THE cGMP-GATED CHANNEL OF PHOTORECEPTOR CELLS: ITS STRUCTURAL PROPERTIES AND ROLE IN PHOTOTRANSDUCTION
Robert S. Molday and Yi-Te Hsu
Introduction:
The cyclic GMP-gated channel of vertebrate rod photoreceptor cells playsa central role in the phototransduction process. In the dark a relatively high concentration of cGMP in the rod outer segment (ROS) maintains a significant number of channels in their open state by the direct, reversible binding of cGMP to the channels (Fensenko et al. 1985; Yau and Nakatani, 1985; Yau and Baylor, 1989). This allows for a steady influx of Na+ and Ca2+ into the outer segment and maintains the cell in a partially depolarized state. The influx of Ca2+ through the channel is balanced by an efflux of Ca2+ by the Na+/Ca2+-K+ exchanger in the ROS plasma membrane, thereby maintaining the intracellular level of Ca2+ at a relatively constant level (Yau and Nakatani, 1984). Low intracellular Na+ concentration is maintained by the balanced extrusion of Na+ by Na+-K+ ATPase localized in the plasma membrane of the rod inner segment.
Photobleaching of rhodopsin in the ROS disk membrane leads to an activation of the visual enzyme cascade system (Stryer, 1986; 1991; Chabre and Deterre, 1989)and a reduction in the concentration of cGMP by a phosphodiesterase catalyzed reaction (Figure1). This results in the closure of the cGMP-gated channels and a transient hyperpolarization ofthe cell. The closure of the channels to Ca2+ results in a reduction in intracellular Ca2+ since the Na+/Ca2+-K+ exchanger continues to extrude Ca2+ from the outer segment. The reduction in Ca2+ has been shown to be important in the recovery of the rod outer segment to its dark state and in light adaptation (Koch and Stryer, 1987; Yau and Nakatani, 1985b; Matthews et al.1988; Kaupp and Koch, 1992). This feedback mechanism is suggested to occur through the interaction of Ca2+- binding proteins with various proteins involved in the phototransduction process, and in particular, guanylate cyclase (Koch and Stryer, 1987).
Cone photoreceptors appear to have a similar visual cascade system and a related cGMP-gated channel (Haynes and Yau, 1985; Watanabe and Murakami, 1991; Picones and Korenbrot, 1992; BF6nigk et al. 1993) The physiological properties of the cGMP-gated channel of ROS have been extensively studied and recently reviewed (Yau and Baylor, 1989; Kaupp, 1991). Generally, cGMP directly and cooperatively bind to and activates the channel with a K1/2 of 10 - 50*M and a Hill coefficient (n) of 1.7-3.5. Analogues of cGMP with substitutents at position 8, such as 8-bromo- cyclic GMP, are also effective activators of the channel in the *M range (Zimmerman et al.,1985). Although the channel is cation selective, it exhibits a low degree of specificity for various monovalent and divalent cations (Yau and Nakatani, 1984b; Hodgkin et al. 1985; Schnetkamp 1987; Furman and Tanaka, 1990).
The rod channel is permeable not only to the physiological ions, Na+, Ca2+ and Mg2+, but also to other monovalent and divalent cationsincluding Li+, K+, Cs+, Rb+, Mn2+ and Ba2+. Under physiological conditions the channel is relatively insensitive to voltage changes. Divalent cations have been shown to block the flow of ions through the channel thereby reducing the effective conductance of the channel (Yau and Baylor, 1989). L-cis diltiazem has been shown to be a stereoselective inhibitor of channel acitivity in the micromolar concentration range (Koch and Kaupp, 1985; Stern et al. 1986). The channel has been localized to the plasma membrane of ROS and has a density of 200 - 500 channels per um2(Bodoia and Detwiler, 1985: Zimmerman and Baylor, 1986; Cook et al. 1989).
Recently, biochemical, immunochemical and molecular biology studies haveprovided insight into the molecular properties of the cGMP-gated channel of rod and cone photoreceptors. In this paper issues related to the molecular composition and structural features of the cGMP- gated channel of photoreceptor cells are addressed, and the regulation of the cGMP-gated channel by calmodulin is discussed in terms of its possible role in phototransduction.
Molecular Composition of the cGMP-gated Channel
Identification and purification of the *-subunit
In the latter half of the 1980s several laboratories had identified different polypeptides as candidates for the cGMP-gated channel subunit of ROS. Cook et al. (1987) isolated a 63 kDa polypeptide from CHAPS detergent-solubilized bovine ROS by ion exchange andred-dye affinity chromatography. This preparation exhibited cGMP-dependent ion flux activitywhen reconstituted into liposomes, but, in contrast to ROS membranes, the channel activity wasnot inhibited by micromolar concentrations of l-cis dilitizem. At about the same time Matesic and Liebman (1987) described the partial purification of a 39 kDa polypeptide from cholate-treated bovine ROS. This 39 kDa protein, which was reported to exhibit immunochemical crossreactivity with rhodopsin, could be photoaffinity labeled with 8'-azido-cGMP and reconstituted into vesicles for measurements of cGMP-dependent channel activity. On this basis, they concluded that the ROS channel was composed of 39 kDa subunits. Shinozawa et al. (1987) suggested that a 250 kDa protein constituted the cGMP-gated channel subunit on the basis of photoaffinity labeling of frog ROS membranes with 3H-cGMP and channel activity measurements in planar lipid bilayers.
Finally, Clack and Stein (1988) reported that purified opsin preparations exhibited cGMP- activated single-channel activity suggesting that the photopigment protein,rhodopsin, itself, may function as the cGMP-gated channel.
Controversy surrounding the identity of this channel subunit was initially resolved when a series of immunochemical studies established the 63 kDa polypeptide as a major subunit of the channel. This subunit is now referred to as the *-subunit. A monoclonal a ntibody which specifically labeled the 63 kDa channel subunit in both ROS membranes and purified channel preparations (Cook et al. 1989) was shown to quantitatively immunoprecipitate both cGMP- gated channel activity and a complex consisting of a 63 kDa and a 240 kDa polypeptide (Molday et al. 1990). In contrast, an antirhodopsin monoclonal antibody which selectively immunoprecipitated rhodopsin did not immunoprecipitate cGMP-dependent channel activity. More recently, a monoclonal antibody generated against a peptide correspon ding to the N- terminal segment of the 63 kDa polypeptide (Molday et al. 1991) has been used to immunoaffinity purify the channel complex from detergent solubilized ROS in a functionally active form. As shown in Figure 2a, the immunoaffinity-purified channel consists of the 63 kDa *-subunit of the channel and an associated 240 kDa polypeptide. The 240 kDa polypeptide had been previously observed by Cook et al. (1987) in some of their earlier channel preparations. The immunoaffinity purified channel complex when reconstituted into liposomes exhibits cGMP-dependent channel activity. The K1/2 of 33 *M and Hill coefficient of 3.3 for cGMP (Figure 2b) are in general agreement with the values obtained for red-dye purified channel preparationas reported by Cook et al. (1987). More recently, Brown et al. (1993) have labeled the 63 kDa*-subunit with 8-p- azidophenacylthio-cGMP, a photoaffinity derivative of cGMP, thus confirming the presence of a cGMP binding site.
A cGMP-Sepharose column has been used by Hurwitz and Holcombe (1991) as an affinity matrix to purify the channel from detergent-solubilized ROS preparations. This channel preparation was reported to consist of an 80 kDa polypeptide which was sensitive to l-cis dilitiazem. Since this polypeptide binds a monoclonal antibody against the 63 kDa channel subunit, it is likely that this 80 kDa polypeptide is related to the 63 kDa*-subunit of channel. The higher apparent molecular weight as determined by SDS gel electrophoresis may be due to anamolies in the migration of this channel subunit under different electrophoresis conditions.
Alternatively, it may be the result of the isolation of the unprocessed form of the channel subunit. The observation that the channel isolated by cGMP-Sepharose affinity chromatography is inhibited by l-cis diltiazem has been intrepreted by Hurwitz and Holcombe (1991) to indicate that l-cis diltiazem sensitivity is a property of the full length 80 kDa channelsubunit, and not the truncated 63 kDa form.
However, recent studies by Chen et al (1993) indicate that the l-cis diltiazem sensitivity of the channel as found in native ROS is not obtainedwhen the full-length 80 kDa *-subunit is expressed, by itself, but instead requires the coexpression of the * and * subunits (see below). This suggests that the channel sensitivity to l-cis d iltiazem is inherent in the *-subunit or the interaction of the * subunit with the * subunit. The differences in apparent molecular weights and l-cis dilitizem sensitivities for the different isolated channel preparations still remain to be clarified. More recently, calmodulin-Sepharose chromatography has been used to obtain a highly enriched channel preparation (Hsu and Molday, 1993). Thechannel isolated by this method was found to contain the 63 kDa and 240 kDa polypeptides as themajor components and several other calmodulin binding proteins as minor contaminants.
These studies taken together indicate that the *-subunit of apparent Mr 63 k is a major component of the native channel complex. Previous reports indicating that a39 kDa polypeptide and rhodopsin exhibit cGMP-dependent channel activity appear to be a resultof contamination of these preparations with this channel complex. An earlier report of Shinozawa et al. (1987) suggesting that the channel subunit is a 250 kDa polypeptide may be relatedto a recent finding that the channel contains a second subunit referred to as the * - subunit (see below).
Cloning and expression of the * - subunit
Direct evidence indicating that the 63 kDa polypeptide is a major subunit of the cyclic GMP-gated channel was obtained from the cloning and expression studies of Kaupp et al. (1989). Oligonucleotide probes prepared from tryptic peptide sequences of the 63 kDa polypeptide were used to screen a bovine retinal cDNA library.
A full-length cDNA clone was obtained which encoded a 79.6 kDa polypeptide containing up to six putative transmembranesegments and a cGMP binding domain. Injection of Xenopus oocytes with mRNA drived from the cloned cDNA resulted in the functional expression of a cGMP-gated channel having many of the electrophysiological properties found for the channel in ROS membranes (Kaupp et al. 1989; Kaupp, 1991). The expressed channel has been reported to be cooperatively activated by cGMP with a K1/2 of 52 *M and a Hill coefficient of 1.75, to display a cation selectivity similar to that found in ROS, and to exhibit a single channel conductance of 20 pS. The expressed channel, however, is considerably less sensitive to l-cis diltizem than the native c hannel found in ROS. The *-subunit expressed in both monkey kidney COS-1 cells and Xenopus oocytes has an apparent Mr of 78 kDa indicating that the full length polypeptide is expressed in these cells (Molday et al. 1991). Dhallan et al. (1992) have cloned and functionally expressed the cDNA for the *-subunit of the human ROS channel in embryonic kidney 293 cells.
Identification and characterization of the *-subunit
The cGMP-gated channel has been generally considered to consist of four or five subunits (Kaupp, 1991), each of which contains a cGMP binding site. This is based onthe finding that cGMP cooperatively activates the channel with a Hill coefficient of 2-3.5. The Hill coefficient gives a minimun number of cGMP molecules required for activation of the channel. Until recently, it had been assumed that the cGMP-gated channel consisted of identical *-subunits. Chen et al. (1993), however, have recently identified a second subunit (*-subunit) by screening a human cDNA library with a partial clone of the *-subunit under low stringency conditions. Sequence analysis of this *-subunit indicates that it exhibits a 30% overall sequence identity to the *-subunit and 50% identity within the cGMP binding domain.
Alternative splicing results in two transcripts which differ in size. The shorter transcript codes for a polypeptide of 623 amino acids with a calculated molecular weight of 70,843 and the longer transcript codes for a polypeptide of 909 amino acids having a molecular weight of 102,330. The two polypeptides are identical in sequence except for an extended N-terminal segment of 286 amino acids for the latter. Patch-clamp studies of human kidney 293 cells transfected with the *-subunit cDNA indicate that neither form of the *-subunits when expressed alone is functionally active (Chen et al. 1993). However, when coexpressed with the *-subunit, cGMP-gated channelactivity can be measured which more closely resembles the activity found in the ROS membranes. In particular, coexpression of the * and *-subunits results in rapid bursts of channel openings or a flickering response and *M sensitivity of the channel to l-cis diltiazem as found for the channel in ROS membranes. In contrast, the expressed *-subunit, by itself, displays a more stable opening and closing behavior and is over an order of magnitude less sensitive to l-cis diltiazem (Kaupp et al. 1989; Chen et al . 1993).
Immunocytochemical studies have confirmed that the *-subunit is present in human rod outer segments (Chen et al. 1993). An antibody raised against a peptide corresponding to the C- terminus of the *-subunit was observed to label the outer segment layer of human rod photoreceptor cells, but not cone photoreceptor cells. However, an antibodygenerated against a peptide segment near the N-terminus of the polypeptide encoded by the longer * transcript and not present in the polypeptide encoded by the shorter transcript did not label photoreceptor outer segments. On the basis of these studies, Chen et al. (1993) have suggestedthat the *-subunit in ROS is encoded by the shorter transcript. Direct studies showing that the shorter transcript is in fact the predominant form expressed in ROS, however, have not been carried out leaving some question as to whether the long or short form of *-subunit is preferentially expressed in rod photoreceptors. Other issues that need to be addressed include the detection and identification of the *-subunit in both isolated ROS membranes and isolated channel preparations and the stoichiometric relationship between the * and *-subunits.
The 240 kDa channel associated polypeptide
In addition to the 63 kDa *-subunit, cGMP-gated channel preparations from bovine ROS also contain another prominant polypeptide having an apparent Mr of 240 k by SDS gel electrophoresis under reducing conditions (Figure 2a). Several monoclonal antibodies have been generated against the 240 kDa polypeptide (Wong and Molday, 1986; Molday etal. 1990). One antibody was found to crossreact with red blood cell spectrin and brain fodrin. On the basis of this crossreactivity, its size, and its ability to bind calmodulin (see be low), it had been initially suggested that the 240 kDa polypeptide may be a member of the spectrin family of cytoskeletal proteins. However, recent rotory shadowing studies have indicated that the240 kDa polypeptide does not have the extended rod-shaped structure observed for spectrin (L. Molday and R. Molday, unpublished results).
Furthermore, initial peptide sequence studies indicate that the 240 kDa polypeptide is not related to spectrin, but instead consists of two components, one of which is the *-subunit of the channel (M. Illing, A. Williams and R. Molday, unpublished results). The finding that the *-subunit is contained within the 240 kDa polypeptide explains the reported findings that cGMP and 8-p-azidophenacylthio-cGMP photoaffinity label a high Mr component (240-250 kDa) in frog and bovine ROS and channel preparations (Shinowara etal.1987; Brown et al. 1993). On the basis of these recent studies the cGMP-gated channel appears to consist of at least two subunits (see Figure 6). The *-subunit appears to be the major functional subunit since cGMP-gated activity can be obtained by expression of this subunit alone; the *-subunit interacts with the *-subunit in rod photoreceptors to alter some of the properties ofthe channel and regulate the cGMP-dependent activity of the channel (see below). The channel complex also contains at least one additional component which together with the *-subunit constitute the 240 kDa polypeptide observed by SDS gel electrophoresis in purified channel preparations. Additional studies are needed to further identity, characterize and quantify these components and to define their role in the structure and function of the channel in rod and cone photoreceptor membranes.
Primary Structural Features of the cGMP-gated Channel Subunits
Structure of the *-subunit
The primary structure of the bovine cGMP-gated channel *-subunit was first determined by cloning and sequence analysis of its cDNA (Kaupp et al. 1989). The full-length cDNA encodes a polypeptide chain of 690 amino acids with a calculated Mr. of 79,601. A stretch of 80- 100 amino acids close to the C-terminus of the channel (Figure 3a) has beenidentified as the cGMP binding domain on the basis of its sequence similarity to the tandem cGMP binding domains of bovine lung cGMP dependent protein kinase and to other cyclic nucleotide binding proteins (Kaupp et al. 1989; Kaupp, 1991) and on the basis of site-directedmutagenesis studies (Altenhofen et al. 1991). The N-terminal region contains a hydrophilic stretch of about 60 amino acids with a lysine rich segment. Hydropathy plots suggest the existence of six relatively hydrophobic segments designated H1 - H6 which are of sufficient length to span the lipid bilayer. With the exception of H4 and H5 the hydrophobicity index of these segments is generally lower than that observed for transmembrane segment of well-characterized membraneproteins.
Accordingly, it remains to be experimentally determined if all or only someof these segments span the membrane. Five consensus sequences (Asn-X-Thr or Asn-X-Ser) for N-linked glycosylation are present at asparagine residues 90, 91, 177, 327, and 423. Lectin binding and endoglycosidase studies have confirmed that the *-subunit is glycosylated at a single site (Wohlfart et al. 1989).
The * subunit of the rod cGMP-gated channel has been cloned from severalspecies. The mouse and human sequences are about 85 % identical to bovine (Pittler et al. 1992; Dhallan et al. 1992), whereas the chicken rod subunit is 75.7% identical (BF6nigk et al. 1993). The chicken cone *-subunit has also been cloned and shown to be 65% identical to the bovine rod subunit (BF6nigk et al. 1993). The cone *-subunit has the same structural features as the rod channel and similar electrophysiological properties when expressed in Xenopus oocytes. The molecular weight of the bovine channel *-subunit calculated from thecDNA-derived protein sequence (79.6 kDa) is considerably larger than the apparent molecular weight of 63 kDa observed by SDS-PAGE.
This difference is not due solely to anomalous migration of the channel subunit on SDS gels since the channel *-subunit expressed in mammalian cells and Xenopus oocytes migrates with an apparent Mr of 78 k (Molday et al. 1991). The lower molecular weight of the channel subunit in ROS is primarily due to the absence of the first 92 amino acids as shown by N-terminal sequence analysis. This truncated form of the channel consisting of 598 amino acids. has a calculated molecular weight of 69.4 k. The difference in molecular weight of the *- subunit as determined.by SDS gel electrophoresis (63 kDa) and that calculated on the basis of the amino acid composition of the truncated channel (69.4 kDa) is attributed tothe inaccuracy of molecular weight determinations of membrane glycoproteins by SDS gel electrophoresis (Molday et al. 1991). Immunochemical labeling studies for light microscopy and electron microscopy employing a monoclonal antibody specific for the N-terminus of the 63 kDa *-subunit has confirmed that this shortened form of the channel is the major form presentin ROS and is not a result of nonspecific proteolysis during the preparation of ROS membranes.It is likely that a photoreceptor cell specific posttranslational cleavage reaction results in the truncated form of the channel subunit found in ROS, although this remains to be determined experimentally. The *- subunit of other mammalian species have also been observed to migrate on SDS gels with an apparent Mr of 63 k suggesting that truncation of the *-subunit is a general characteristic of the mammalian rod channel (Molday et al. 1991). Comparison of the rod and cone *-subunits in chicken retinal cell extracts with that of the *-subunits transiently expressed in COS-1 kidney cells indicates that a similar cleavage reaction occurs in chicken rod and cone photoreceptor cells (BF6nigk et al. 1993). The cleavage site between serine 92 and serine 93 in the bovine sequence N-N-S-S-N-K-E (N - Asn, S - Ser, K - Lys, E - Glu) is conserved in the human and mouse channel *-subunit (Figure 3b). Related, although nonidentical sequences, are found in the chicken rod and cone channel subunits; it reamins to be determined if this segment is also the site of cleavage for the chicken channel rod and cone *-subunits.
Sequence analysis of the rod and cone channel *-subunit has also revealed two structural features found in voltage-gated channels (Figure 3b, BF6ngik et al. 1993).A voltage sensor motif related to the S-4 segment of the voltage-gated shaker K+ channel is found in photoreceptor, as well as olfactory, cyclic nucleotide-gated channel subunits (Jan and Jan, 1990; Goulding et al. 1992). This motif consists of a repeating sequence of a positively chargedArg (R) or Lys (K) residue separated by two predominantly hydrophobic amino acids.
Voltage-gated channel subunits generally have seven repeating sequences, whereas the nucleotide-gated channels have four or less (Figure 3b). In voltage-gated channels this sequence has been suggested tospan the membrane bilayer. A change in the voltage across the membrane is thought to induce a conformational change in this segment which in turn affects the ion translocating properties of the channel. In the photoreceptor and olfactory cyclic nucleotide-gated channel this voltage-sensor-like motif is more limited and contains several negatively charged residues. These negatively-charged residues and/or ineffective coupling of this segment to the pore region may be responsible for the insensitivity of these cyclic nucleotide-gated channels to physiological changes in voltage (Yau and Baylor, 1989; Kaupp, 1991).
Photoreceptor and olfactory channel subunits also contain a segment which has considerable sequence similarity to the pore region of the voltage-gated K+channel (Goulding et al. 1992; Heginbotham et al. 1992; BF6nigk et al. 1993). For voltage-gatedchannels this region consists of a stretch of about 21 amino acids which is considered to extendinto the lipid bilayer as two antiparallel * strands connected by a hairpin turn (Yellen et al. 1991;Durell and Guy, 1992). Toxin binding studies and site-directed mutagenesis studies of the K+ chann el have provided support for the functional role of this segment in the translocation of ions across membranes.
Yool and Schwarz (1991) have reported that mutations in this pore region ofthe K+ alters the ion selectivity of the channel without affecting the gating properties. Heginbotham et al. (1992) have also shown that deletion of two amino acids (Tyr-Gly-) in the pore region o f the K+ channel which are absent in cyclic nucleotide-gated channels results in a loss of K+ selectivity and introduction of a divalent ion blockage of the channel as found in cGMP-gated channels. In the case of cyclic nucleotide-gated channels exchange of the pore sequence of the rod *-subunit with the pore sequence of the olfactory subunit has been shown to impart ion conducting properties of the olfactory channel onto the rod channel (Goulding et al. 1993). Site-directed mutagenesis studies have also defined the residue in the pore region which is responsible for the observed divalent cation block of conductance (Root and McKinnon, 1993). In these studies mutation of glutamic acid 363 to a glutamine residue has been shown to effectively eliminate the blockage of the channel conductance by external Mg2+ and Ca2+. These studies support the role of this segment of the channel as the primary structural domain involved in ion tra nslocation through the channel.
Structure of the *-subunit
The *-subunit of the rod channel has many of the structural features of the *-subunit (Chen et al. 1993). In particular this subunit has a modified voltage sensor-like motif and a pore region in positions similar to that of the *-subunit (figure 3b). The putative pore region of *- subunit, however, unlike the pore region of the *-subunit, does not have a negatively-charged residue. Hydropathy plots also show the presence of up to six hydrophobic segments although some of these are less hydrophobic than corresponding segments of the *-sub unit. Unlike the *- subunit, the *-subunit does not contain a consensus sequence for N-linked glycosylation suggesting that this subunit is either not glycosylated or is O-glycosylated on serine or threonine residues.
Proposed Topological Model for the Channel Subunits in the Membrane
Immunocytochemical labeling studies have led to some insight into the organization of the *-subunit of the channel in ROS membranes. Monoclonal antibodies directed against epitopes near the N-terminus and the C-terminus have been observed to label the cytoplasmic surface of the ROS plasma membrane (Cook et al. 1989; Molday et al. 1991, 1992). Thesestudies indicate that the N and C terminus of the *-subunit of the native channel are localized on the cytoplasmic side of the ROS plasma membrane and support the view that this subunit cont ains an even number of membrane spanning segments.
The identity and orientation of the glycosylation site of the *-subunit has been determined using peptide-directed antibodies (Wohlfart et al. 1992). In the truncatedform of the *-subunit as found in bovine ROS membranes, there are three potential glycosylation sites at positions 177, 327 and 423. Position 177 is within the H1 hydrophobic segment and is not considered as a likely site for glycosylation. Immunocytochemical and biochemical studies using antibodies generated against synthetic peptides encompassing glycosylation sites at 327 and 423 have confirmed that asparagine 327 contains the N-glycosylation site and is localized on the extracellular surface of the ROS plasma membrane.
On the basis of labeling studies and sequence analysis, we have proposed a working model for the organization of the *-subunit of the channel within the membrane as shown in Figure 4. In this model the N-terminal and C-terminal segments are oriented on the cytoplasmic side and the glycosylation site at Asn 327 is exposed on the extracellular surface of the ROS plasma membrane. The voltage sensor motif S4 as found in voltage-gated channels and hydrophobic segments H1 - H5 are viewed as transmembrane segments. The poreregion located between H4 and H5 is viewed as extending into the membrane in an antiparallel * conformation as suggested for the K+ channel (Durell and Guy, 1992). The cGMP binding domain is located near the C-terminus and likely interacts with the pore region to control the iontranslocation properties of the channel. This model has many general structural features which are common to voltage- gated channels and suggests that the two families of ion channels may have evolved from the same ancestral channel. Similar models have been developed independently by other groups on the basis of sequence similarities of the cyclic nucleotide-gated channels to voltage-gated channels (Goulding et al. 1992; Heginbotham et al. 1992).
The *-subunit is envisioned to have a similar topography as the *-su bunit with both a voltage sensor motif and a pore region (Chen et al. 1993). The poreregions of both the * and * subunits would likely line the central cavity of the channel and be responsible for many of the ion translocating properties of the channel. There are, however, some sequence differences in the * and *-subunits. Of particular interest is the absence of a negatively charged glutamic acid residue in the *-subunit. This may affect the binding affinity of divalentcations to the channel and alter divalent cation blockage properties found when the *-subunit alone is expressed for structure-function studies (Root and MacKinnon, 1993).
The Rod Cyclic-GMP-gated Channel is a Member of a Family of Nucleotide- gated Ion Channels
Two major classes of ion channels include the voltage-gated channels andthe ligand gated channels (Miller, 1989, 1991; Jan and Jan 1989). Voltage gated channels respond to changes in voltage across a membrane and are represented by K+ selective channels, Na+selective channels and Ca2+ selective channels. Ligand gated channels respond to the binding of extracellular ligands and are represented by such complexes as the acetylcholine receptor, GABA receptor and glycine receptor. Cyclic nucleotide-gated channels constitute another family of channels which respond to intracellular cyclic nucleotides. In addition to cGMP-gated channels ofrod and cone photoreceptor cells (Kaupp et al. 1989; Pittler et al. 1992; BF6nigk et al, 1993), the olfactory neurons contain channels which respond to cAMP as well as cGMP (Nakamura and Gold, 1987). The *-subunit of olfactory channels has been cloned from bovine (Ludwig et al. 1990), rat (Dhallan et al. 1990) and catfish (Goulding et al. 1992) olfactory neurons.They share a high degree of sequence identity (~57%) to the *-subunit of rod and cone photoreceptor channels and contain a cyclic nucleotide binding domain, a voltage sensor-like motif anda pore regeion (Figure 3a and 3b). It is likely that the olfactory channel like the rod and cone c hannels contains more than one subunit, although this remains to be determined.
Recent molecular biology and electrophysiological studies indicate that cyclic nucleotide gated channels are also present in other cells and tissues including pineal(Dryer and Henderson, 1991), heart (Biel et al. 1993), kidney (Ahmad et al. 1990; Light et al. ,1990), and bipolar cells (Nawy and Jahr, 1990). Initial cloning studies indicate that the corresponding *-subunits from different tissues show a high degree of sequence similarity to the rod, cone or olfactory channel *- subunit (Hundal et al. 1993; Ahmad et al. 1992; Biel et al. 1993). Detailed studies are needed to define the subunit composition, structure, location and role of these channels in cell function.
Interaction of Calmodulin with the cGMP-gated Channel
Binding of calmodulin to the channel
Earlier studies had indicated that ROS contain significant amounts of the calcium binding protein calmodulin (Kohnken et al. 1981), but the identity of the target proteins for calmodulin in ROS was not determined. Recently, we have shown by calmodulin affinity chr omatography and Western blotting that the cGMP-gated channel complex is a major calmodulin binding protein of ROS membranes (Hsu and Molday, 1993). When detergent solubilized ROS membrane proteins are passed through a calmodulin-Sepharose column in the presence of Ca2+ and are subsequently eluted in Ca2+-free buffer containing EDTA, the cGMP-gated channel complex consisting of the 63 kDa *-subunit and the 240 kDa protein is observed as the major componentas visualized by SDS gel electrophoresis. Western blots of the calmodulin purified and immu noaffinity-purified channel complex labeled with 125I-calmodulin indicate that calmodulin bindsto the 240 kDa polypeptide, but not the 63 kDa *-subunit.
Calmodulin modulation of cGMP-gated channel activity in ROS membranes
The effect of calmodulin on cGMP-gated channel ion flux activity was measured in ROS vesicles loaded with the Ca2+ sensitive dye Arsenazo III (Hsu and Molday, 1993). In the absence of calmodulin, the channel in ROS membrane vesicles was observed to be cooperatively activated by cGMP with a Km of 19 *M and a Hill coefficient of 3.7 for cGMP (Figure 5). In the presence of Ca2+-calmodulin the dose-response curve for cGMP was shifted to the right such that the Km increased to 33 *M, but the Hill coefficient and the maximun rate (Vmax) of ion influx essentially remained unchanged. Although this shift in Km in the presence of Ca2+-calmodulin is modest, since the cooperativity is high, calmodulin can result in a 5-6 fold decrease in channel activity at relatively low cGMP (~ 12 *M) as may be found under physiological conditions. This change in the apparent Km likely reflects an effect of Ca2+-calmodulin on the bindingaffinity of the channel for cGMP and not on ion translocation properties of the channel. This is supported by the finding that the calmodulin effect is observed for different cations and does not affect the maximum velocity of ion influx (Hsu and Molday, 1993). Caretta et al (1988) had earlier reported a similar effect of Ca2+ on the binding of fluorescent-labeled cGMP to ROS membranes.It is likely that endogeneous calmodulin was present in the ROS preparations used in this study and was responsible for this observed Ca2+ dependent decrease in cGMP-binding. Calmodulin modulation of the channel activity has been found to be dependent on Ca2+ (Hsu and Molday, 1993). In the absence of calmodulin, Ca2+ has no effect on channel activity as measured by ion flux assays. However, in the presence of calmodulin, Ca2+ suppresses the channel activity over Ca2+ concentration range of 50-300 nM. The calmodulin effectis also inhibited by excess mastoparen, a inhibitor of calmodulin ( Y.-T. Hsu and R. Molday, unp ublished results).
These studies indicate that at the level of isolated ROS membranes, Ca2+-calmodulin alters the sensitivity of the channel for cGMP. Modulation of the affinity of the channel for cGMP by Ca2+- calmodulin is analogous to modulation of the affinity of hemoglobin for oxygen by changes in pH as defined by the Bohr effect.
Recently, patch clamp studies have indicated that the sensitivity of theolfactory channel for cyclic nucleotides is also modulated by Ca2+ (Kramer and Siegelbaum, 1992). The calcium binding protein which interacts with the olfactory channel, however, has not yet been identified. It is possible that calmodulin may be involved in this modulation of the olfactory channel. A light activated channel encoded by the trp gene in Drosophila photoreceptors has also been shown to contain a calmodulin binding domain (Hardie and Minke, 1992). Its role in invertebrate phototransduction, however, has not yet been defined. It remains to be determined if the Ca2+- calmodulin modulation is a feature of many cation channels and in particular other members of the family of nucleotide-gated channels. It also remains to be determined if other endogeneous Ca2+- binding proteins interact with the channel, and if the channel is regulatedby other mechanisms such as phosphorylation as suggested in the studies of Gordon et al. (1992).
Possible Role of Ca2+-calmodulin Regulation of the Channel in Phototransduction
If Ca2+-calmodulin binds to and modulates the channel activity in intactphotoreceptors under physiological conditions as suggested by the in vitro studies, then one may envision a mechanism in which the channel switches between a low affinity and high affinity state during photoactivation and recovery (Figure 6). In the dark free cGMP concentration of 4-10 *M maintains a significant number of channels in their open state allowing forthe influx of Na+ and Ca2+ into the outer segment. The balanced efflux of Ca2+ by the Na+/Ca2+-K+ exchanger maintains Ca2+ levels at about 300 nM. Under these conditions, Ca2+-calmodulin would bind to the channel and maintain it in its low affinity state (Figure 7a). Photobleaching of rhodopsin and activation of phosphodiestease via the visual cascade system results in a decrease in cGMP and a closure of the cGMP-gated channels (Figure 7b). This will result in a lowering of intracellular Ca2+ concentration below 100 nM as Ca2+ is extruded through the Na+/Ca2+-K+exchanger.
Reduced Ca2+ concentration will cause Ca2+ to dissociate from calmodulin and cause the channel to switch from its low affinity state to its high affinity state (Figure 7b). This will enable the channel to open at lower cGMP concentrations. Reduced intracellular Ca2+ levels will also activate guanylate cyclase through a guanylate cyclase activating protein (Koch and Stryer, 1988).
These two processes, activation of guanylate cyclase and conversion of the channel from a low to a high affinity state for cGMP, will facilitate photorecovery to dark levels (Figure 7b). An increased intracellular Ca2+ concentration resulting from reopening of the channels will return guanylate cyclase to its basal level of activity and the channel to its lowaffinity state by rebinding Ca2+-calmodulin (figure 7a).
The presence of several Ca2+-binding proteins in ROS suggest that changes in Ca2+ levels during photoactivation and recovery may affect several reactions. These include regulation of guanylate cyclase activity by a putative guanylate cyclase activator protein (Koch and Stryer, 1988), modulation of the affinity of the channel for cGMP by calmodulin (Hsu and Molday, 1993) and regulation of the light activation of PDE through the effect of S-modulin (recoverin) on rhodopsin phosphorylation (Kawamura, 1993).
Summary
The cGMP-gated channel of rod and cone photoreceptor cells is a member of a family of cyclic nucleotide-gated channels found in many different cells. Primary structural analysis, site- directed mutagenesis, biochemical and immunochemical studies have indicated that the channel consists of two major subunits and one or more associated proteins. The * and * subunits have a cGMP binding domain near the C-terminus, an even number of transmembrane segments, possibly as many as six, a voltage sensor-like motif and a pore region. The latter two features are found in voltage-gated channels and suggest that the nucleotide-gated channels and the voltage-gated channels have evolved from the same ancestral channel. In rod and cone photoreceptor cells the * -subunit undergoes a post-translational cleavage reaction involving the removal of a segment at the N-terminus. The *-subunit appears to be associated with another polype ptide which together constitute a 240 kDa polypeptide observed in purified channel preparations by SDS gel electrophoresis. The basis for these modifications is not yet known. Calmodulin has also been shown to bind to the 240 kDa polypeptide and modulate the activity of the rod photoreceptor channel in a Ca2+-dependent manner in in vitro systems. This Ca2+ dependentinteraction of the channel with calmodulin alters the affinity of the channel for cGMP. Together with other Ca2+ dependent processes this Ca2+ dependent regulation of the channel is suggested to play a role in the recovery of the photoreceptor cell following photoactivation. Althoughsignificant progress has been made over the past several years on analysis of the molecular properties of cyclic nucleotide-gated channels, much remains to be understood about the molecular composition, structure, regulation and localization of these channels in various cell systems and the role of these channels in cell processes.
Acknowledgement: This work was supported by grants from NIH (EY-02422),MRC (MT 9588) and the RP Foundation of Canada.
Ahmad, I., Redmond, L.J. and Barnstable, C.J. (1990) Developmental and tissue-specific expression of rod photoreceptor cGMP-gated ion channel gene. Biochem. Biophys. Res. Commun. 173:463- 470.
Ahmad, I., Korbmacher,C., Segal, A.S., Cheung, P., Boulpaep, E.L., and Barnstable, C.J. (1992) Mouse cortical collecting duct cells show nonselective cation channel activity and express a gene related to the cGMP-gated rod photreceptor channel. Proc. Natl. Acad. Sci.U.S.A. 89: 10262- 10266.
Altenhofen, W., Ludwig, J., Eismann, E., Kraus, W., BF6nigk, W., and Kaupp, U.B. (1991) Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium. Proc. Natl. Acad. Sci. U.S.A. 88: 9868-987.
Biel, M., Altenhofen, W., Hullin, R., Ludwig, J., Freichel, M., Flocherzi, V., Dascal, N., Kaupp, U.B. and Hofmann, F. (1993) Primary structure and functional expression of a cyclic nucleotide- gated channel from rabbit aorta. FEBS Letters 329:134-138.
Bodoia, R.D. and Detwiler, P.B. (1985) Patch-clamp recordings of the light-sensitive dark noise in retinal rods from the lizard and frog. J. Physiol. 367:183-216.
BF6nigk, W., Altenhofen, W., Muller, F., Dose, A., Illing, M., Molday, R.S. and Kaupp, U.B. (1993) Rod and cone photoreceptor cells express distinct genes for cyclic GMP-gated channels. Neuron 10: 865-877.
Brown, R.L., Gerber, W.V. and Karpen, J.W. (1993) Specific labeling and permanent activation of the retinal rod cGMP-activated channel by the photaffinity analog 8-p-azidophenacylthio-cGMP. Proc. Natl. Acad. Sci. U.S.A. 90: 5369-5373.
Caretta, A., Cavaggioni, A., Grimaldi, R. and Sorbi, R. (1988) Regulation of cyclic GMP binding to retinal rod membranes by calcium. Eur. J. Biochem. 177: 139-146.
Chabre, M. and Deterre, P. (1989) Molecular mechanism of visual transduction. Eur. J. Biochem. 179: 255-266.
Chen, T.-Y., Peng, Y.-W., Dhallan, R.S., Ahamed, B., Reed, R.R., and Yau, K .-W. (1993) A new subunit of the cyclic nucleotide-gated cation channel in retinal rods. Nature 362, 764-767.
Clack, J.W. and Stein, P.J. (1988) Opsin exhibits cGMP-activated single-channel activity. Proc. Natl. Acad. Sci. U.S.A. 85, 9806-9810.
Cook, N.J., Hanke, W., and Kaupp, U.B. (1987) Identification, purification and functional reconstitution of the cyclic GMP-dependent channel from rod photoreceptors.Proc. Natl. Acad. Sci. U.S.A. 84: 585-589.
Cook, N.J., Molday, L.L., Reid, D., Kaupp, U.B. and Molday, R.S. (1989) ThecGMP-gated Channel of bovine rod photoreceptors is localized exclusively in the plasmamembrane. J. Biol. Chem. 264:6996-6999.
Dhallan, R.S., Yau, K.W., Schrader, K.A. and Reed, R.R. (1990) Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons.Nature 347: 184-187.
Dhallen, R.S., Macke, J.P., Eddy, R.L., Shows, T.B., Reed, R.R., Yau, K.W. and Nathans, J. (1992) Human rod photoreceptor cGMP-gated channel: amino acid sequence, gene structure, and functional expression. J. Neurosci. 12: 3248-3256.
Dryer, S.E. and Henderson, D. (1991) A cyclic GMP-activated channel in dissociated cells of the chick pineal gland. Nature 353: 756-758.
Durell, S.R. and Guy, R. (1992) Atomic scale structure and functional models of voltage-gated potassium channels. Biophys. J. 62: 238-250.
Fesenko, E.E., Kolesnikov, S.S., and Lyubarsky, A.L. (1985) Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segments. Nature 313: 310-313.
Furman, R.E. and Tanaka, J.C. (1990) Monovalent selectivity of the cyclic guanosine monophosphate-activated ion channel. J. Gen. Physiol. 96: 57-82.
Gordon, S.E., Brautigan, D.L.,and Zimmerman, A.L. (1992) Protein phosphatases modulate the apparent agonist affinity of the ligand-regulated ion channel of retinal rods. Neuron 8:739-748.
Goulding, E.H., Ngai, J., Kramer, R.H., Colicos, S., Axel, R., Siegelbaum, S.A., and Chess, A. (1992) Molecular cloning and single channel properties of the cyclic nucleo tide-gated channel from catfish olfactory neurons. Neuron 8:45-58.
Goulding, E.H., Tibbs,G.R., Liu, D. and Siegelbaum, S.A. (1993) Role of theH5 domain in determining pore diameter and ion permeation through cyclic nucleotide-gated channels. Nature 364: 61-64.
Hardie, R.C. and Minke, B. (1992) The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors. Neuron 8:643-651.
Haynes, L.W. and Yau, K.-W. (1985) Cyclic GMP-sensitive conductance in outer segment membrane of catfish cones. Nature 317:61-64.
Heginbotham, L., Abramson, T., and MacKinnon, R. (1992) A functional connection between the pores of distantly-related ion channels as revealed by mutant K+channels. Science 258: 1152- 1155.
Hodgkin, A.L., McNaughton, P.A., and Nunn, B.J. (1985) The ionic selectivity and calcium dependence of the light-sensitive pathway in toad rods. J. Physiol. 391: Hundal, S.P., Difrancesco, D., Mangoni, M., Grammar, W.J., and Conley, E.C.(1993) An isolform of the cGMP-gated retinal photoreceptor channel gene expressed in the sinoa trial node (pacemaker) region of rabbit heart. Biochem. Soc. Trans. 21: 119S.
Hurwitz, R. and Holcombe, V. (1991) Affinity purification of the photoreceptor cGMP-gated cation channel. J. Biol. Chem. 266: 7975-7977.
Jan, L.Y. and Jan, Y.N. (1989) Voltage-sensitive ion channels. Cell 56:13-25.
Jan, L.Y. and Jan, Y.N. (1990) A superfamily of ion channels. Nature 345: 672.
Kaupp, U.B., Niidome, T., Tanabe, T., Terada, S., BF6nigk, W., StFChmer, W., Cook, N.J., Kangawa, K., Matsuo, H., Hirose, T., Miyata, T. and Numa, S. (1989) Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gatedchannel. Nature 342, 762-766.
Kaupp, U.B. (1991) The cyclic nucleotide-gated channels of vertebrate photoreceptors and olfactory epithelium. TINS 14: 150-157.
Kaupp, U.B. and Koch, W.-H. (1992) Role of cGMP and Ca2+ in vertebrate photoreceptor excitation and adaptation. Ann. Rev. Physiol. 54: 153-175.
Kawamura, S. Rhodopsin phosphorylation as a mechanism of cyclic GMP phospho diesterase regulation by S-modulin. Nature 362: 855-857.
Koch, W.-H. and Stryer, L. (1988) Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions. Nature 334: 64-66.
Koch, K.-W. and Kaupp, U.B. (1985) Cyclic GMP directly regulates a cationicconductance in membranes of bovine rods by a cooperative mechanism. J. Biol. Chem. 260: 6788-6800.
Kohnken, R.E., Chafouleas, J.G., Eadie, D.M., Means, A.R. and McConnell, D.G. (1981) Calmodulin in bovine rod outer segments. J.Biol.Chem. 256: 12517-12522.
Kramer, R. H. and Siegelbaum, S.A. (1992) Intracellular Ca2+ regulates the sensitivity of cyclic nucleotide-gated channels in olfactory receptor neurons. Neuron 9: 897-906.
Light, D.B., Corbin, J.D. and Stanton, B.A. (1990) Dual ion-channel regulation by cyclic GMP and cyclic GMP-dependent protein kinase. Nature 344: 336-339.
Ludwig, J., Marglit, T., Eismann, E., Lancet, D. and Kaupp, U.B. (1990) Primary structure of a cAMP-gated channel from bovine olfactory epithelium. FEBS Lett. 270: 24-29.
Matesic, D. and Liebman, P.A. (1987) cGMP-dependent cation channel of retinal rod outer segments. Nature 326: 600-603.
Matthews, H.R., Murphy, R.L., Fain, G.L., and Lamb, T.D. (1988) Photoreceptor light adaptation is medicated by cytoplasmic calcium concentration. Nature 334, 67-69.
Miller, C. (1989) Genetic manipulation of ion channels: A new approach to structure and mechanism. Neuron 2: 1195-1205.
Miller, C. (1991) Annus Mirabilis of potassium channels. Science 252: 1092-1096.
Molday, L.L., Cook, N.J., Kaupp, U.B., and Molday, R.S. (1990) The cGMP-gated cation channel of bovine rod photoreceptor cells is associated with a 240 kDa protein exhibiting immunochemical cross-reactivity with spectrin. J. Biol. Chem. 265: 18690-18695.
Molday, R.S., Molday, L.L., Dose, A., Clark-Lewis, I., Illing, M., Cook, N.J., Eismann, E., and Kaupp, U.B. (1991) The cGMP-gated channel of the rod photoreceptor cell. Characterization and orientation of the amino terminus. J.Biol. Chem. 266: 2197-21922.
Molday, R.S., Reid, D.M., Connell, G. and Molday, L.L.) Molecular properties of the cGMP-gated channel of rod photoreceptor cells as probed with monoclonal antibodies. In: Signal Transduction in Photoreceptor Cells. (eds. P.A. Hargrave, K.P. Hofmann, U.B. Kaupp) Springer-Verlag. 180- 194.
Nawy, S. and Jahr, C.E. (1990) Suppression by glutamate of cGMP-activated conductance in retinal bipolar cells. Nature 346: 269-271.
Picones, A. and Korenbrot, J.I. (1992) Permeation and interaction of monovalent cations with the cGMP-gated channel of cone photreceptors. J. Gen. Physiol. 100: 647-673.
Pittler, S.J., Lee, A.K., Altherr, M.R., Howard, T.A., Seldin, M.F., Hurwitz, R.L., Wasmuth, J.J., and Baehr, W. (1992) Primary structure and chromosomal localization of human and mouse rod photoreceptor cGMP-gated cation channel. J.Biol. Chem. 267: 6257-6262.
Root, M.J. and MacKinnon, R. (1993) Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel. Neuron 11, 459-466.
Schnetkamp, P.P.M. (1990) Cation selectivity of and cation binding to the cGMP-dependent channel in bovine rod outer segment membranes. J. Gen. Physiol. 96:7-534.
Shinozawa, T., Sokabe, M., Terada, S., Matsusaka, H. and Yoshizawa, T. (1987). Detection of cyclic GMP binding protein and ion channel activity in frog rod outer segments. J. Biochem. 102: 281-290.
Stern, J.H., Kaupp, U.B. and MacLeish, P.R. (1986) control of the light-regulated current in rod photoreceptors by cyclic GMP, calcium and L-cis-diltiazem. Proc. Natl. Acad . Sci. U.S.A. 83: 1167.
Stryer, L. (1986) Cyclic GMP cascade of vision. Ann. Rev. Neurosci. 9: 87-119.
Stryer, L. (1991) Visual excitation and recovery. J. Biol. Chem. 266, 10711-10714.
Watanabe, S.I. and Murakami, M. (1991) Similar properties of the cGMP-activated channels between cones and rods in the carp retina. Visual Neuroscience 6: 563-568.
Wohlfart, P., Muller, H., and Cook, N.J. (1989) Lectin binding and enzymatic deglycosylation of cGMP-gated channel from bovine rod photoreceptors. J. Biol. Chem. 264: 20934-20939.
Wohlfart, P., Haase, H., Molday, R.S. and Cook, N.J. (1992) Antibodies against synthetic peptides used to determine the topology and site of glycosylation of the cGMP-gated channel from bovine rod photoreceptors. J. Biol. Chem. 267, 644-648.
Wong, S. and Molday, R.S. (1986) A spectrin-like protein in retinal rod outer segments. Biochemistry 25, 6294-6300.
Yau, K.-W and Nakatani, K. (1984a) Electrogenic Na-Ca exchange in retinal rod outer segment. Nature 311: 661-663.
Yau, K.-W. and Nakatani, K. (1984b) Cation selectivity of light-sensitive conductance in retinal rods. Nature 309: 352-354.
Yau, K.-W. and Nakatani, K. (1985a) Light-suppressible, cyclic GMP-sensitive conductance in the plasma membrane of a truncated rod outer segment. Nature 317: 252-255.
Yau, K.-W. and Nakatani, K. (1985b) Light-induced reduction of cytoplasmic free calcium in retinal rod outer segment. Nature 313: 579-582.
Yau, K.-W. and Baylor, D.A. (1989) Cyclic GMP-activated conductance of retinal photoreceptor cells. Ann. Rev. Neurosci. 12: 289-327.
Yellen, G., Jurman, M.E., Abramson, T., MacKinnon, R. (1991) Mutations affecting internal TEA blockage identify the probable pore-forming region of K+ channels. Science 251:939-942.
Yool, A.J. and Schwartz, T.L. (1991) Alteration of ionic selectivity of a K+ channel by mutation of the H5 region. Nature 349:700-704.
Zimmerman, A.L. and Baylor, D.A. (1986) Cyclic GMP-sensitive conductance ofretinal rods consists of aqueous pores. Nature 321: 70-72.
Zimmmerman, A.L., Yamanaka, G., Eckstein, F., Baylor, D.A., and Stryer, L. (1985) Interaction of hydrolysis-resistant analogs of cyclic GMP with the phosphodiesterase and l ight-sensitive channel of retinal rod outer segments. Proc. Natl. Acad. Sci. U.S.A. 82: 8813-17.
Figure Legends: [figures only available in paper version]
Figure 1. A diagram showing the basic reactions of the visual transductionpathway. In the dark, elevated cGMP concentrations maintain a relevant number of cGMP-gated channels in their open state. This allows the influx of Na+ and Ca2+ into the outer segment and maintains the photoreceptor cell in a partially depolarized state. Photobleaching of rhodopsin, involving the photoisomerization of 11-cis retinal to its all-trans isomer, results in the formation of activated (meta II) rhodopsin which catalyzes the exchange of bound GDP for GTP on transducin. Transducin will in turn activate phosphodiesterase which catalyzes the hydrolysis of cGMP to 5'-GMP. The decrease in free cGMP concentration will cause the channel to close as cGMP to dissociates from the channel. Closure of the channel to the influx of Na+ and Ca2+ will result in a transient hyperpolar ization of the cell. The recovery of the rod outer segment to its dark state occurs through 1) the inactivation of rhodopsin by a rhodopsin kinase (RK) catalyzed phosphorylation reaction and the binding of arrestin (Ar); 2) inactivation of transducin by hydrolysis of bound GTP to GDP; 3) inhibition of phosphodiesterase by the rebinding of the inhibitory subunits to the catalytic subunits of phosphodiesterase; 4) resynthesis of cGMP from GTP by guanylate cyclase (GC); and 5) the reopening of the cGMP-gated channel as cGMP levels increase.
Figure 2. Immunoaffinity purification and functional reconstitution of the cGMP-gated channel complex of bovine rod outer segments (ROS). a) SDS gels of ROS membranes (lane a) and the immunoaffinity-purified channel (lane b) stained with Coomassie Blue (CB) or transferred to Immobilon membranes and labeled with monoclonal antibodies PMs 5E11 against the 240 kDa channel protein and PMc 1D1 against the *-subunit of the channel. b) cGMP-dependent Ca2+ efflux from liposomes reconstituted with the immunoaffinity purified channel. The sigmoidal curve was calculated using a Km of 33 *M and a Hill coefficient of 3.3 for cGMP. For these studies, CHAPS detergent-solubilized ROS membranes were added to a PMc 6E7 anti-channel monoclonal antibody-Sepharose column and eluted with a N-terminal peptide corresponding to the epitope for this antibody (Molday et al., 1991). The purified channel complex was reconstituted into lipid vesicles by the procedure of Cook et al. (1987).
Figure 3. Sequence alignment of various structural domains of cyclic nucleotide-gated channels from different species. a) Alignment of the potential cyclic nucleotide binding sites of the bovine rod *-subunit (Kaupp et al. 1989), human and mouse rod *-subunit (Pittler et al. 1992; the chicken rod and cone *-subunits (BF6ngik et al. 1993), the bovine olfactory channel subunit (Ludwig et al. 1990), and the rod *-subunit (Chen et al. 1993). A high degree of sequence identity within the nucleotide binding domain is observed. b) Sequence alignment of the voltage sensor-like motifs (S4), pore regions, and putative N- terminal cleavage sites.
Figure 4. Working model for the organization of the *-subunit of bovine rod cGMP-gated channel within the lipid bilayer. The first 92 amino acids (dashed line) as predicted from cDNA sequence analysis are absent in the channel in ROS membranes possibly by a post- translational cleavage reaction. Segments labeled H1 - H5 and the S4 voltage sensor-like motif are considered as transmembrane segments and the segment between H4 and H5 is the pore region. Immunogold labeling studies have established the N and C terminus on the cytoplasmic side (Cook et al. 1989; Molday et al. 1991) and asparagine Asn-327 containing a N-linked oligosaccharide chain on the extracellular side (Wohlfart et al. 1992). The cGMP binding domain is located near the C-terminus. The *-subunit is suggested to have a similar topographical organization.
Figure 5. The effect of calmodulin on the activation of the rod channel by cGMP. Calcium influx assays were carried out using ROS membrane vesicles containing Arsenazo III dye either in the absence (*) or presence (*) of calmodulin. Calmodulin is shown to increase the Km of the channel for cGMP from 19 *M to 33 *M without affecting its maximum velocity or cooperativity for cGMP. The solid lines were drawn from a sigmoidal binding isotherm using the indicated Km and a Hill coefficient of 3.6 (Hsu and Molday, 1993).
Figure 6. Schematic model depicting the interaction of calmodulin with the channel complex. In the presence of high intracellular Ca2+ (~300 nM) as found in dark adapted rod photoreceptors, Ca2+-calmodulin binds to the 240 kDa protein of the channel complex and maintains the channel in a low affinity state (high K') for cGMP. Under conditions of low intracellular Ca2+ (<100 nM) as found after photoexcitation, Ca2+ and possibly calmodulin dissociate from the channel complex and cause the channel to switch to its high affinity state (low K) for cGMP. Calmodulin binds to either the *-subunit of the channel or an unidentified polypeptide(s) (X) which make up the 240 kDa polypeptide. In this model, the channel is viewed as a complex consist ing of three *-subunits (one subunit is omitted for simplicity) and two *-subunits. The actual number of subunits and associated polypeptides, however, remains to be determined.
Figure 7. Possible role for calmodulin modulation of the channel during the visual transduction process. a) In the dark, an elevated level of cGMP maintains a significant number ofcGMP-gated channels in their open state and allows the influx of Na+ and Ca2+ into the outer se gment. The balanced influx of Ca2+ through the channel with the efflux of Ca2+ through the Na+/Ca2+-K+ exchanger results in a relatively high level of Ca2+ (~300 nM) within the outer segment. Under these conditions calmodulin associates with the channel complex and maintains thechannel in its low affinity state for cGMP. The channel in its low affinity state will respond to a decrease in the level of cGMP during photoexcitation of the outer segment. b) Photobleaching of rhodopsin (Rho*) results in the activation of phosphodiesterase (PDE) and a decrease in the level of cGMP *. This causes the closure of the cGMP-gated channel * and a decrease in intracellular Ca2+ * due to the continuous extrusion of Ca2+ by Na+/Ca2+-K+ exchanger. This drop in Ca2+ will cause calmodulin to dissociate from the channel complex and shift the channel from its low affinity state to its high affinity state for cGMP. The low level of Ca2+ will also activate guanylate cyclase through a Ca2+-dependent guanylate cyclase activating protein. The combined effect of resynthesizing cGMP * and making the channel more sensitive to cGMP levels would facilitate the recovery of the outer segment to its dark level. As the channels reopen, the Ca2+ level in the outer segment is restored *. This results in the rebinding of calmodulin to the channel and conversion of the channel to its low affinity state. Guanylate cyclase is also restored to its basal level of activity.