Molecular cloning, functional expression and chromosomal localization of a human homolog of the cyclic nucleotide‐gated ion channel of retinal cone photoreceptors

Yu, Wei; Grunwald, Maria E.; Yau, King Wai

doi:10.1016/0014-5793(96)00889-7

Cited by 56 publications

(49 citation statements)

References 44 publications

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“…A similar, but far less potent, inhibition by Ca 2ϩ -CaM exists for a homologous cGMP-gated channel mediating phototransduction in retinal rods (ROD) (7)(8)(9)(10)28), but in this case the binding site for Ca 2ϩ -CaM is localized to a corresponding region on the channel's ␤-subunit (ROD␤) (29,30). Another cGMP-gated channel mediates phototransduction in retinal cones (CONE) (31)(32)(33). We report here that human CONE's ␣-subunit (CONE␣) also has a Ca 2ϩ -CaM binding site that is in roughly the same location as on OLF␣.…”

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confidence: 84%

Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin

Grunwald

Zhong

Lai

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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The action of calmodulin (CaM) on target proteins is important for a variety of cellular functions. We demonstrate here, however, that the presence of a CaM-binding site on a protein does not necessarily imply a functional effect. The ␣-subunit of the cGMP-gated cation channel of human retinal cones has a CaM-binding site on its cytoplasmic N-terminal region, but the homomeric channel that it forms is not functionally modulated by CaM. Mutational analysis based on comparison to the highly homologous olfactory cyclic nucleotide-gated channel ␣-subunit, which does form a CaMmodulated channel, indicates that residues downstream of the CaM-binding domain on these channels are also important for CaM to have an effect. These findings suggest that a CaM-binding site and complementary structural features in a protein probably evolve independently, and an effect caused by CaM occurs only in the presence of both elements. More generally, the same may be true for other recognized binding sites on proteins for modulators or activators, so that a demonstrated physical interaction does not necessarily imply functional consequence. C almodulin (CaM) plays an important role in a variety of cellular processes. Typically, it acts by first binding Ca 2ϩ , with the Ca 2ϩ -CaM complex then binding to diverse targets such as protein kinase and phosphatase, adenylyl cyclase, cyclicnucleotide phosphodiesterase, nitric oxide synthase, cytoskeletal proteins, ion transporters, and ion channels (1-3). The direct effects of CaM on ion channels have been recognized only recently (4-18). One ion channel that shows very strong Ca 2ϩ -CaM modulation and has been studied quite extensively is the cyclic nucleotide-gated, nonselective cation channel mediating olfactory transduction in olfactory receptor neurons (OLF) (11,12,19). The opening of this channel generates the olfactory receptor potential (20-23). Because of its high Ca 2ϩ permeability (24, 25), the opening of OLF leads to a rise in cytosolic Ca 2ϩ , which in turn strongly inhibits the channel via Ca 2ϩ -CaM to produce negative feedback (11), making the cell adapt to the olfactory stimulus (24,26,27). This inhibition by Ca 2ϩ -CaM consists of a favoring of the closed state of the channel, manifested as an increase in the half-activation constant, K 1/2 , of the channel for cyclic nucleotide (i.e., a shift of the doseresponse relation to higher ligand concentrations) (11, 12). The binding site on OLF for Ca 2ϩ -CaM has been localized to the cytoplasmic N-terminal region of its ␣-subunit (OLF␣) (12). A similar, but far less potent, inhibition by Ca 2ϩ -CaM exists for a homologous cGMP-gated channel mediating phototransduction in retinal rods (ROD) (7-10, 28), but in this case the binding site for Ca 2ϩ -CaM is localized to a corresponding region on the channel's ␤-subunit (ROD␤) (29,30). Another cGMP-gated channel mediates phototransduction in retinal cones (CONE) (31-33). We report here that human CONE's ␣-subunit (CONE␣) also has a Ca 2ϩ -CaM binding site that is in roughly the same loca...

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confidence: 84%

Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin

Grunwald

Zhong

Lai

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…Since the NH 2 -terminal sequence of CNGgust has not been definitely determined, a precise comparison of the NH 2 -terminal regions is not achieved. In terms of overall homology, CNGgust shows the highest similarity (82%) to bovine testis CNG channel (20,21) and the same degree of similarity to human cone CNG channel (22). However, it shows lower similarities (50 -70%) to other CNG channels: 66% to rat olfactory CNG channel (6) and 63% to rat rod CNG channel (23).…”

Section: A Cng Channel Expressed In Rat Tonguementioning

confidence: 94%

Taste Buds Have a Cyclic Nucleotide-activated Channel, CNGgust

Misaka¹,

Kusakabe²,

Emori³

et al. 1997

Journal of Biological Chemistry

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Cyclic nucleotide-gated (CNG) channels have been characterized as important factors involved in physiological processes including sensory reception for vision and olfaction. The possibility thus exists that a certain CNG channel functions in gustation as well. In the present study, we carried out reverse transcription-polymerase chain reaction and genomic DNA cloning and characterized a CNG channel (CNGgust) as a cyclic nucleotide-activated species expressed in rat tongue epithelial tissues where taste reception takes place. Several types of 5-rapid amplification of cDNA ends clones of CNGgust cDNA were obtained with various 5-terminal sequences. As the CNGgust gene was a single copy, the formation of such CNGgust variants should result from alternative splicing. The encoded protein was homologous to known vertebrate CNG channels with 50 -80% similarities in amino acid sequence, and particularly homologous to bovine testis CNG channel and human cone CNG channel with 82% similarities. CNGgust was functional when expressed in human embryonic kidney cells, where it opened upon the addition of cGMP or cAMP. Immunohistochemical analysis using an antibody raised against a CNGgust peptide demonstrated the channel to be localized on the pore side of each taste bud in the circumvallate papillae, with no signal observed for degenerated taste buds after denervation of the glossopharyngeal nerve. All these results, together with the indication that cyclic nucleotides play a role gustatory signaling pathway(s), strongly suggest the involvement of CNGgust in taste signal transduction.

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“…44; see also Ref. 45). 2 Taken together, these observations suggest an evolutionary path in which the ancestral channel that gave rise to the various ␣-and ␤-subunits of the cyclic nucleotide-gated channels might already have a CaM-binding site-like domain on its N terminus.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Domain on the β-Subunit of the Rod cGMP-gated Cation Channel That Mediates Inhibition by Calcium-Calmodulin

Grunwald

et al. 1998

Journal of Biological Chemistry

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The cGMP-gated cation channel mediating phototransduction in retinal rods has recently been shown to be inhibited by calcium-calmodulin, through direct binding of the latter to the ␤-subunit of the heterotetrameric channel complex. Here, we report the characterization of this inhibition and the identification of a domain crucial for this modulation. Heterologous expression of the ␣-and ␤-subunits of the human rod channel in HEK 293 cells produced a cGMP-gated current that was highly sensitive to calcium-calmodulin, with half-maximal inhibition at approximately 4 nM. In biochemical and electrophysiological experiments on deletion mutants of the ␤-subunit, we have identified a region on its cytoplasmic N terminus that binds calmodulin and is necessary for the calmodulin-mediated inhibition of the channel. However, in gel shift assays and fluorescence emission experiments, peptides derived from this region indicated a low calmodulin affinity, with dissociation constants of approximately 3-10 M. On the C terminus, a region was also found to bind calmodulin, but it was likewise of low affinity, and its deletion did not abolish the calmodulin-mediated inhibition. We suggest that although the identified region on the N terminus of the ␤-subunit is crucial for the calmodulin effect, other regions are likely to be involved as well. In this respect, the rod channel appears to differ from the olfactory cyclic nucleotide-gated channel, which is also modulated by calcium-calmodulin.

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Molecular cloning, functional expression and chromosomal localization of a human homolog of the cyclic nucleotide‐gated ion channel of retinal cone photoreceptors

Cited by 56 publications

References 44 publications

Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin

Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin

Taste Buds Have a Cyclic Nucleotide-activated Channel, CNGgust

Identification of a Domain on the β-Subunit of the Rod cGMP-gated Cation Channel That Mediates Inhibition by Calcium-Calmodulin

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