445 Calmodulin regulation of cyclic-nucleotide-gated channels Robert S Molday Cyclic-nucleotide-gated (CNG) channels play key roles in photoreceptor and olfactory signal-transduction p_athways. Recent studies have focused on the molecular characterization of CNG channel subunits and on the identification of the structural domains that contribute to ligand selectivity and affinity, ion gating and permeation, and regulation of channel activity. Calmodulin has been shown to bind directly to the rod and olfactory channels and to modulate their sensitivity to cyclic nucleotides. This Cal+-dependent regulation of channel activity appears to play a role in the termination of the signal-transduction pathway in olfactory neurons and rod photoreceptor cells. It remains to be determined whether calmodulin also regulates the activity of related channels in other cells. Addresses Department of Biochemistry and Molecular Biology, 2146 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia, V6T 123 Canada; e-mail: molday@unixg.ubc.ca Abbreviations CaM calmodulin CAP catabolite gene activator protein CNG cyclic-nucleotide-gated GARP glutamic-acid-rich protein ROS rod outer segment S membrane-spanning segment Current Opinion in Neurobiology 1996, 6~445-452 0 Current Biology Ltd ISSN 0959-4368 Introduction Cyclic-nucleotide-gated (CNG) channels comprise a fam- ily of cation-selective channels that are directly activated by cyclic nucleotides (CAMP and cGMP). In photoreceptor cells and olfactory neurons, CNG channels play a central role in signal-transduction pathways by controlling the flow of Na+ and Ca2+ into these cells in response to signal-induced changes in intracellular levels of cGMP or CAMP [1,2]. Related CNG channels have also been detected in a variety of other vertebrate and invertebrate cells, including the pineal gland, kidney and heart cells, ganglion cells, bipolar cells, and spermatozoa (for a review, see [3*]); however, in most cases, their role in cell function remains to be determined. Since the initial isolation, cloning and functional expres- sion of the cGMP-gated channel of rod photoreceptors [4], considerable progress has been made toward identifying and characterizing subunits of the rod and olfactory CNG channels, and defining determinants that contribute to ligand selectivity and affinity, ion permeation, gating properties and inhibition by divalent ions [3*,5*]. Recent studies also indicate that the activity of both rod and olfactory CNG channels is modulated by calmodulin (CaM) [6,7], a ubiquitous Caz+-binding protein known to regulate a wide variety of enzymes, receptors, cytoskeletal proteins and ion transporters. This review covers recent structure/function studies of the rod and olfactory CNG channels, and focuses on recent findings related to the interaction of CaM with these channels and its role in rod and olfactory signaling mechanisms. Molecular properties of CNG channels Recent biochemical, molecular cloning and expression studies indicate that the CNG channels zyxwvutsrqponmlkjihgfed of rod photore- ceptor cells and olfactory neurons consist of two subunits, generally designated as 01 and p subunits (or subunits 1 and 2), that assemble into a hetero-oligomeric complex [4,&11,12**]. Each subunit contains similar structural features, including six hydrophobic segments (Sl-S6) that probably serve as membrane-spanning segments, a cyclic-nucleotide-binding domain close to the carboxyl terminus, a voltage-sensor-like motif comprising the S4 transmembrane segment, and a pore region that lines the central cavity of the channel and plays a central role in ion permeation. As the voltage-sensor motif and the pore region are characteristic features of voltage-gated channels, it has been suggested that CNG and voltage-gated channels are distant relatives [13]. A topological model for the CNG channel subunits (Figure 1) has been proposed based on the similarity of CNG channels to voltage-gated channels and on immunocytochemical labeling studies using site-directed antibodies [13-171. Recently, additional support for this model has been obtained using gene-fu- sion techniques [ 181. The ~1 subunits of vertebrate photoreceptor and olfactory CNG channels were the first subunits to be cloned and have been the most extensively studied because they can assemble into functional homo-oligomeric CNG channels [4,8]. The photoreceptor and olfactory c1 sub- units are similar in size (M, -70-85 K) and are -57% identical in sequence [4,8,14,15,19,20]. They all contain a consensus sequence for N-linked glycosylation within the hydrophilic segment linking the S5 transmembrane segment to the pore region (Figure la). This site has been shown to he glycosylated in the rod zyxwvutsrqponmlkjihgfe a subunit [17]; corresponding sites in the cone and olfactory subunits are also likely to be glycosylated. A photoreceptor-cell-specific post-translational reaction removes the amino-terminal segment from the rod and cone a subunit [l&16]. As a result, the bovine rod a subunit migrates on SDS gels at -63 kDa, instead of the - 79 kDa observed for the heterologously expressed subunit. The reason for this