Molecular cloning and functional characterization of a new modulatory cyclic nucleotide-gated channel subunit from mouse retina

Abstract

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.

Fig. 1.

Primary structure of murine CNG6.A, Alignment of the amino acid sequence of mCNG6 with rCNG4.3 and a partial sequence of rCNG6 (the prefix indicates the species: m = mouse; r = rat). Amino acids identical between mCNG6 and rCNG4.3 or all three proteins are boxed. The putative transmembrane segments (S1–S6), the pore region,and the CNBD are underlined. Gaps in the sequences are represented by dashes.Arrowheads in the N terminus of mCNG6 indicate the region corresponding to probe c used for in situhybridization. Arrows mark the sequence corresponding to the EST HSA12972. An asterisk indicates the methionine at position 337 in the sequence of CNG6 that was replaced by leucine in the CNG6 expression vector. The sequence of mCNG6 is available from the European Molecular Biology Laboratory database under the accession number AJ243572. B, Phylogenetic tree of the mammalian CNG channel subunits. The tree was calculated based on the pairwise comparison with the transmembrane domains and the cyclic nucleotide binding domain of the respective subunits. The sequences are derived from mouse [CNG1 (Pittler et al., 1992); CNG3 (Biel et al., 1999b); CNG6 (this paper)] or rat [CNG2 (Dhallan et al., 1990); CNG4 (Sautter et al., 1998); CNG5 (Bradley et al., 1994)].

Fig. 2.

Expression of CNG6 transcripts in mouse tissues.A, Scheme of the mCNG6 cDNA. The protein coding region of CNG6 is represented by a box. The transmembrane segments (1–6), the pore (P), and the CNBD are indicated. The 3′ and 5′ untranslated sequences are shown by athin line. The location of probes a andb used for Northern analysis are indicated below the sequence. B, Northern analysis of CNG6 expression.First lane, Left, Six micrograms of poly(A⁺) RNA isolated from mouse eyes was hybridized with probe b. Right blot, A mouse multiple tissue blot (Clontech) was analyzed with probea. Each lane contained ∼2 μg of poly(A⁺) RNA from the following tissues: heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis. Audioradiographic exposure was for 7 d at −70°C.

Fig. 3.

Expression of CNG channel subunits in mouse retina as determined by in situ hybridization. Shown are dark-field photographs of cryosections of mouse retina hybridized with³⁵S-labeled antisense riboprobes specific for CNG1 (A), CNG4 (B), CNG3 (C), and CNG6 (probe c) (D, F) and with a sense riboprobe specific for CNG6 (E). CNG1- and CNG4-specific probes detected strong expression in retinal photoreceptors as indicated by the reaction product localized to the inner segments (IS) of these cells. CNG3- and CNG6-specific probes only labeled a subset of photoreceptors and also hybridized weakly with the inner nuclear layer (INL). A sense probe directed against CNG6 produced no signal (E). Exposure to film emulsion was 2 weeks for CNG1 and CNG4 and 5 weeks for CNG3 and CNG6. OS, Outer segment; IS, inner segment; ONL, outer nuclear layer; IPL, inner plexiform layer;OPL, outer plexiform layer; GCL, ganglion cell layer. Scale bars, 30 μm.

Fig. 4.

Single-channel activity of homomeric and heteromeric channels. The recordings show single-channel currents from an inside-out patch of HEK293 cells transfected either with an expression vector encoding CNG3 (A) or equimolar amounts of expression plasmids encoding CNG3 and CNG6 (B). The currents were evoked by 1 μm cGMP at a membrane potential of +80 mV.o, Open channel; c, closed channel.

Fig. 5.

Current–voltage relations of CNG3 and CNG3/CNG6 channels in the presence of 2 mm extracellular Ca²⁺. The currents were activated by 300 μm cGMP and normalized to the current at +60 mV (I₊₆₀). The points represent means ± SEM from four (CNG3) or six (CNG3/CNG6) patches.

Fig. 6.

Sensitivity of homomeric and heteromeric channels to l-cis diltiazem. Current traces of the CNG3 (A) and the CNG3/CNG6 channels (B) induced at ±80 mV in the absence (○) and presence (●) of 10 μm intracellularl-cis-diltiazem (Dilt.).C, D, Steady-state current–voltage relations of the CNG3 (C) and CNG3/CNG6 currents (D) in the presence and absence of 10 μml-cis diltiazem. cGMP (300 μm) was used to activate the channels inA–D.

Fig. 7.

Increase of cAMP efficacy induced by CNG6. Currents were induced at +80 mV in excised inside-out patches containing either CNG3 (A) or CNG3/CNG6 (B) by a saturating concentration of cGMP (1 mm) or cAMP (10 mm). Note that the initial decay of the cGMP current in B does not occur in the homomeric channel (A). C, Dose–response curves for CNG3 (open symbols) and CNG3/CNG6 (closed symbols) currents induced by cGMP (circles) or cAMP (triangles). Curves were normalized to the response at 1 mm of cGMP. Each point represents the mean ± SEM from five to seven patches. Thesolid lines are best fits calculated by the Hill equation (see Materials and Methods). Concentrations are as follows (in μm): CNG3, cGMP: K_a = 16.6 ± 2.2, ν = 2.4 ± 0.1 (n = 6); cAMP: K_a = 1690 ± 70, ν = 1.8 ± 0.1 (n = 5); CNG3/CNG6, cGMP: K_a = 20.2 ± 0.6, ν = 2.2 ± 0.1 (n = 7); cAMP:K_a = 735 ± 60, ν = 1.5 ± 0.1 (n = 6).

Fig. 8.

Ligand-independent openings of homomeric and heteromeric channels. In two individual patches containing approximately 60 channels of either CNG3 (A) or CNG3/CNG6 (B), currents were activated by applying 200 msec pulses at 0.5 Hz from a holding potential of 0 to +80 mV. For each patch 50 consecutive pulses, 10 of which are shown, were analyzed. The mean spontaneous current (I_sp) was defined as the average current measured from 50 pulses. After the ligand-independent activation, the patches were perfused with a saturating cGMP concentration and again depolarized to +80 mV to activate the maximal current (I_max) of the CNG3 (C) and CNG3/CNG6 (D) channel. o, Open channel; c, closed channel.