Stoichiometry and arrangement of subunits in rod cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) ion channels mediate the response to light in retinal rods. They are tetramers of two homologous subunits (alpha and beta), each of which is essential for the function of the channels in vivo. We have investigated the stoichiometry and arrangement of these two subunits to determine how they come together within an individual channel complex. We exploited the very specific geometric and spatial requirements for forming a high-affinity Ni2+-binding site to examine the number and relative positions of the subunits. We found that only an order of alpha/alpha/beta/beta could account qualitatively and quantitatively for the observed intersubunit coordination of Ni2+ in wild-type and mutant alpha/beta channels. Furthermore, our results suggest a structural dimerization among like subunits, at least at the level of the Ni2+-binding site.     

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 (Ca2+/CaM) inhibits CNG channels, thereby having a putative role in sensory adaptation. Functional differences in Ca2+/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 Ca2+/CaM. In contrast, two subunit types (CNGA1 and CNGB1) appear sufficient to mirror the native properties of rod CNG channels, including the inhibition by Ca2+/CaM. Within CNG channel tetramers, specific subunit interactions also mediate Ca2+/CaM-dependent inhibition. In olfactory CNGA2 channels, Ca2+/CaM binds to an N-terminal region and disrupts an interaction between the N- and C-terminal regions, causing inhibition. Ca2+/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 Ca2+/CaM-dependent inhibition in olfactory and rod CNG channels.     

Structural basis for ligand selectivity of heteromeric olfactory cyclic nucleotide-gated channels

In vertebrate olfactory receptors, cAMP produced by odorants opens cyclic nucleotide-gated (CNG) channels, which allow Ca(2+) entry and depolarization of the cell. These CNG channels are composed of alpha subunits and at least two types of beta subunits that are required for increased cAMP selectivity. We studied the molecular basis for the altered cAMP selectivity produced by one of the beta subunits (CNG5, CNCalpha4, OCNC2) using cloned rat olfactory CNG channels expressed in Xenopus oocytes. Compared with alpha subunit homomultimers (alpha channels), channels composed of alpha and beta subunits (alpha+beta channels) were half-activated (K(1/2)) by eightfold less cAMP and fivefold less cIMP, but similar concentrations of cGMP. The K(1/2) values for heteromultimers of the alpha subunit and a chimeric beta subunit with the alpha subunit cyclic nucleotide-binding region (CNBR) (alpha+beta-CNBRalpha channels) were restored to near the values for alpha channels. Furthermore, a single residue in the CNBR could account for the altered ligand selectivity. Mutation of the methionine residue at position 475 in the beta subunit to a glutamic acid as in the alpha subunit (beta-M475E) reverted the K(1/2,cAMP)/K(1/2,cGMP) and K(1/2, cIMP)/K(1/2,cGMP) ratios of alpha+beta-M475E channels to be very similar to those of alpha channels. In addition, comparison of alpha+beta-CNBRalpha channels with alpha+beta-M475E channels suggests that the CNBR of the beta subunit contains amino acid differences at positions other than 475 that produce an increase in the apparent affinity for each ligand. Like the wild-type beta subunit, the chimeric beta/alpha subunits conferred a shallow slope to the dose-response curves, increased voltage dependence, and caused desensitization. In addition, as for alpha+beta channels, block of alpha+betaCNBRalpha channels by internal Mg(2+) was not steeply voltage-dependent (zdelta approximately 1e(-)) as compared to block of alpha channels (zdelta 2.7e(-)). Thus, the ligand-independent effects localize outside of the CNBR. We propose a molecular model to explain how the beta subunit alters ligand selectivity of the heteromeric channels.     

Regulation of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels are found in several cell types, and are best studied in photoreceptors and olfactory sensory neurons. There, CNG channels are gated by the second messengers of the visual and olfactory signalling cascades, cGMP and cAMP respectively, and operate as transduction channels generating the stimulus-induced receptor potentials. In visual and olfactory sensory cells CNG channels conduct cationic currents. Calcium can contribute a large fraction of this current, and calcium influx serves a modulatory role in CNG-channel mediated signal transduction. There have been recent developments in our understanding of how the regulation of CNG channels contributes to the physiological properties of photoreceptors and olfactory sensory cells, and in particular on the role of calcium-mediated feedback.     

Presynaptic cyclic nucleotide-gated ion channels modulate neurotransmission in the mammalian olfactory bulb

Cyclic nucleotide-gated channels (CNGCs) on the dendritic cilia of olfactory receptor neurons (ORNs) are critical for sensory transduction in the olfactory system. Do CNGCs also play a role in the axons and/or nerve terminals of ORNs? We find that the cyclic nucleotides cAMP and cGMP can both facilitate and depress synaptic transmission between olfactory nerve fibers and their targets in olfactory bulb glomeruli. Cyclic nucleotides increase intracellular Ca(2+) in ORN terminals and enhance spontaneous transmitter release; at higher concentrations, cyclic nucleotides depress evoked transmission by altering olfactory nerve excitability. Cyclic nucleotides have no effect on transmission or nerve excitability, however, in mice lacking olfactory CNGCs. Taken together, our results identify a novel role for presynaptic CNGCs in modulating neurotransmission.     

Odorant inhibition of the olfactory cyclic nucleotide-gated channel with a native molecular assembly

Human olfaction comprises the opposing actions of excitation and inhibition triggered by odorant molecules. In olfactory receptor neurons, odorant molecules not only trigger a G-protein-coupled signaling cascade but also generate various mechanisms to fine tune the odorant-induced current, including a low-selective odorant inhibition of the olfactory signal. This wide-range olfactory inhibition has been suggested to be at the level of ion channels, but definitive evidence is not available. Here, we report that the cyclic nucleotide-gated (CNG) cation channel, which is a key element that converts odorant stimuli into electrical signals, is inhibited by structurally unrelated odorants, consistent with the expression of wide-range olfactory inhibition. Interestingly, the inhibitory effect was small in the homo-oligomeric CNG channel composed only of the principal channel subunit, CNGA2, but became larger in channels consisting of multiple types of subunits. However, even in the channel containing all native subunits, the potency of the suppression on the cloned CNG channel appeared to be smaller than that previously shown in native olfactory neurons. Nonetheless, our results further showed that odorant suppressions are small in native neurons if the subsequent molecular steps mediated by Ca(2+) are removed. Thus, the present work also suggests that CNG channels switch on and off the olfactory signaling pathway, and that the on and off signals may both be amplified by the subsequent olfactory signaling steps.     

Molecular mechanisms of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels are highly specialized to carry out their unique role in cell signaling. Significant progress has been made in the last several years determining the molecular mechanisms for these specializations. The activation of the channels begins with the binding of cyclic nucleotide to a domain in the carboxyl terminal region. This binding, in turn, produces an induced fit of the protein that involves a movement of the C-helix portion of the binding domain. The induced fit of the binding domain is coupled to an allosteric conformational change that opens the channel pore. The pore is formed primarily from the sequence between the S5 and S6 segments. A single glutamic acid in the pore represents the binding site for multiple monovalent cations, the blocking site for external divalent cations, and the site for the effect of protons on permeation.     

Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.

Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.     

Anomalous mole-fraction effects in recombinant and native cyclic nucleotide-gated channels in rat olfactory receptor neurons.

Anomalous mole-fraction effects (AMFE) were studied, using the inside-out configuration of the patchclamp technique, in both recombinant wild-type alpha-homomeric rat olfactory adenosine 3',5'-cyclic monophosphate (cAMP)-gated channels (rOCNC1) expressed in human embryonic kidney cells (HEK 293) and native cyclic nucleotide-gated (CNG) channels in acutely isolated rat olfactory receptor neurons. Single-channel and macroscopic currents were activated by 200 microM and 500 microM cAMP, respectively. Macroscopic currents, measured with mixtures of Na(+)-NH(4)(+) or Cs(+)-Li(+) in the cytoplasmic bathing solution, displayed AMFE in the rOCNC1 channels at both positive and negative membrane potentials. The rOCNC1 single-channel conductance showed a distinct minimum (or maximum) in an 80% Na(+)-20% NH(4)(+) mixture (or a 60% Cs(+)-40% Li(+) mixture), but only at positive membrane potentials. Macroscopic measurements in native olfactory CNG channels with mixtures of Na(+)-NH(4)(+) indicated similar AMFE. These results suggest that both native CNG channels and recombinant alpha-homomeric channels allow several ions to be present simultaneously within the channel pore. They also further validate the dominant role of the alpha-subunit in permeation through these channels, provide the first evidence to suggest that rOCNC1 channels have multi-ion properties and further justify the use of the rOCNC1 channel as an effective model for structure-function studies of ion permeation and selectivity in olfactory CNG channels.     

Bimodal agonism in heteromeric cyclic nucleotide-gated channels

Direct binding of cGMP or cAMP to tetrameric cyclic nucleotide-gated (CNG) channels will normally promote the open (conductive) conformation. However, the catfish CNGA2 subtype exhibits bimodal agonism, whereby open probability (P_o) increases with initial cGMP binding events ("pro" action) but decreases with subsequent cGMP binding events ("con" action) that occur at concentrations above 3 mM. We constructed, and heterologously expressed, chimeric CNG channel subunits with sequence substitutions in the binding domain (BD), and tested their activation using patch-clamp of cell-free membranes. A normal subunit with the rat CNGA4 BD (with only pro action) could be converted into a bimodal subunit (both pro and con action) by replacing the N-terminal portion of the BD with catfish CNGA2 sequence. We then fused two bimodal and two normal subunits in tandem tetramers, to form heteromeric CNG channels with bimodal pseudo-subunits either adjacent (cis) or diagonally opposite (trans). The cis tetramer showed con action, with a mean ratio of steady-state conductances g_(30mMcGMP) / g_(3mMcGMP) = 0.87, demonstrating bimodal agonism in a heteromeric CNG channel for the first time. In contrast, trans tetramers showed normal cGMP agonism up to 30 mM cGMP with mean g_(30mMcGMP) / g_(3mMcGMP) = 1.02, although a minority of oocytes (4 of 15) expressed anomalous channel populations with con action. Rearranging subunits in a heteromer thus influences a channel's P_o at high cGMP concentration. The sensitivity of con action to neighbouring subunits implies a cooperative mechanism.     

Intracellular Ca2+ regulates the sensitivity of cyclic nucleotide-gated channels in olfactory receptor neurons.

In olfactory receptor neurons, odorants stimulate production of cAMP, which directly activates cyclic nucleotide-gated (CNG) channels. Olfactory adaptation is thought to result from a rise in intracellular Ca2+. To determine whether inhibition of CNG channels plays a role in adaptation, we have investigated the action of Ca2+ on these channels in inside-out "macro" patches from the dendrite and cilia of catfish olfactory neurons. Internal Ca2+, with a K1/2 of 3 microM, profoundly inhibits CNG channels by shifting the dose-response relationship to higher cAMP levels without altering the maximal response. The inhibition does not appear to result from a direct interaction of Ca2+ with the CNG channel. Thus, the inhibition washes out after excision of the inside-out patch, and Ca2+ does not inhibit the cloned catfish CNG channel expressed in Xenopus oocytes. Hence we propose that a regulatory Ca(2+)-binding protein, distinct from the CNG channel, controls the gain of signal transduction and contributes to olfactory adaptation by decreasing the sensitivity of the CNG channel to cAMP.     

Hyperpolarization-activated and cyclic nucleotide-gated channels are differentially expressed in juxtaglomerular cells in the olfactory bulb of mice

In the olfactory bulb, input from olfactory receptor neurons is processed by neuronal networks before it is relayed to higher brain regions. In many neurons, hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels generate and control oscillations of the membrane potential. Oscillations also appear crucial for information processing in the olfactory bulb. Four channel isoforms exist (HCN1–HCN4) that can form homo- or heteromers. Here, we describe the expression pattern of HCN isoforms in the olfactory bulb of mice by using a novel and comprehensive set of antibodies against all four isoforms. HCN isoforms are abundantly expressed in the olfactory bulb. HCN channels can be detected in most cell populations identified by commonly used marker antibodies. The combination of staining with marker and HCN antibodies has revealed at least 17 different staining patterns in juxtaglomerular cells. Furthermore, HCN isoforms give rise to an unexpected wealth of co-expression patterns but are rarely expressed in the same combination and at the same level in two given cell populations. Therefore, heteromeric HCN channels may exist in several cell populations in vivo. Our results suggest that HCN channels play an important role in olfactory information processing. The staining patterns are consistent with the possibility that both homomeric and heteromeric HCN channels are involved in oscillations of the membrane potential of juxtaglomerular cells.     

Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K(+) and other monovalent cations, but exclude Na(+); this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K(+)-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K(+) currents.     

Subunit configuration of heteromeric cone cyclic nucleotide-gated channels

Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to be tetrameric assemblies of CNGB3 (B3) and CNGA3 (A3) subunits. We have used functional and biochemical approaches to investigate the stoichiometry and arrangement of these subunits in recombinant channels. First, tandem dimers of linked subunits were used to constrain the order of CNGB3 and CNGA3 subunits; the properties of channels formed by B3/B3+A3/A3 dimers, or A3/B3+B3/A3 dimers, closely resembled those of channels arising from B3+A3 monomers. Functional markers in B3/B3 (or A3/A3) dimers confirmed that both B3 subunits (and both A3 subunits) gained membership into the pore-forming tetramer and that like subunits were positioned adjacent to each other. Second, chemical crosslinking and co-immunoprecipitation studies using epitope-tagged monomer subunits both demonstrated the presence of two CNGB3 subunits in cone channels. Together, these data support a preferred subunit arrangement for cone CNG channels (B3-B3-A3-A3) that is distinct from the 3A:1B configuration of rod channels.     

Dissecting intersubunit contacts in cyclic nucleotide-gated ion channels

In cyclic nucleotide-gated (CNG) ion channels, binding of cGMP or cAMP drives a conformational change that leads to opening of an ion-conducting pore. One region implicated in the coupling of ligand binding to opening of the pore is the C linker region. Here, we used crosslinking of endogenous cysteines to study interregion proximity. We demonstrate that an individual amino acid--C481--in the C linker region of each of two neighboring subunits can form a disulfide bond. Further, using tandem dimers, we show that a disulfide bond between C35 in the N-terminal region and C481 in the C linker region can form either within a subunit or between subunits. From our data on proximity between individual amino acids and previous studies, a picture emerges of the C linker as a potential dimerization interface.     

Different roles for the cyclic nucleotide binding domain and amino terminus in assembly and expression of hyperpolarization-activated,cyclic nucleotide-gated channels

In mammalian heart and brain, pacemaker currents are produced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which probably exist as heteromeric assemblies of different subunit isoforms. To investigate the molecular domains that participate in assembly and membrane trafficking of HCN channels, we have used the yeast two-hybrid system, patch clamp electrophysiology, and confocal microscopy. We show here that the N termini of the HCN1 and HCN2 isoforms interacted and were essential for expression of functional homo- or heteromeric channels on the plasma membrane of Chinese hamster ovary cells. We also show that the cyclic nucleotide binding domain (CNBD) of HCN2 was required for the expression of functional homomeric channels. This expression was dependent on a 12-amino acid domain corresponding to the B-helix in the CNBD of the catabolite activator protein. However, co-expression with HCN1 of an HCN2 deletion mutant lacking the CNBD rescued surface immunofluorescence and currents, indicating that a CNBD need not be present in each subunit of a heteromeric HCN channel. Furthermore, neither CNBDs nor other COOH-terminal domains of HCN1 and HCN2 interacted in yeast two-hybrid assays. Thus, interaction between NH(2)-terminal domains is important for HCN subunit assembly, whereas the CNBD is important for functional expression, but its absence from some subunits will still allow for the assembly of functional channels.