Subject(s)
Calmodulin/metabolism , Cyclic GMP/physiology , Ion Channels/physiology , Retinal Rod Photoreceptor Cells/physiology , Amino Acid Sequence , Animals , Binding Sites , Cattle , Cell Line , Cyclic Nucleotide-Gated Cation Channels , Ion Channels/chemistry , Ion Channels/metabolism , Macromolecular Substances , Membrane Potentials/physiology , Molecular Sequence Data , Patch-Clamp Techniques , Peptide Fragments/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Fluorescence/methods , TransfectionABSTRACT
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 alpha-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 alpha-subunit, which does form a CaM-modulated 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.
Subject(s)
Calmodulin/physiology , Cyclic GMP/physiology , Heterotrimeric GTP-Binding Proteins/physiology , Ion Channels/physiology , Amino Acid Sequence , Animals , Binding Sites , Calmodulin/metabolism , Cyclic Nucleotide-Gated Cation Channels , GTP-Binding Protein alpha Subunits , Heterotrimeric GTP-Binding Proteins/chemistry , Heterotrimeric GTP-Binding Proteins/metabolism , Humans , Ion Channels/chemistry , Ion Channels/metabolism , Molecular Sequence Data , Rats , Sequence Homology, Amino AcidABSTRACT
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 beta-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 alpha- and beta-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 beta-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 microM. 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 beta-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.
Subject(s)
Calcium/pharmacology , Calmodulin/pharmacology , Cyclic GMP/metabolism , Ion Channels/antagonists & inhibitors , Ion Channels/chemistry , Retinal Rod Photoreceptor Cells/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cattle , Cloning, Molecular , Cyclic GMP/pharmacology , Cyclic Nucleotide-Gated Cation Channels , Diltiazem/pharmacology , Egtazic Acid/pharmacology , Humans , Macromolecular Substances , Molecular Sequence Data , Peptide Fragments/chemistry , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
We have cloned from human retina a cyclic nucleotide-gated (CNG) ion channel that is distinct from the one found in rod photoreceptors. This channel protein is highly homologous to the CNG channel that recently has been cloned from bovine testis and kidney and has been shown to be present in retinal cone photoreceptors. When expressed in human embryonic kidney cells, the protein forms functional ion channels with properties broadly similar to those described for the cloned bovine channel. The gene for this channel resides on chromosome 2.
Subject(s)
Chromosomes, Human, Pair 2 , Ion Channels/genetics , Ion Channels/physiology , Retina/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Amino Acid Sequence , Animals , Cattle , Cell Line , Chromosome Mapping , Cloning, Molecular , Cyclic AMP/pharmacology , Cyclic GMP/pharmacology , Cyclic Nucleotide-Gated Cation Channels , DNA Primers , Electrophysiology , Embryo, Mammalian , Gene Expression , Humans , Ion Channels/chemistry , Kidney , Membrane Potentials/drug effects , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , TransfectionABSTRACT
An ion channel directly activated by cGMP was first discovered about ten years ago. Since then, a number of ion channels with the same property (cyclic nucleotide-activated channels) have been reported that are involved in a variety of cell functions. In addition, other channels have been found that are not primarily controlled by cyclic nucleotides but are modulated by them (cyclic nucleotide-modulated channels). These channels likewise have diverse functions and tissue distributions. Both channel classes are reviewed here. Coverage includes the cyclic-nucleotide binding site on these channels, ion permeation, pharmacological blockers, channel gating and modulation, and physiological functions of the channels.