.005 (tetrameric, n = 9). d, Saturated cGMP current produced by the co-expression of wild-type or mutant hCNGA3 with hCNGB1. The values (means ± s.e.m.) are 184 ± 67 pA (hCNGA3, n = 13), 16.3 ± 5.1 pA (dimeric, n = 14), 168 ± 86 pA (trimeric, n = 14) and 26.8 ± 8.4 pA (termeric, n = 14).

Figure 5

Analysis of the A and B subunits of the native rod CNG channel. The CNG channel was purified from detergent-solubilized bovine rod outer segments on a PMc 1D1–Sepharose immunoaffinity column and analysed by SDS–PAGE and western blotting. a, Coomassie Blue-stained gel. b, Fluorescence from tryptophan residues modified by an ultraviolet-induced reaction of trichloroethanol and the channel. c, Fluorescence from Oregon Green maleimide-modified cysteine residues of the channel. d, Western blot labelled with a mixture of monoclonal antibodies, PMb 3C9 and PMc 1D1, against the B (M_r ~240K) and A subunits (M_r ~68K), respectively. Lanes 1–4 contained about 4, 3, 2 and 1 μg of protein, respectively.