Calcium/Calmodulin Modulation of Olfactory and Rod Cyclic Nucleotide-gated Ion Channels*

Cyclic nucleotide-gated (CNG) ion channels mediate sensory transduction in olfactory sensory neurons and retinal photoreceptor cells. In these systems, internal calcium/calmodulin (Ca 2 (cid:1) /CaM) inhibits CNG channels, thereby having a putative role in sensory adaptation. Functional differences in Ca 2 (cid:1) /CaM-dependent inhibition depend on the different subunit composition of olfactory and rod CNG channels. Recent evidence shows that three subunit types (CNGA2, CNGA4, and CNGB1b) make up native olfactory CNG channels and account for the fast inhibition of native channels by Ca 2 (cid:1) /CaM. In contrast, two subunit types (CNGA1 and CNGB1) appear sufficient to mirror the native properties of rod CNG channels, including the inhibition by Ca 2 (cid:1) /CaM. Within CNG channel tetramers, specific subunit interactions also mediate Ca 2 (cid:1) /CaM-dependent inhibition. In olfactory CNGA2 channels, Ca 2 (cid:1) /CaM binds to an N-terminal region and disrupts an interaction between the N- and C-terminal regions, causing inhibition. Ca 2 (cid:1) /CaM also binds the N-terminal region of CNGB1 subunits and disrupts an intersubunit, N- and C-terminal interaction between CNGB1 and CNGA1 subunits in rod channels. However, the precise N- and C-terminal regions that form these interactions in olfactory channels are different from those in rod channels. Here, we will review recent advances in understanding the subunit composition and the mechanisms and roles for Ca 2 (cid:1)

creases the cytosolic concentration of cAMP, thus opening previously closed CNG channels and causing membrane depolarization (Fig. 1). In rod photoreceptor cells, light activates rhodopsin, which begins a cascade that decreases the cytosolic concentration of cGMP, resulting in closure of previously open CNG channels and membrane hyperpolarization (Fig. 1). Thus, odorants in the olfactory system and light in the visual system produce opposite effects on membrane voltage. For both systems, Ca 2ϩ ions feed back to down-regulate the enzymatic cascade (Fig. 1) (2,7,8). In the olfactory system Ca 2ϩ ions bind to calmodulin (CaM), and the Ca 2ϩ /CaM complex directly inhibits olfactory CNG channels ( Fig. 1) (9), constituting a major mechanism underlying olfactory adaptation (7,10,11). In the visual system, Ca 2ϩ ions interact with several members of the phototransduction cascade to cause negative feedback and visual adaptation (8,12). Ca 2ϩ /CaM inhibits native rod CNG channels (13,14); however, the role for Ca 2ϩ /CaM in visual adaptation is apparently not as large as in olfactory adaptation (15). Ca 2ϩ /CaM also inhibits CNG channels in retinal cones (16,17); however, this topic is outside of our present scope. We will focus on recent, intriguing similarities and differences in the molecular mechanisms underlying Ca 2ϩ /CaM-dependent inhibition in olfactory and rod CNG channels.
Native olfactory and rod CNG channels are inhibited by nanomolar levels of CaM in a Ca 2ϩ -and time-dependent manner (9,13,14). For both channels Ca 2ϩ /CaM decreases the apparent affinity for cyclic nucleotide, i.e. more cyclic nucleotide is required to open the same number of Ca 2ϩ /CaM-bound channels than for unbound channels. One way for this to occur is if Ca 2ϩ /CaM destabilizes the opening allosteric transition, as proposed for olfactory channels (9). Quantitatively, however, inhibition is different between the channel types. Ca 2ϩ /CaM decreases the apparent affinity of olfactory channels for cyclic nucleotide about 10-fold (9), whereas that for rod channels decreases about 2-fold (13,14). A mechanistic explanation for this difference will be discussed below.

Physiological Role for Ca 2؉ /CaM Modulation of CNG Channels
Olfactory adaptation, a decrease in the electrical response of the cell to repeated application of odorants, depends on the concentration of internal Ca 2ϩ ions. The time courses of adaptation to either pulses of odorant or to photolysis of caged cAMP are the same, suggesting that adaptation works though olfactory CNG channels (10). Moreover, adapted channels and Ca 2ϩ /CaM-inhibited channels have a similar apparent affinity for cAMP, suggesting that odorant adaptation is due to Ca 2ϩ /CaM-dependent inhibition of CNG channels (Fig. 1). Long term olfactory adaptation may work though a different pathway (19,20).
Unlike the case for olfactory neurons, the physiological role for Ca 2ϩ /CaM modulation of CNG channels in rod photoreceptors is not well established. In theory, the interaction of Ca 2ϩ /CaM with CNG channels is sufficient to form a negative feedback loop in native rods (13). In the dark, in high levels of Ca 2ϩ and cGMP, Ca 2ϩ /CaM-bound channels would be inhibited, thereby having a lower apparent affinity for cGMP. In the light, Ca 2ϩ and cGMP levels drop, Ca 2ϩ /CaM would not be bound, and channels would exhibit a higher apparent affinity for cGMP. Through Ca 2ϩ /CaM, rod channels would be perfectly tuned to respond to changes in the cGMP concentration in different levels of light, thereby extending the range of the photoresponse and aiding in visual adaptation. However, the significance of such a mechanism in native cells has been questioned because Ca 2ϩ / CaM alters the apparent affinity of rod channels by about 2-fold, which is a relatively small change compared with the 10,000-fold range in intensity over which visual adaptation occurs (15,21,22). Also, in a computed model of the response-intensity relation in rods the contribution of direct Ca 2ϩ /CaM inhibition of CNG channels in light adaptation was minimal (15,21). One major target for Ca 2ϩ -dependent adaptation in rods is guanylate cyclase-activating protein. Its down-regulation by Ca 2ϩ reduces the activity of guanylate cyclase, which reduces the rate of formation of cGMP and in turn closes CNG channels (8,12,15,23). A second major target for Ca 2ϩ ion is the Ca 2ϩ -binding protein recoverin; its activation inhibits the rhodopsin kinase, which keeps light-activated rhodopsin and ultimately phosphodiesterase active, and that decreases the cGMP concentration and leads to CNG channel closure (15,24).

Cloned CNG Channel Subunits
Advances in understanding the molecular mechanism(s) of Ca 2ϩ / CaM inhibition have been made by studies of cloned CNG channels. Currently, six types of mammalian CNG channel subunits are divided into two classes; the CNGA class contains CNGA1, CNGA2, CNGA3, and CNGA4 subunits, and the CNGB class contains the CNGB1 and CNGB3 subunits. CNGB also contains CNGB1b, an olfactory-specific splice variant of CNGB1. There is no clone designated CNGB2 (25). Channel subunits are 35-75% similar, and all have the same proposed transmembrane arrangement with intracellular N-and C-terminal regions and six transmembrane domains (Fig. 2). Four subunits coassemble to form a tetrameric channel with a central pore region (26,27).

Subunit Composition of Olfactory and Rod CNG Channels
Functional inhibition of CNG channels by Ca 2ϩ /CaM depends on the subunit composition of CNG channels. CNG channels in olfactory sensory neurons are formed by three different channel subunits, CNGA2, CNGA4, and CNGB1b (Fig. 2). These three subunits are all present in olfactory neurons as determined by molecular and biochemical studies (28,29). All three subunits are necessary to form channels that reproduce key functional properties of native olfactory channels, including the apparent affinities for cGMP and cAMP, the single channel kinetics, the presence of substrates at the single-channel level, and the fast kinetics of Ca 2ϩ /CaM inhibition (Table I) (11, 28 -30). Expressed alone, CNGA2 subunits form functional homomeric CNG channels but lack some properties of native channels (Table I). CNGA2 homomers are inhibited by Ca 2ϩ /CaM but with slow, non-native kinetics (30,31). The CNGA4 and CNGB1b subunits do not form functional homomeric channels when expressed alone but rather form heteromeric channels (with CNGA2) and are thus considered modulatory subunits (28,29,32).
The role of individual olfactory CNG channel subunits in adaptation has recently become clearer by studies with a mouse containing an engineered deletion of the CNGA4 subunit (11). Olfactory cells from these mutant mice do not adapt to repeated application of odorants, unlike cells from wild-type mice. In addition, the cells from CNGA4-deficient mice have 200-fold slower kinetics of modulation by Ca 2ϩ /CaM than do wild-type mice. In a complementary study, CNGA2/CNGA4/CNGB1b channels exhibit native-like kinetics of Ca 2ϩ /CaM inhibition, whereas CNGA2/CNGB1b channels have much slower kinetics, similar to those of the CNGA4 knock-out mouse (30). Although it does not bind Ca 2ϩ /CaM directly, the CNGA4 subunit allows for state-independent association of Ca 2ϩ /CaM with the CNGA2/CNGA4/CNGB1b channel complex. The on-rate for Ca 2ϩ /CaM does not decrease as the channel open probability (P o ) changes from 0 to 1 in CNGA2/CNGA4/CNGB1b channels whereas the on-rate decreases Ͼ10-fold in homomeric CNGA2 channels over the same change in P o . This allows channels with a high open probability to be inhibited by Ca 2ϩ /CaM, a property that enables negative feedback by Ca 2ϩ /CaM in a system where the opening of channels controls membrane depolarization (11,30). Consistent with these findings, the behaving CNGA4-deficient mouse has impaired odor detection in the presence of an adapting odor (33).
The native retinal rod CNG channel is formed exclusively by co-assembly of CNGA1 and CNGB1 subunits into heteromeric channels (Fig. 2). Molecular and biochemical evidence shows the presence and interaction of these two proteins in rod cells (34 -36). In functional expression studies, CNGA1/CNGB1 heteromers have properties similar to those of native channels, including similar Minireview: Cyclic Nucleotide-gated Ion Channels 18706 apparent affinity for cGMP and cAMP, sensitivity to L-cis-diltiazem, fast single channel gating, and inhibition by Ca 2ϩ /CaM (Table I) (34 -37). Heteromeric rod channels contain three CNGA1 subunits and one CNGB1 subunit (Fig. 2) (38 -41). CNGA1 subunits form functional homomeric CNG channels in heterologous systems but lack several features of native rod channels (Table I) (37,42). CNGB1 subunits are considered modulatory subunits as they do not form functional channels in heterologous expression systems but co-assemble with CNGA1 to form channels with native-like properties, including inhibition by Ca 2ϩ /CaM. Thus, although both are tetrameric, olfactory and rod channels differ in the type (CNGA2, CNGA4, and CNGB1b versus CNGA1 and CNGB1) and number (3 versus 2, respectively) of component subunits. Further, the olfactory CNGA2 subunit contains sufficient machinery for an elementary form of Ca 2ϩ /CaM modulation, whereas the rod CNGA1 subunit requires the CNGB1 subunit for Ca 2ϩ /CaM modulation (Table I). Although not reviewed here, cone CNG channels are likely formed by CNGA3 and CNGB3 subunits (43,44).

Molecular Mechanisms of Ca 2؉ /CaM Inhibition
The mechanisms that underlie Ca 2ϩ /CaM inhibition of olfactory and rod channels are broadly similar. For both channels, Ca 2ϩ / CaM binds to an N-terminal region of the channel and disrupts an interaction between this region and a C-terminal region, causing inhibition. Upon comparison, however, the precise molecular mechanisms of inhibition are quite different and we review those here.

Ca 2؉ /CaM-binding Sites in the N-terminal Regions of CNG
Channel Subunits Olfactory CNGA2 subunits contain a site in their N-terminal region ( 68 FQRIVRLVGVIRDW 81 ) that is necessary and sufficient to bind to Ca 2ϩ /CaM (9). Deletion of this site results in CNGA2 channels that are insensitive to Ca 2ϩ /CaM, suggesting a critical role for this site in functional inhibition (9,45). The CaM-binding region in CNGA2 is an archetypal "1-8-14" site, characterized by hydrophobic residues at positions 1 and 14 and long chain aliphatic residues at position 8 (as underlined) (46). Rod CNGA1 subunits do not contain a Ca 2ϩ /CaM-binding site; however, CNGB1 subunits have an N-terminal site ( 682 LQELVKLFKERTEKVKEKLI 701 ) that is necessary for Ca 2ϩ / CaM binding (47)(48)(49). This site is critical for functional inhibition as its deletion in CNGB1 subunits results in heteromeric channels (after co-expression with CNGA1) that are insensitive to Ca 2ϩ /CaM (47)(48)(49). Several key residues (underlined) are similar to those in the IQ type of CaM binding motifs (IQXXXRGXXXRXX(F/W)); however, the CNGB1 region is "unconventional" as it lacks the central glycine, and the final hydrophobic residue is not amphipathic and requires Ca 2ϩ for CaM binding, unlike IQ motifs (46). CNGB1 also contains a C-terminal region that binds to Ca 2ϩ /CaM in biochemical assays, but the functional significance of this site is unclear, because when deleted, channels retain wild-type Ca 2ϩ /CaM dependence (47,48).

Differential Role in Gating of the N-terminal Region in Olfactory and Rod Channels
In addition to binding to Ca 2ϩ /CaM, the 1-8-14 site from the N-terminal region of CNGA2 has an autoexcitatory effect on channel gating. When this site is deleted, the apparent affinity for cyclic nucleotides and the fractional activation by cAMP in CNGA2 homomers decreases about 10-fold (9,45). In addition, transplanting this region to CNGA1 subunits increases the apparent affinity, which can be completely explained by an autoexcitatory effect of the Nterminal region on the final opening transition (50,51). This effect is closely linked with the Ca 2ϩ /CaM binding ability of this region; mutations that disrupt binding also disrupt autoexcitatory properties (52).
In CNGA1/CNGB1 channels, however, the Ca 2ϩ /CaM site from CNGB1 does not appear to have an autoexcitatory role in channel gating. Deletion of this site from CNGB1 subunits yields channels (after co-expression with CNGA1) that have the same apparent affinity for cGMP and fractional activation by cAMP as found in wildtype channels (49). This suggests a fundamentally different mechanism for Ca 2ϩ /CaM-dependent inhibition in olfactory and rod channels.

N-and C-terminal Interactions in CNG Channels
In CNGA2 channels the N-terminal region forms an interaction with the C-terminal region (45). Specifically, the 1-8-14 site is necessary to form an interaction with the C-linker region (which connects the S6 to the CNBD) and the CNBD (Fig. 3). This interdomain interaction may promote channel opening by helping to stabilize conformations of the C-linker and CNBD that are permissive of the open conformation of the channel.
The cytoplasmic N-and C-terminal regions also form an interaction in rod CNGA1/CNGB1 channels (49,53). The Ca 2ϩ /CaM-binding site in the N-terminal region of CNGB1 is necessary to form an interaction with a short C-terminal region of CNGA1 that is distal to the CNBD. The C-linker and CNBD of CNGA1 are not involved in the interaction, unlike the case in olfactory channels (Fig. 3). Thus, the N-and C-terminal regions that interact in olfactory versus rod channels are fundamentally different on the molecular level (Fig. 3).

Ca 2؉ /CaM Disrupts Interdomain Interactions in CNG Channels
Ca 2ϩ /CaM disrupts the N-and C-terminal interactions in both olfactory CNGA2 and rod CNGA1/CNGB1 channels (45,49). This result suggests a mechanism for inhibition in both channels; Ca 2ϩ / CaM binds to the CaM-binding site in the N-terminal region and disrupts the interaction with the C-terminal region. For CNGA2 channels, Ca 2ϩ /CaM disruption removes an autoexcitatory interaction between the N-and C-terminal regions, which accounts for inhibition. In CNGA1/CNGB1 channels, the N-terminal region of CNGB1 does not have an autoexcitatory effect, suggesting a different mechanism of inhibition. Ca 2ϩ /CaM may directly inhibit CNGA1/CNGB1 channels.

Ca 2؉ /CaM Modulation of Cone CNG Channels and
Other Ion Channels Like native olfactory and rod CNG channels, native cone CNG channels are inhibited by internal Ca 2ϩ (17,54). Although exogenous Ca 2ϩ /CaM inhibits native cone channels, Ca 2ϩ appears to operate through an unidentified factor that diffuses from channels even at elevated Ca 2ϩ levels, arguing against a physiological role for CaM (17,54). Ca 2ϩ /CaM does bind to cloned cone (CNGA3) channels from  (56). Identification of a cone modulatory subunit (CNGB3) (44) may help sort out a role for Ca 2ϩ /CaM in heteromeric CNGA3/CNGB3 channels. Ca 2ϩ /CaM also regulates several other ion channels via mechanisms distinct from those in olfactory and rod CNG channels (57). The SK class of Ca 2ϩ -activated K ϩ channels is activated by Ca 2ϩ binding to a pre-existing CaM channel complex (58,59). L-type Ca 2ϩ channels bind Ca 2ϩ -free CaM and inactivate upon binding Ca 2ϩ ion (60). N-Methyl-D-aspartate receptors inactivate when Ca 2ϩ /CaM binds and displaces a C-terminal region that interacts with the cytoskeleton (61).

Conclusions
Several recent studies in olfactory and rod CNG channels highlight intriguing similarities and differences in the mechanisms underlying Ca 2ϩ /CaM inhibition. Many interesting questions and implications continue to arise from these studies. In olfactory CNG channels much of the original investigations into mechanism focused on CNGA2 homomeric channels. As native channels have now been shown to contain three different subunit types (CNGA2/ CNGA4/CNGB1b), two of which have Ca 2ϩ /CaM-binding sites (CNGA2 and CNGB1b), it will be of interest to see whether the CNGB1b site also plays a role in the fast Ca 2ϩ /CaM-dependent modulation of heteromeric olfactory channels.
The unusual subunit stoichiometry of rod CNG channels (three CNGA1 and one CNGB1 subunit) suggests that CNGB1 subunits have a special impact on Ca 2ϩ /CaM-dependent modulation. Because CNGB1 is the only subunit in the tetramer that binds Ca 2ϩ /CaM, rod channels may be inhibited by a single Ca 2ϩ /CaM molecule.