ABSTRACT
Cyclic nucleotide-gated (CNG) channels have been well characterized in the sensory receptors of vision and olfaction, but their characteristics in other tissues remain largely unknown. Here, we report characterization of a novel brain-specific CNG channel from zebrafish. Unique among CNG channels, the transcript is expressed mainly in the brain. When expressed in Xenopus oocytes, the channel's electrophysiological properties are distinct compared to CNG channels from either rods (CNGA1), olfactory receptors (CNGA2), or cones (CNGA3). The channel is less sensitive to cAMP than cGMP (K(1/2) of 280 and 7 microM, respectively), with a maximum cAMP efficacy at least 80% of that with saturating levels of cGMP. The single-channel conductance of 58pS is larger than most other CNG channels. Like other CNG channels the channel is relatively nonselective among monovalent cations. However, unlike other CNG channels, there was rundown of the macroscopic current within 30-100 min after patch excision.
Subject(s)
Brain Chemistry , Ion Channels/physiology , Zebrafish Proteins/physiology , Amino Acid Sequence , Animals , Cyclic AMP/pharmacology , Cyclic GMP/pharmacology , Ion Channels/chemistry , Ion Channels/drug effects , Molecular Sequence Data , Sequence Alignment , Zebrafish , Zebrafish Proteins/chemistry , Zebrafish Proteins/drug effectsABSTRACT
PURPOSE: To determine whether inhibition of cyclic nucleotide-gated (CNG) ion channels by retinoids might be useful in treating degenerative retinal diseases in which either the CNG channels are hypersensitive to 3',5'-cyclic guanosine monophosphate (cGMP) or the photoreceptor cGMP concentration is elevated. METHODS: Patch clamp (electrophysiological) methods were used to measure activation by cGMP of wild-type human cone (hCNGA3), mutant cone (hCNGA3-N471S), and wild-type bovine rod (bCNGA1) CNG channels heterologously expressed in Xenopus oocytes. Cyclic GMP-activated currents were measured in excised, inside-out membrane patches before and after treatment with either all-trans retinal (ATR) or all-trans C22 aldehyde, which is too long to fit into the chromophore binding pocket of opsin and therefore cannot activate the visual transduction cascade. RESULTS: At physiological cGMP concentrations, 150 nM ATR reduced the open probability of the mutant cone CNG channel by reducing its apparent cGMP affinity to that of the normal cone channel. Furthermore, all-trans C22 aldehyde similarly inhibited the mutant cone channel as well as normal rod and cone CNG channels. CONCLUSIONS: Our results raise the possibility that retinoids, such as all-trans C22 aldehyde, that inhibit CNG channels without affecting the transduction cascade, may be useful in treating degenerative retinal diseases in which either the cGMP concentration is elevated or the CNG channels are hypersensitive to cGMP.
Subject(s)
Cyclic GMP/pharmacology , Ion Channels/genetics , Ion Channels/metabolism , Mutation , Retinitis Pigmentosa/genetics , Retinoids/pharmacology , Animals , Asparagine , Cattle , Cyclic Nucleotide-Gated Cation Channels , Humans , Ion Channels/antagonists & inhibitors , Ion Channels/drug effects , Oocytes , Patch-Clamp Techniques , Retinitis Pigmentosa/drug therapy , Serine , Transduction, Genetic , Vitamin A/pharmacology , XenopusABSTRACT
Rod vision is initiated when 11-cis-retinal, bound within rhodopsin, absorbs a photon and isomerizes to all-trans-retinal (ATR). This triggers an enzyme cascade that lowers cGMP, thereby closing cyclic nucleotide-gated (CNG) channels. ATR then dissociates from rhodopsin, with bright light releasing millimolar levels of ATR. We have recently shown that ATR is a potent closed-state inhibitor of the rod CNG channel, and that it requires access to the cytosolic face of the channel (McCabe, S.L., D.M. Pelosi, M. Tetreault, A. Miri, W. Nguitragool, P. Kovithvathanaphong, R. Mahajan, and A.L. Zimmerman. 2004. J. Gen. Physiol. 123:521-531). However, the details of the interaction between the channel and ATR have not been resolved. Here, we explore the nature of this interaction by taking advantage of specific retinoids and retinoid analogues, namely, beta-ionone, all-trans-C15 aldehyde, all-trans-C17 aldehyde, all-trans-C22 aldehyde, all-trans-retinol, all-trans-retinoic acid, and all-trans-retinylidene-n-butylamine. These retinoids differ in polyene chain length, chemical functionality, and charge. Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid-channel interaction. We propose that the cytoplasmic face of the channel contains a retinoid binding site. This binding site likely contains a hydrophobic region that allows the ionone ring and polyene tail to sit in an optimal position to promote interaction of the terminal functional group with residues approximately 15 A away from the ionone ring. Based on our functional data with retinoids possessing either a positive or a negative charge, we speculate that these amino acid residues may be polar and/or aromatic.
