Cyclic Nucleotide-modulated Ion Channels Research Articles

Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel

Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate.

Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel

SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that the cis/trans conformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms: trans Pro300 SthK with high ligand binding affinity and fast activation, and cis Pro300 SthK with low affinity and slow activation. Following channel activation, the cis/trans equilibrium, catalyzed by prolyl isomerases, is shifted towards trans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells.

N6-modified cAMP derivatives that activate protein kinase A also act as full agonists of murine HCN2 channels

cAMP acts as a second messenger in many cellular processes. Three protein types mainly mediate cAMP-induced effects: PKA, exchange protein directly activated by cAMP (Epac), and cyclic nucleotide-modulated channels (cyclic nucleotide-gated or hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels). Discrimination among these cAMP signaling pathways requires specific targeting of only one protein. Previously, cAMP modifications at position N6 of the adenine ring (PKA) and position 2'-OH of the ribose (Epac) have been used to produce target-selective compounds. However, cyclic nucleotide-modulated ion channels were usually outside of the scope of these previous studies. These channels are widely distributed, so possible channel cross-activation by PKA- or Epac-selective agonists warrants serious consideration. Here we demonstrate the agonistic effects of three PKA-selective cAMP derivatives, N6-phenyladenosine-3',5'-cyclic monophosphate (N6-Phe-cAMP), N6-benzyladenosine-3',5'-cyclic monophosphate (N6-Bn-cAMP), and N6-benzoyl-adenosine-3',5'-cyclic monophosphate (N6-Bnz-cAMP), on murine HCN2 pacemaker channels. Electrophysiological characterization in Xenopus oocytes revealed that these derivatives differ in apparent affinities depending on the modification type but that their efficacy and effects on HCN2 activation kinetics are similar to those of cAMP. Docking experiments suggested a pivotal role of Arg-635 at the entrance of the binding pocket in HCN2, either causing stabilizing cation-π interactions with the aromatic ring in N6-Phe-cAMP or N6-Bn-cAMP or a steric clash with the aromatic ring in N6-Bnz-cAMP. A reduced apparent affinity of N6-Phe-cAMP toward the variants R635A and R635E strengthened that notion. We conclude that some PKA activators also effectively activate HCN2 channels. Hence, when studying PKA-mediated cAMP signaling with cAMP derivatives in a native environment, activation of HCN channels should be considered.

Voltage vs. Ligand II: Structural insights of the intrinsic flexibility in cyclic nucleotide-gated channels

ABSTRACTIn the preceding article, we present a flexibility analysis of the voltage-gated ion channel (VGIC) superfamily. In this study, we describe in detail the flexibility profile of the voltage-sensor domain (VSD) and the pore domain (PD) concerning the evolution of 6TM ion channels. In particular, we highlight the role of flexibility in the emergence of CNG channels and describe a significant level of sequence similarity between the archetypical VSD and the TolQ proteins. A highly flexible S4-like segment exhibiting Lys instead Arg for these membrane proteins is reported. Sequence analysis indicates that, in addition to this S4-like segment, TolQ proteins also show similarity with specific motifs in S2 and S3 from typical V-sensors. Notably, S3 flexibility profiles from typical VSDs and S3-like in TolQ proteins are also similar. Interestingly, TolQ from early divergent prokaryotes are comparatively more flexible than those in modern counterparts or true V-sensors. Regarding the PD, we also found that 2TM K+-channels in early prokaryotes are considerably more flexible than the ones in modern microbes, and such flexibility is comparable to the one present in CNG channels. Voltage dependence is mainly exhibited in prokaryotic CNG channels whose VSD is rigid whereas the eukaryotic CNG channels are considerably more flexible and poorly V-dependent. The implication of the flexibility present in CNG channels, their sensitivity to cyclic nucleotides and the cation selectivity are discussed. Finally, we generated a structural model of the putative cyclic nucleotide-modulated ion channel, which we coined here as AqK, from the thermophilic bacteria Aquifex aeolicus, one of the earliest diverging prokaryotes known. Overall, our analysis suggests that V-sensors in CNG-like channels were essentially rigid in early prokaryotes but raises the possibility that this module was probably part of a very flexible stator protein of the bacterial flagellum motor complex.

Free energy landscape remodeling of the cardiac pacemaker channel explains the molecular basis of familial sinus bradycardia

The hyperpolarization-activated and cyclic nucleotide-modulated ion channel (HCN) drives the pacemaker activity in the heart, and its malfunction can result in heart disorders. One such disorder, familial sinus bradycardia, is caused by the S672R mutation in HCN, whose electrophysiological phenotypes include a negative shift in the channel activation voltage and an accelerated HCN deactivation. The outcomes of these changes are abnormally low resting heart rates. However, the molecular mechanism underlying these electrophysiological changes is currently not fully understood. Crystallographic investigations indicate that the S672R mutation causes limited changes in the structure of the HCN intracellular gating tetramer, but its effects on protein dynamics are unknown. Here, we utilize comparative S672R versus WT NMR analyses to show that the S672R mutation results in extensive perturbations of the dynamics in both apo- and holo-forms of the HCN4 isoform, reflecting how S672R remodels the free energy landscape for the modulation of HCN4 by cAMP, i.e. the primary cyclic nucleotide modulator of HCN channels. We show that the S672R mutation results in a constitutive shift of the dynamic auto-inhibitory equilibrium toward inactive states of HCN4 and broadens the free-energy well of the apo-form, enhancing the millisecond to microsecond dynamics of the holo-form at sites critical for gating cAMP binding. These S672R-induced variations in dynamics provide a molecular basis for the electrophysiological phenotypes of this mutation and demonstrate that the pathogenic effects of the S672R mutation can be rationalized primarily in terms of modulations of protein dynamics.

Structure of the SthK carboxy-terminal region reveals a gating mechanism for cyclic nucleotide-modulated ion channels.

Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.

Family of prokaryote cyclic nucleotide-modulated ion channels.

Cyclic nucleotide-modulated ion channels are molecular pores that mediate the passage of ions across the cell membrane in response to cAMP or GMP. Structural insight into this class of ion channels currently comes from a related homolog, MloK1, that contains six transmembrane domains and a cytoplasmic cyclic nucleotide binding domain. However, unlike eukaryote hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, MloK1 lacks a C-linker region, which critically contributes to the molecular coupling between ligand binding and channel opening. In this study, we report the identification and characterization of five previously unidentified prokaryote homologs with high sequence similarity (24-32%) to eukaryote HCN and CNG channels and that contain a C-linker region. Biochemical characterization shows that two homologs, termed AmaK and SthK, can be expressed and purified as detergent-solubilized protein from Escherichia coli membranes. Expression of SthK channels in Xenopus laevis oocytes and functional characterization using the patch-clamp technique revealed that the channels are gated by cAMP, but not cGMP, are highly selective for K(+) ions over Na(+) ions, generate a large unitary conductance, and are only weakly voltage dependent. These properties resemble essential properties of various eukaryote HCN or CNG channels. Our results contribute to an understanding of the evolutionary origin of cyclic nucleotide-modulated ion channels and pave the way for future structural and functional studies.

A KcsA/MloK1 Chimeric Ion Channel Has Lipid-dependent Ligand-binding Energetics

Cyclic nucleotide-modulated ion channels play crucial roles in signal transduction in eukaryotes. The molecular mechanism by which ligand binding leads to channel opening remains poorly understood, due in part to the lack of a robust method for preparing sufficient amounts of purified, stable protein required for structural and biochemical characterization. To overcome this limitation, we designed a stable, highly expressed chimeric ion channel consisting of the transmembrane domains of the well characterized potassium channel KcsA and the cyclic nucleotide-binding domains of the prokaryotic cyclic nucleotide-modulated channel MloK1. This chimera demonstrates KcsA-like pH-sensitive activity which is modulated by cAMP, reminiscent of the dual modulation in hyperpolarization-activated and cyclic nucleotide-gated channels that display voltage-dependent activity that is also modulated by cAMP. Using this chimeric construct, we were able to measure for the first time the binding thermodynamics of cAMP to an intact cyclic nucleotide-modulated ion channel using isothermal titration calorimetry. The energetics of ligand binding to channels reconstituted in lipid bilayers are substantially different from those observed in detergent micelles, suggesting that the conformation of the chimera's transmembrane domain is sensitive to its (lipid or lipid-mimetic) environment and that ligand binding induces conformational changes in the transmembrane domain. Nevertheless, because cAMP on its own does not activate these chimeric channels, cAMP binding likely has a smaller energetic contribution to gating than proton binding suggesting that there is only a small difference in cAMP binding energy between the open and closed states of the channel.

Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1

Cyclic nucleotide-modulated ion channels are important for signal transduction and pacemaking in eukaryotes. The molecular determinants of ligand gating in these channels are still unknown, mainly because of a lack of direct structural information. Here we report ligand-induced conformational changes in full-length MloK1, a cyclic nucleotide-modulated potassium channel from the bacterium Mesorhizobium loti, analysed by electron crystallography and atomic force microscopy. Upon cAMP binding, the cyclic nucleotide-binding domains move vertically towards the membrane, and directly contact the S1–S4 voltage sensor domains. This is accompanied by a significant shift and tilt of the voltage sensor domain helices. In both states, the inner pore-lining helices are in an ‘open’ conformation. We propose a mechanism in which ligand binding can favour pore opening via a direct interaction between the cyclic nucleotide-binding domains and voltage sensors. This offers a simple mechanistic hypothesis for the coupling between ligand gating and voltage sensing in eukaryotic HCN channels.

Spectroscopic Investigation of Agonist-Induced Rearrangements of Cyclic Nucleotide-Modulated Ion Channels

Cyclic nucleotide-gated (CNG) and hyperpolarization activated cyclic nucleotide-modulated (HCN) ion channels are activated by the direct binding of cyclic nucleotides, e.g. adenosine 3',5'-cyclic monophosphate (cAMP), to a conserved, cytoplasmic domain. The structure of the cyclic nucleotide binding domain (CNBD) is similar to those found in other cyclic nucleotide-activated proteins, including the kinases PKA and PKG, the transcription factor CAP, and the guanine nucleotide exchange factor Epac. The core of this structure contains an eight-stranded β-barrel followed by two helices (the B and C helices). Cyclic nucleotides initially bind to residues in the β-barrel. Subsequent to binding, the C helix of HCN and CNG channels undergoes a translation toward the binding pocket as well as a stabilization of its helical structure. This conformational rearrangement is coupled to opening of the ion channel pore. We have extended our previous studies using transition metal ion fluorescence resonance energy transfer (tmFRET) in the purified C-terminal domain of HCN2 to demonstrate that the B helix also reorganizes relative to the β-barrel subsequent to agonist binding. Furthermore, we have used electron paramagnetic resonance (EPR) on the spin-labeled HCN2 C- terminus to investigate the reorientation and stabilization of the CNBD induced by cAMP binding. These studies further extend our knowledge of the conformational changes in the CNBD of HCN and CNG channels and may provide a general picture of the activation of other families of cyclic nucleotide-regulated proteins.

Identification and experimental verification of a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels

Studies of bacterial ion channels have provided significant insights into the structure–function relationships of mechanosensitive and voltage-gated ion channels. However, to date, very few bacterial channels that respond to small molecules have been identified, cloned, and characterized. Here, we use bioinformatics to identify a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels containing a channel domain related by sequence homology to the mechanosensitive channel of small conductance (MscS). In this initial report, we clone selected members of this channel family, use electrophysiological measurements to verify their ability to directly gate in response to cyclic nucleotides, and use osmotic downshock to demonstrate their lack of mechanosensitivity. In addition to providing insight into bacterial physiology, these channels will provide researchers with a useful model system to investigate the role of ligand-gated ion channels (LGICs) in the signaling processes of higher organisms. The identification of these channels provides a foundation for structural and functional studies of LGICs that would be difficult to perform on mammalian channels. Moreover, the discovery of bCNG channels implies that bacteria have cyclic nucleotide-gated and cyclic nucleotide-modulated ion channels, which are analogous to the ion channels involved in eukaryotic secondary messenger signaling pathways.

The Structure of the Prokaryotic Cyclic Nucleotide-Modulated Potassium Channel MloK1 at 16 Å Resolution

The gating ring of cyclic nucleotide-modulated channels is proposed to be either a two-fold symmetric dimer of dimers or a four-fold symmetric tetramer based on high-resolution structure data of soluble cyclic nucleotide-binding domains and functional data on intact channels. We addressed this controversy by obtaining structural data on an intact, full-length, cyclic nucleotide-modulated potassium channel, MloK1, from Mesorhizobium loti, which also features a putative voltage-sensor. We present here the 3D single-particle structure by transmission electron microscopy and the projection map of membrane-reconstituted 2D crystals of MloK1 in the presence of cAMP. Our data show a four-fold symmetric arrangement of the CNBDs, separated by discrete gaps. A homology model for full-length MloK1 suggests a vertical orientation for the CNBDs. The 2D crystal packing in the membrane-embedded state is compatible with the S1-S4 domains in the vertical "up" state.

Ligand Binding and Activation in a Prokaryotic Cyclic Nucleotide-modulated Channel

We designed a technique that directly determines binding of cyclic nucleotides to the prokaryotic cyclic nucleotide modulated ion channel MloK1. The ability to purify large quantities of MloK1 facilitated equilibrium binding assays, which avoided the inherent problem of relatively low affinity binding which hindered the use of eukaryotic channels. We found that MloK1 specifically binds cAMP and cGMP with affinity values in the range of those observed for activity assays for eukaryotic channels. Notably, the concentration of ligand that elicited 50% of maximum response in 86Rb flux assays ( K 1/2), also referred to as ligand sensitivity, was smaller than the corresponding value obtained from binding assays ( K d) potentially indicating significant channel activity in partially liganded states. To gain further insight into the mechanism of binding and activation of these channels, we mutated several amino acids in the ligand-binding pocket of MloK1, known from electrophysiological studies of homologous eukaryotic channels to affect ligand selectivity and binding efficacy. The S308V MloK1 mutant (a mutation which decreases cGMP selectivity in eukaryotic channels) decreased both the observed cGMP binding affinity and the sensitivity to cGMP relative to the wild-type (WT) channel, leaving those for cAMP unchanged. Conversely, the A352D MloK1 mutant (a mutation which increases cGMP selectivity in eukaryotic channels) increased both the affinity and the sensitivity for cGMP relative to the WT channel, again leaving those for cAMP unchanged. Mutations at R307 in MloK1, the most conserved residue in the binding pocket of cyclic nucleotide-binding proteins, were not tolerated as these mutants do not form functional channels. Furthermore, for each mutation, changes in binding affinities were mirrored by equivalent changes in ligand sensitivity. These data, together with the evidence that partially liganded channels open significantly, suggested strong coupling between cyclic nucleotide binding and MloK1 channel opening.

The VGL-chanome: a protein superfamily specialized for electrical signaling and ionic homeostasis.

Complex multicellular organisms require rapid and accurate transmission of information among cells and tissues and tight coordination of distant functions. Electrical signals and resulting intracellular calcium transients, in vertebrates, control contraction of muscle, secretion of hormones, sensation of the environment, processing of information in the brain, and output from the brain to peripheral tissues. In nonexcitable cells, calcium transients signal many key cellular events, including secretion, gene expression, and cell division. In epithelial cells, huge ion fluxes are conducted across tissue boundaries. All of these physiological processes are mediated in part by members of the voltage-gated ion channel protein superfamily. This protein superfamily of 143 members is one of the largest groups of signal transduction proteins, ranking third after the G protein-coupled receptors and the protein kinases in number. Each member of this superfamily contains a similar pore structure, usually covalently attached to regulatory domains that respond to changes in membrane voltage, intracellular signaling molecules, or both. Eight families are included in this protein superfamily-voltage-gated sodium, calcium, and potassium channels; calcium-activated potassium channels; cyclic nucleotide-modulated ion channels; transient receptor potential (TRP) channels; inwardly rectifying potassium channels; and two-pore potassium channels. This article identifies all of the members of this protein superfamily in the human genome, reviews the molecular and evolutionary relations among these ion channels, and describes their functional roles in cell physiology.

A cyclic nucleotide modulated prokaryotic K+ channel.

A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K+ channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus. The gene was cloned from genomic DNA, and the protein, denoted MloK1, was overexpressed in Escherichia coli and purified. Gel filtration analysis revealed a heterogeneous distribution of protein sizes which, upon inclusion of cyclic nucleotide, coalesces into a homogeneous population, eluting at the size expected for a homotetramer. As followed by a radioactive 86Rb+ flux assay, the putative channel protein catalyzes ionic flux with a selectivity expected for a K+ channel. Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations. Since this bacterial homologue does not have the “C-linker” sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening.