Altered cyclic nucleotide binding and pore opening in a diseased human HCN4 channel.

A growing number of nonsynonymous mutations in the human HCN4 channel gene, the major component of the funny channel of the sinoatrial node, are associated with disease but how they impact channel structure and function, and, thus, how they result in disease, is not clear for any of them. Here, we study the S672R mutation, in the cyclic nucleotide-binding domain of the channel, which has been associated with an inherited bradycardia in an Italian family. This may be the best studied of all known mutations, yet the underlying molecular and atomistic mechanisms remain unclear and controversial. We combine measurements of binding by isothermal titration calorimetry to a naturally occurring tetramer of the HCN4 C-terminal region with a mathematical model to show that weaker binding of cAMP to the mutant channel contributes to a lower level of facilitation of channel opening at submicromolar ligand concentrations but that, in general, facilitation occurs over a range that is similar between the mutant and wild-type because of enhanced opening of the mutant channel when liganded. We also show that the binding affinity for cGMP, which produces the same maximum facilitation of HCN4 opening as cAMP, is weaker in the mutant HCN4 channel but that, for both wild-type and mutant, high-affinity binding of cGMP occurs in a range of concentrations below 1 µM. Thus, binding of cGMP to the HCN4 channel may be relevant normally in vivo and reduced binding of cGMP, as well as cAMP, to the mutant channel may contribute to the reduced resting heart rate observed in the affected family.

A mechanism for the auto-inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel opening and its relief by cAMP.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1-4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic "C-linker," which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or "tetrameric" C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated.

Energetics of cyclic AMP binding to HCN channel C terminus reveal negative cooperativity.

Cyclic AMP binds to the HCN channel C terminus and variably stabilizes its open state. Using isothermal titration calorimetry, we show that cAMP binds to one subunit of tetrameric HCN2 and HCN4 C termini with high affinity (∼0.12 µM) and subsequently with low affinity (∼1 µM) to the remaining three subunits. Changes induced by high affinity binding already exist in both a constrained HCN2 tetramer and the unconstrained HCN1 tetramer. Natural "preactivation" of HCN1 may explain both the smaller effect of cAMP on stabilizing its open state and the opening of unliganded HCN1, which occurs as though already disinhibited.

Mechanism of accelerated current decay caused by an episodic ataxia type-1-associated mutant in a potassium channel pore.

In Kv1.1, single point mutants found below the channel activation gate at residue V408 are associated with human episodic ataxia type-1, and impair channel function by accelerating decay of outward current during periods of membrane depolarization and channel opening. This decay is usually attributed to C-type inactivation, but here we provide evidence that this is not the case. Using voltage-clamp fluorimetry in Xenopus oocytes, and single-channel patch clamp in mouse ltk- cells, of the homologous Shaker channel (with the equivalent mutation V478A), we have determined that the mutation may cause current decay through a local effect at the activation gate, by destabilizing channel opening. We demonstrate that the effect of the mutant is similar to that of trapped 4-aminopyridine in antagonizing channel opening, as the mutation and 10 mm 4-AP had similar, nonadditive effects on fluorescence recorded from the voltage-sensitive S4 helix. We propose a model where the Kv1.1 activation gate fails to enter a stabilized open conformation, from which the channel would normally C-type inactivate. Instead, the lower pore lining helix is able to enter an activated-not-open conformation during depolarization. These results provide an understanding of the molecular etiology underlying episodic ataxia type-1 due to V408A, as well as biophysical insights into the links between the potassium channel activation gate, the voltage sensor and the selectivity filter.

Regulation of sinoatrial funny channels by cyclic nucleotides: From adrenaline and IK2 to direct binding of ligands to protein subunits.

The funny current, and the HCN channels that form it, are affected by the direct binding of cyclic nucleotides. Binding of these second messengers causes a depolarizing shift of the activation curve, which leads to greater availability of current at physiological membrane voltages. This review outlines a brief history on this regulation and provides some evidence that other cyclic nucleotides, especially cGMP, may be important for the regulation of the funny channel in the heart. Current understanding of the molecular mechanism of cyclic nucleotide regulation is also presented, which includes the notions that full and partial agonism occur as a consequence of negatively cooperative binding. Knowledge gaps, including a potential role of cyclic nucleotide-regulation of the funny current under pathophysiological conditions, are included. The work highlighted here is in dedication to Dario DiFrancesco on his retirement.

Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression.

All four mammalian hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel isoforms have been shown to undergo N-linked glycosylation in the brain. With the mouse HCN2 isoform as a prototype, HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. This difference between HCN1 and HCN2 is consistent with evolutionary trajectories that diverged in an isoform-specific manner after gene duplication from a common HCN ancestor that lacked N-glycosylation and was able to localize efficiently to the cell surface.

In situ co-distribution and functional interactions of SAP97 with sinoatrial isoforms of HCN channels.

The sinoatrial node is a region of specialized cardiomyocytes that is responsible for the repetitive activity of the adult heart. The sinoatrial node is heavily innervated compared to the other regions of the heart, and the specialized cardiomyocytes of this region receive neural and hormonal input from the autonomic nervous system, which leads to changes in heart rate. A key regulator of sinoatrial beating frequency in response to autonomic input is the hyperpolarization-activated cyclic nucleotide gated (HCN) channel, a mixed cationic channel whose activity is increased by the binding of cAMP to its cytoplasmic side. HCN channels localize to distinct regions or "hot spots" on the cell surface of sinoatrial myocytes, but how these regions are formed, whether they correspond to specific signaling domains and the specific HCN isoforms and other proteins therein are not known. In this paper, we show that both HCN2 and HCN4 isoforms co-distribute with the adapter protein SAP97, an important component of distinct punctae in the sinoatrial node of the rabbit heart. HCN4, but not HCN2, also co-distributes with the post-synaptic marker beta-catenin, thus identifying diverse organized domains within this tissue. Furthermore, we show, using heterologous expression systems, whole-cell patch clamp electrophysiology and imaging, that SAP97 interacts functionally with HCN in a manner that depends upon the PDZ compatible binding motif of the C-terminus, but that its effects on I(f) behaviour are HCN isoform and context dependent. Together, the data suggest that SAP97 contributes to isoform specific organization of HCN channels within specific domains in the sinoatrial node of the rabbit.

Binding and structural asymmetry governs ligand sensitivity in a cyclic nucleotide-gated ion channel.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open more easily when cAMP or cGMP bind to a domain in the intracellular C-terminus in each of four identical subunits. How sensitivity of the channels to these ligands is determined is not well understood. Here, we apply a mathematical model, which incorporates negative cooperativity, to gating and mutagenesis data available in the literature and combine the results with binding data collected using isothermal titration calorimetry. This model recapitulates the concentration-response data for the effects of cAMP and cGMP on wild-type HCN2 channel opening and, remarkably, predicts the concentration-response data for a subset of mutants with single-point amino acid substitutions in the binding site. Our results suggest that ligand sensitivity is determined by negative cooperativity and asymmetric effects on structure and channel opening, which are tuned by ligand-specific interactions and residues within the binding site.

Hyperpolarization-activated cyclic nucleotide-modulated 'HCN' channels confer regular and faster rhythmicity to beating mouse embryonic stem cells.

The hyperpolarization-activated cation current (I(f)), and the hyperpolarization-activated cyclic nucleotide-modulated 'HCN' subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell-derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. I(f) and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole-cell voltage- and current-clamp techniques. Our data show that the proportion of cells expressing I(f), and the density of I(f) in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The I(f) blocker ZD7288 (0.3 microm) reduced I(f) and the beating rate of embryoid bodies. Taken together, the activation kinetics of I(f) and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of I(f) to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation.

Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations.

BACKGROUND: Mutations in HERG and KCNQ1 potassium channels have been associated with Long QT syndrome and atrial fibrillation, and more recently with sudden infant death syndrome and sudden unexplained death. In other proteins, disease-associated amino acid mutations have been analyzed according to the chemical severity of the changes and the locations of the altered amino acids according to their conservation over metazoan evolution. Here, we present the first such analysis of arrhythmia-associated mutations (AAMs) in the HERG and KCNQ1 potassium channels. RESULTS: Using evolutionary analyses, AAMs in HERG and KCNQ1 were preferentially found at evolutionarily conserved sites and unevenly distributed among functionally conserved domains. Non-synonymous single nucleotide polymorphisms (nsSNPs) are under-represented at evolutionarily conserved sites in HERG, but distribute randomly in KCNQ1. AAMs are chemically more severe, according to Grantham's Scale, than changes observed in evolution and their severity correlates with the expected chemical severity of the involved codon. Expected chemical severity of a given amino acid also correlates with its relative contribution to arrhythmias. At evolutionarily variable sites, the chemical severity of the changes is also correlated with the expected chemical severity of the involved codon. CONCLUSION: Unlike nsSNPs, AAMs preferentially locate to evolutionarily conserved, and functionally important, sites and regions within HERG and KCNQ1, and are chemically more severe than changes which occur in evolution. Expected chemical severity may contribute to the overrepresentation of certain residues in AAMs, as well as to evolutionary change.

Using Bioluminescence Resonance Energy Transfer to measure ion channel assembly.

Bioluminescence Resonance Energy Transfer (BRET) measures protein interactions within 10 nm of each other. Aside from its ability to probe for interactions at high resolution, this technique operates in live, intact cells, and offers a high throughput method of detection. Thus far, BRET has been widely used in measuring G protein receptor dimerization. In this chapter, we describe the BRET methodology in detail and apply this technique to the measurement of ion channel assembly. In addition, we discuss how BRET can be used to compare the extent of homomeric and heteromeric channel assembly.

Evolution and structural diversification of hyperpolarization-activated cyclic nucleotide-gated channel genes.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are members of the voltage-gated channel superfamily and play a critical role in cellular pace-making. Overall sequence conservation is high throughout the family, and channel functions are similar but not identical. Phylogenetic analyses are imperative to understand how these genes have evolved and to make informed comparisons of HCN structure and function. These have been previously limited, however, by the small number of available sequences, from a minimal number of species unevenly distributed over evolutionary time. We have now identified and annotated 31 novel genes from invertebrates, urochordates, fish, amphibians, birds, and mammals. With increased sequence numbers and a broader species representation, a more precise sequence comparison was performed and an evolutionary history for these genes was constructed. Our data confirm the existence of at least four vertebrate paralogs and suggest that these arose via three duplication and diversification events from a single ancestral gene. Additional lineage-specific duplications appear to have occurred in urochordate and fish genomes. Based on exon boundary conservation and phylogenetic analyses, we hypothesize that mammalian gene structure was established, and duplication events occurred, after the divergence of urochordates and before the divergence of fish from the tetrapod lineage. In addition, we identified highly conserved sequence regions that are likely important for general HCN functions, as well as regions with differences conserved among each of the individual paralogs. The latter may underlie more subtle isoform-specific properties that are otherwise masked by the high identity among mammalian orthologs and/or inaccurate alignments between paralogs.

Cation and voltage dependence of lidocaine inhibition of the hyperpolarization-activated cyclic nucleotide-gated HCN1 channel.

Lidocaine is known to inhibit the hyperpolarization-activated mixed cation current (Ih) in cardiac myocytes and neurons, as well in cells transfected with cloned Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels. However, the molecular mechanism of Ih inhibition by this drug has been limitedly explored. Here, we show that inhibition of Ih by lidocaine, recorded from Chinese hamster ovary (CHO) cells expressing the HCN1 channel, reached a steady state within one minute and was reversible. Lidocaine inhibition of Ih was greater at less negative voltages and smaller current amplitudes whereas the voltage-dependence of Ih activation was unchanged. Lidocaine inhibition of Ih measured at -130 mV (a voltage at which Ih is fully activated) was reduced, and Ih amplitude was increased, when the concentration of extracellular potassium was raised to 60 mM from 5.4 mM. By contrast, neither Ih inhibition by the drug nor Ih amplitude at +30 mV (following a test voltage-pulse to -130 mV) were affected by this rise in extracellular potassium. Together, these data indicate that lidocaine inhibition of Ih involves a mechanism which is antagonized by hyperpolarizing voltages and current flow.

The Ih Channel Gene Promotes Synaptic Transmission and Coordinated Movement in Drosophila melanogaster.

Hyperpolarization-activated cyclic nucleotide-gated "HCN" channels, which underlie the hyperpolarization-activated current (Ih), have been proposed to play diverse roles in neurons. The presynaptic HCN channel is thought to both promote and inhibit neurotransmitter release from synapses, depending upon its interactions with other presynaptic ion channels. In larvae of Drosophila melanogaster, inhibition of the presynaptic HCN channel by the drug ZD7288 reduces the enhancement of neurotransmitter release at motor terminals by serotonin but this drug has no effect on basal neurotransmitter release, implying that the channel does not contribute to firing under basal conditions. Here, we show that genetic disruption of the sole HCN gene (Ih) reduces the amplitude of the evoked response at the neuromuscular junction (NMJ) of third instar larvae by decreasing the number of released vesicles. The anatomy of the (NMJ) is not notably affected by disruption of the Ih gene. We propose that the presynaptic HCN channel is active under basal conditions and promotes neurotransmission at larval motor terminals. Finally, we demonstrate that Ih partial loss-of-function mutant adult flies have impaired locomotion, and, thus, we hypothesize that the presynaptic HCN channel at the (NMJ) may contribute to coordinated movement.

Cyclic Purine and Pyrimidine Nucleotides Bind to the HCN2 Ion Channel and Variably Promote C-Terminal Domain Interactions and Opening.

Cyclic AMP is thought to facilitate the opening of the HCN2 channel by binding to a C-terminal domain and promoting or inhibiting interactions between subunits. Here, we correlated the ability of cyclic nucleotides to promote interactions of isolated HCN2 C-terminal domains in solution with their ability to facilitate channel opening. Cyclic IMP, a cyclic purine nucleotide, and cCMP, a cyclic pyrimidine nucleotide, bind to a C-terminal domain containing the cyclic nucleotide-binding domain but, in contrast to other cyclic nucleotides examined, fail to promote its oligomerization, and produce only modest facilitation of opening of the full-length channel. Comparisons between ligand bound structures identify a region between the sixth and seventh ß strands and the distal C helix as important for facilitation and tight binding. We propose that promotion of interactions between the C-terminal domains by a given ligand contribute to its ability to facilitate opening of the full-length channel.

Allosteric coupling of the inner activation gate to the outer pore of a potassium channel.

In potassium channels, functional coupling of the inner and outer pore gates may result from energetic interactions between residues and conformational rearrangements that occur along a structural path between them. Here, we show that conservative mutations of a residue near the inner activation gate of the Shaker potassium channel (I470) modify the rate of C-type inactivation at the outer pore, pointing to this residue as part of a pathway that couples inner gate opening to changes in outer pore structure and reduction of ion flow. Because they remain equally sensitive to rises in extracellular potassium, altered inactivation rates of the mutant channels are not secondary to modified binding of potassium to the outer pore. Conservative mutations of I470 also influence the interaction of the Shaker N-terminus with the inner gate, which separately affects the outer pore.

Architecture of the HCN selectivity filter and control of cation permeation.

Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) channels are similar in structure and function to voltage-gated potassium channels. Sequence similarity and functional analyses suggest that the HCN pore is potassium channel-like, consisting of a selectivity filter and an activation gate at the outer and inner ends, respectively. In GYG-containing potassium channels, the selectivity filter sequence is 'T/S-V/I/L/T-GYG', forming a row of four binding sites through which potassium ions flow. In HCNs, the equivalent residues are 'C-I-GYG', but whether they also form four cation binding sites is not known. Here, we focus on the anomalous filter residue of HCNs, the cysteine located at the inner side of the selectivity filter. In potassium channels, this position is occupied by threonine or serine and forms the fourth and most internal ion binding site of the selectivity filter. We find that this cysteine in HCNs does not contribute to permeation or form a fourth binding site.

Regulation of GIP and GLP1 receptor cell surface expression by N-glycosylation and receptor heteromerization.

In response to a meal, Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) are released from gut endocrine cells into the circulation and interact with their cognate G-protein coupled receptors (GPCRs). Receptor activation results in tissue-selective pleiotropic responses that include augmentation of glucose-induced insulin secretion from pancreatic beta cells. N-glycosylation and receptor oligomerization are co-translational processes that are thought to regulate the exit of functional GPCRs from the ER and their maintenance at the plasma membrane. Despite the importance of these regulatory processes, their impact on functional expression of GIP and GLP-1 receptors has not been well studied. Like many family B GPCRs, both the GIP and GLP-1 receptors possess a large extracellular N-terminus with multiple consensus sites for Asn-linked (N)-glycosylation. Here, we show that each of these Asn residues is glycosylated when either human receptor is expressed in Chinese hamster ovary cells. N-glycosylation enhances cell surface expression and function in parallel but exerts stronger control over the GIP receptor than the GLP-1 receptor. N-glycosylation mainly lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. N-glycosylation is also required for expression of the GIP receptor at the plasma membrane and efficient GIP potentiation of glucose-induced insulin secretion from the INS-1 pancreatic beta cell line. Functional expression of a GIP receptor mutant lacking N-glycosylation is rescued by co-expressed wild type GLP1 receptor, which, together with data obtained using Bioluminescence Resonance Energy Transfer, suggests formation of a GIP-GLP1 receptor heteromer.

Asymmetric divergence in structure and function of HCN channel duplicates in Ciona intestinalis.

Hyperpolarization-activated Cyclic Nucleotide (HCN) channels are voltage-gated cation channels and are critical for regulation of membrane potential in electrically active cells. To understand the evolution of these channels at the molecular level, we cloned and examined two of three HCN homologs of the urochordate Ciona intestinalis (ciHCNa and ciHCNb). ciHCNa is like mammalian HCNs in that it possesses similar electrical function and undergoes N-glycosylation of a sequon near the pore. ciHCNb lacks the pore-associated N-glycosylation sequon and is predictably not N-glycosylated, and it also has an unusual gating phenotype in which the channel's voltage-sensitive gate appears to close incompletely. Together with previous findings, the data support an evolutionary trajectory in which an HCN ancestor underwent lineage-specific duplication in Ciona, to yield one HCN with most features that are conserved with the mammalian HCNs and another HCN that has been uniquely altered.

Alanine scanning of the S6 segment reveals a unique and cAMP-sensitive association between the pore and voltage-dependent opening in HCN channels.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels resemble Shaker K+ channels in structure and function. In both, changes in membrane voltage produce directionally similar movement of positively charged residues in the voltage sensor to alter the pore structure at the intracellular side and gate ion flow. However, HCNs open when hyperpolarized, whereas Shaker opens when depolarized. Thus, electromechanical coupling between the voltage sensor and gate is opposite. A key determinant of this coupling is the intrinsic stability of the pore. In Shaker, an alanine/valine scan of residues across the pore, by single point mutation, showed that most mutations made the channel easier to open and steepened the response of the channel to changes in voltage. Because most mutations likely destabilize protein packing, the Shaker pore is most stable when closed, and the voltage sensor works to open it. In HCN channels, the pore energetics and vector of work by the voltage sensor are unknown. Accordingly, we performed a 22-residue alanine/valine scan of the distal pore of the HCN2 isoform and show that the effects of mutations on channel opening and on the steepness of the response of the channel to voltage are mixed and smaller than those in Shaker. These data imply that the stabilities of the open and closed pore are similar, the voltage sensor must apply force to close the pore, and the interactions between the pore and voltage sensor are weak. Moreover, cAMP binding to the channel heightens the effects of the mutations, indicating stronger interactions between the pore and voltage sensor, and tips the energetic balance toward a more stable open state.