Cyclic Nucleotide-gated Channel Pore Research Articles

Insights into the Ion Selectivity Mechanism of CNG Channels from Mutants of NaK: Structural and Functional Studies

Cyclic nucleotide-gated (CNG) channels are non-selective cation channels that play crucial roles in visual and olfactory signal transduction. They are members of the tetrameric cation channel family that include voltage-gated K+, Na+ and Ca2+ channels. However, while other members exhibit high degree of ion selectivity, CNG channels are noted for their lack of specificity. CNG channels conduct all alkali metal ions and some alkaline earths, most notably Ca2+. How the CNG channel pore can conduct these various cations which have substantially different ionic radii and formal charges is not well understood. Here we report high-resolution crystal structures of mutants of the NaK channel that mimic the selectivity filter of CNG channels, along with supporting functional analyses. Within the NaK selectivity filter (63TVGDGNFS70) the DGNFS sequence was replaced with ETPP, ETPT and DTPS, each of which represent a CNG α-subunit sequence. The mutant structures exhibit selectivity filter architecture and ion binding profiles different from either NaK or K+ channel structures, having three ion binding sites in their selectivity filters. Two of the sites correspond to sites 3 and 4 in KcsA and NaK, while the third site corresponds to site 2 in KcsA, but is a vestibule in NaK. Similar to CNG channels these mutants exhibit calcium binding, which depends on the presence of the conserved acidic residues (E or D). Mutating the acidic residues on these mutants to neutral residues (E◊Q or D◊N) abolishes calcium binding. Functional analyses using Rb-86 flux assay revealed ion conduction behavior similar to CNG channels. These results provide strong evidence that these NaK mutants exhibit the same properties of CNG channels in ion conduction and selectivity and their structures provide insight into understanding ion selectivity in CNG channels.

Access of Quaternary Ammonium Blockers to the Internal Pore of Cyclic Nucleotide-gated Channels: Implications for the Location of the Gate

Cyclic nucleotide-gated (CNG) channels play important roles in the transduction of visual and olfactory information by sensing changes in the intracellular concentration of cyclic nucleotides. We have investigated the interactions between intracellularly applied quaternary ammonium (QA) ions and the α subunit of rod cyclic nucleotide-gated channels. We have used a family of alkyl-triethylammonium derivatives in which the length of one chain is altered. These QA derivatives blocked the permeation pathway of CNG channels in a concentration- and voltage-dependent manner. For QA compounds with tails longer than six methylene groups, increasing the length of the chain resulted in higher apparent affinities of ∼1.2 RT per methylene group added, which is consistent with the presence of a hydrophobic pocket within the intracellular mouth of the channel that serves as part of the receptor binding site. At the single channel level, decyltriethyl ammonium (C10-TEA) ions did not change the unitary conductance but they did reduce the apparent mean open time, suggesting that the blocker binds to open channels. We provide four lines of evidence suggesting that QA ions can also bind to closed channels: (1) the extent of C10-TEA blockade at subsaturating [cGMP] was larger than at saturating agonist concentration, (2) under saturating concentrations of cGMP, cIMP, or cAMP, blockade levels were inversely correlated with the maximal probability of opening achieved by each agonist, (3) in the closed state, MTS reagents of comparable sizes to QA ions were able to modify V391C in the inner vestibule of the channel, and (4) in the closed state, C10-TEA was able to slow the Cd2+ inhibition observed in V391C channels. These results are in stark contrast to the well-established QA blockade mechanism in Kv channels, where these compounds can only access the inner vestibule in the open state because the gate that opens and closes the channel is located cytoplasmically with respect to the binding site of QA ions. Therefore, in the context of Kv channels, our observations suggest that the regions involved in opening and closing the permeation pathways in these two types of channels are different.

Structural basis of gating of CNG channels

Cyclic nucleotide-gated (CNG) ion channels, underlying sensory transduction in vertebrate photoreceptors and olfactory sensory neurons, require cyclic nucleotides to open. Here, we present structural models of the tetrameric CNG channel pore from bovine rod in both open and closed states, as obtained by combining homology modeling-based techniques, experimentally derived spatial constraints and structural patterns present in the PDB database. Gating is initiated by an anticlockwise rotation of the N-terminal region of the C-linker, which is then, transmitted through the S6 transmembrane helices to the P-helix, and in turn from this to the pore lumen, which opens up from 2 to 5 Å thus allowing for ion permeation. The approach, here presented, is expected to provide a general methodology for model ion channels and their gating when structural templates are available and an extensive electrophysiological analysis has been performed.

Binding symmetry of extracellular divalent cations to conduction pore studied using tandem dimers of a CNG channel

Cyclic nucleotide-gated (CNG) channels are composed of the tetramer of α-subunit alone or α- and β-subunits. The α-subunits of these channels have a conserved glutamate (Glu) residue within the pore-forming region and the residue determines the selectivity as well as the affinity for the extracellular divalent cations. Using the high-affinity mutant (E363D) of bovine retinal CNG channel in which the Glu at position 363 was replaced to Asp, we constructed tandem dimers and investigated the binding characteristics of divalent cations to the site. The gating and permeation characteristics of individual homomeric tandem dimers are indistinguishable to those of homo-tetramers formed by parental monomers. The heteromeric tandem dimers showed the binding affinity for Sr 2+ identical to the geometric mean of the affinities for two parent channels, indicating the energy additive and thus the simultaneous interaction. On the other hand, the binding affinity for Mg 2+ followed the harmonic mean of those parent channels indicating that Mg 2+ interacts more strongly with the subunit bearing Asp residue at the position. Thus the results strongly suggest that the Glu363 residues in the CNG channel pore be flexible enough to adapt different binding symmetries for different divalent cations. Moreover, the simultaneous interaction between the four Glu residues and Sr 2+ provides an important structural constraint to the CNG channel outer vestibule of unknown structure.

The Interaction of Na + and K + in the Pore of CyclicNucleotide-Gated Channels

The permeability ratio between K + and Na + ions in cyclic nucleotide-gated channels is close to 1, and the single channel conductance has almost the same value in the presence of K + or Na +. Therefore, K + and Na + ions are thought to permeate with identical properties. In the α-subunit from bovine rods there is a loop of three prolines at positions 365 to 367. When proline 365 is mutated to a threonine, a cysteine, or an alanine, mutant channels exhibit a complex interaction between K + and Na + ions. Indeed K +, Rb + and Cs + ions do not carry any significant macroscopic current through mutant channels P365T, P365C and P365A and block the current carried by Na + ions. Moreover in mutant P365T the presence of K + in the intracellular (or extracellular) medium caused the appearance of a large transient inward (or outward) current carried by Na + when the voltage command was quickly stepped to large negative (or positive) membrane voltages. This transient current is caused by a transient potentiation, i.e., an increase of the open probability. The permeation of organic cations through these mutant channels is almost identical to that through the wild type (w.t.) channel. Also in the w.t. channel a similar but smaller transient current is observed, associated to a slowing down of the channel gating evident when intracellular Na + is replaced with K +. As a consequence, a rather simple mechanism can explain the complex behavior here described: when a K + ion is occupying the pore there is a profound blockage of the channel and a potentiation of gating immediately after the K + ion is driven out. Potentiation occurs because K + ions slow down the rate constant K off controlling channel closure. These results indicate that K + and Na + ions do not permeate through CNG channels in the same way and that K + ions influence the channel gating.

Extracellular proton alters the divalent cation binding affinity in a cyclic nucleotide-gated channel pore

Extracellular protons in the range of 10 −9 to 10 −5 M effectively suppressed Na + current ( K 1/2=10 −6.1) through the bovine retinal guanosine 3′,5′-cyclic mononucleotide-gated ion channel expressed in Xenopus oocytes. The reduction of channel current was mediated by a single glutamate residue (Glu363) within the pore-forming region of the channel, also involved in extracellular divalent cation binding. Increasing the concentration of extracellular proton decreased the binding affinity of the extracellular divalent cation (e.g. Sr 2+) and the large difference of binding affinity previously observed between the wild-type and E363D mutant channel disappeared. These results indicate that the permeation characteristics of cyclic nucleotide-gated ion channel can be altered by extracellular pH through a single acidic residue in the channel conduction pathway.

Tetracaine reports a conformational change in the pore of cyclic nucleotide-gated channels.

Local anesthetics are a diverse group of clinically useful compounds that act as pore blockers of both voltage- and cyclic nucleotide-gated (CNG) ion channels. We used the local anesthetic tetracaine to probe the nature of the conformational change that occurs in the pore of CNG channels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the alpha subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed states than to open states. Here we show that neutralization of a glutamic acid in the conserved P region (E363G) eliminated this state dependence of tetracaine block. Tetracaine blocked E363G channels with the same effectiveness at high concentrations of cGMP, when the channel spent more time open, and at low concentrations of cGMP, when the channel spent more time closed. In addition, Ni2+, which promotes the opening allosteric transition, decreased the effectiveness of tetracaine block of wild-type but not E363G channels. Similar results were obtained in a chimeric CNG channel that exhibits a more favorable opening allosteric transition. These results suggest that E363 is accessible to internal tetracaine in the closed but not the open configuration of the pore and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.

Mechanism of tetracaine block of cyclic nucleotide-gated channels.

Local anesthetics are a diverse group of ion channel blockers that can be used to probe conformational changes in the pore. We examined the effects of the local anesthetic tetracaine on rod and olfactory cyclic nucleotide-gated channels expressed from subunit 1 in Xenopus oocytes. We found that 40 microM tetracaine effectively blocked the bovine rod channel but not the rat olfactory channel at saturating concentrations of cGMP. By testing chimeric channels containing regions of sequence from both rod and olfactory channels, we found that determinants of apparent affinity for tetracaine at saturating cGMP did not map to any one region of the channel sequence. Rather, the differences in apparent affinity could be explained by differences between the chimeras in the free energy of the opening allosteric transition. If a channel construct (such as the rod channel) spent appreciable time in the closed state at saturating cGMP, then it had a high apparent affinity for tetracaine. If, on the other hand, a channel construct (such as the olfactory channel) spent little time in the closed state at saturating cGMP, then it had a low apparent affinity for tetracaine. Furthermore, tetracaine became more effective at low concentrations of cGMP and at saturating concentrations of cAMP, conditions which permit the channels to spend more time in the closed configuration. These results were well fit by a model in which tetracaine binds more tightly to the closed channel than to the open channel. Dose-response curves for tetracaine in the presence of saturating cGMP are well fit with a Michaelis-Menten binding scheme indicating that a single tetracaine molecule is sufficient to produce block. In addition, tetracaine block is voltage dependent with an effective z delta of +0.56. These data are consistent with a pore-block hypothesis. The finding that tetracaine is a state-dependent pore blocker suggests that the inner mouth of the pore of cyclic nucleotide-gated channels undergoes a conformational change during channel opening.

Exposure of Residues in the Cyclic Nucleotide–Gated Channel Pore: P Region Structure and Function in Gating

In voltage-gated ion channels and in the homologous cyclic nucleotide–gated (CNG) channels, the loop between the S5 and S6 transmembrane segments (P region) is thought to form the lining of the pore. To investigate the structure and the role in gating of the P region of the bovine retinal CNG channel, we determined the accessibility of 11 cysteine-substituted P region residues to small, charged sulfhydryl reagents applied to the inside and outside of membrane patches in the open and closed states of the channel. The results suggest that the P region forms a loop that extends toward the central axis of the channel, analogous to the L3 loop of bacterial porin channels. Furthermore, the P region, in addition to forming the ion selectivity filter, functions as the channel gate, the structure of which changes when the channel opens.

Block of cyclic nucleotide-gated channels in salamander olfactory receptor neurons by the guanylyl cyclase inhibitor LY83583.

1. Using whole cell voltage-clamp recordings, the guanylyl cyclase inhibitor LY83583 [6-(phenylamino)-5,8-quinolinedione] is shown to act as a potent blocker of cyclic nucleotide-gated (CNG) channels in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. Under our experimental conditions, onset of the blockade by LY83583 occurs on the time scale of seconds and is completely reversed upon wash-out of the drug. Dose-response curves reveal a Kd of 1.4 microM (at -60 mV). Other data suggest that LY83583 acts within the CNG channel pore and that the channels must be in an activated state before the drug can exert its effect. 3. It appears that LY83583 can act on both CNG channels and soluble guanylyl cyclase (sGC) and that these two effects can be distinguished by their different recovery behaviors. The LY83583-induced blockade of CNG channels activated directly by guanosine 3',5' cyclic monophosphate (cGMP) is rapidly reversible (with a recovery time constant of approximately 3 s), whereas previous results have shown that no recovery is obtained during minute-long washing periods when the channels are activated indirectly through exogenous carbon monoxide application, which acts as a stimulator of sGC in ORNs. 4. LY83583 appears to be a novel and useful agent in examining neural functions due to CNG channel responses.