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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels 1 are essential for pacemaking activity and neural signalling 2,3 . Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain 4 and epileptic seizures 5 . The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor 6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs.  7,8 ). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Neuropathic pain impairs quality of life, is widely prevalent, and incurs significant costs. Current pharmacological therapies have poor/no efficacy and significant adverse effects; safe and effective alternatives are needed. Hyperpolarisation-activated cyclic nucleotide-regulated (HCN) channels are causally implicated in some forms of peripherally mediated neuropathic pain. Whilst 2,6-substituted phenols, such as 2,6-di-tert-butylphenol (26DTB-P), selectively inhibit HCN1 gating and are antihyperalgesic, the development of therapeutically tolerable, HCN-selective antihyperalgesics based on their inverse agonist activity requires that such drugs spare the cardiac isoforms and do not cross the blood-brain barrier., Methods: In silico molecular dynamics simulation, in vitro electrophysiology, and in vivo rat spared nerve injury methods were used to test whether 'hindered' variants of 26DTB-P (wherein a hydrophilic 'anchor' is attached in the para-position of 26DTB-P via an acyl chain 'tether') had the desired properties., Results: Molecular dynamics simulation showed that membrane penetration of hindered 26DTB-Ps is controlled by a tethered diol anchor without elimination of head group rotational freedom. In vitro and in vivo analysis showed that BP4L-18:1:1, a variant wherein a diol anchor is attached to 26DTB-P via an 18-carbon tether, is an HCN1 inverse agonist and an orally available antihyperalgesic. With a CNS multiparameter optimisation score of 2.25, a >100-fold lower drug load in the brain vs blood, and an absence of adverse cardiovascular or CNS effects, BP4L-18:1:1 was shown to be poorly CNS penetrant and cardiac sparing., Conclusions: These findings provide a proof-of-concept demonstration that anchor-tethered drugs are a new chemotype for treatment of disorders involving membrane targets., (Copyright © 2023 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

2,6-di- tert -butylphenol (2,6-DTBP) ameliorates mechanical allodynia and thermal hyperalgesia produced by partial sciatic nerve ligation in mice, and selectively inhibits HCN1 channel gating. We hypothesized that the clinically utilized non-anesthetic dimerized congener of 2,6-DTBP, probucol (2,6-di- tert -butyl-4-[2-(3,5-di- tert -butyl-4-hydroxyphenyl)sulfanylpropan-2-ylsulfanyl]phenol), would relieve the neuropathic phenotype that results from peripheral nerve damage, and that the anti-hyperalgesic efficacy in vivo would correlate with HCN1 channel inhibition in vitro. A single oral dose of probucol (800 mg/kg) relieved mechanical allodynia and thermal hyperalgesia in a mouse spared-nerve injury neuropathic pain model. While the low aqueous solubility of probucol precluded assessment of its possible interaction with HCN1 channels, our results, in conjunction with recent data demonstrating that probucol reduces lipopolysaccharide-induced mechanical allodynia and thermal hyperalgesia, support the testing/development of probucol as a non-opioid, oral antihyperalgesic albeit one of unknown mechanistic action., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Peter A. Goldstein reports financial support was provided by US Department of Defense. Olaf A. Andersen reports financial support was provided by National Institutes of Health. Dianna E. Willis reports financial support was provided by National Institutes of Health. Peter A. Goldstein reports a relationship with Akelos, Inc. that includes: board membership and non-financial support. Gareth R. Tibbs reports a relationship with Akelos, Inc. that includes: board membership and non-financial support. Dianna E. Willis reports a relationship with Akelos, Inc. that includes: board membership and non-financial support. J. David Warren reports a relationship with Akelos, Inc. that includes: board membership and non-financial support. Peter A. Goldstein, Rebecca L. Joyce, and Gareth R. Tibbs are co-inventors on patents related to the development of alkylphenols for the treatment of neuropathic pain. J. David Warren, Dianna E. Willis, Gareth R. Tibbs, and Peter A. Goldstein serve on the Scientific Advisory Board for Akelos, Inc., a research-based biotechnology company that has secured a licensing agreement for the use of those patents., (© 2023 The Authors.)

Background and Purpose: In models of neuropathic pain, inhibition of HCN1 is anti-hyperalgesic. 2,6-di-iso-propyl phenol (propofol) and its non-anesthetic congener, 2,6-di-tert-butyl phenol, inhibit HCN1 channels by stabilizing closed state(s)., Experimental Approach: Using in vitro electrophysiology and kinetic modeling, we systematically explore the contribution of ligand architecture to alkylphenol-channel coupling., Key Results: When corrected for changes in hydrophobicity (and propensity for intra-membrane partitioning), the decrease in potency upon 1-position substitution (NCO∼OH >> SH >>> F) mirrors the ligands' H-bond acceptor (NCO > OH > SH >>> F) but not donor profile (OH > SH >>> NCO∼F). H-bond elimination (OH to F) corresponds to a ΔΔG of ∼4.5 kCal mol -1 loss of potency with little or no disruption of efficacy. Substitution of compact alkyl groups (iso-propyl, tert-butyl) with shorter (ethyl, methyl) or more extended (sec-butyl) adducts disrupts both potency and efficacy. Ring saturation (with the obligate loss of both planarity and π electrons) primarily disrupts efficacy., Conclusions and Implications: A hydrophobicity-independent decrement in potency at higher volumes suggests the alkylbenzene site has a volume of ≥800 Å 3 . Within this, a relatively static (with respect to ligand) H-bond donor contributes to initial binding with little involvement in generation of coupling energy. The influence of π electrons/ring planarity and alkyl adducts on efficacy reveals these aspects of the ligand present towards a face of the channel that undergoes structural changes during opening. The site's characteristics suggest it is "druggable"; introduction of other adducts on the ring may generate higher potency inverse agonists., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The ability of a diverse group of agents to produce general anesthesia has long been an area of intense speculation and investigation. Over the past century, we have seen a paradigm shift from proposing that the anesthetized state arises from nonspecific interaction of anesthetics with the lipid membrane to the recognition that the function of distinct, and identifiable, membrane-embedded proteins is dramatically altered in the presence of intravenous and inhaled agents. Among proteinaceous targets, metabotropic and ionotropic receptors garnered much of the attention over the last 30 years, and it is only relatively recently that voltage-gated ion channels have clearly and rigorously been shown to be important molecular targets. In this review, we will consider the experimental issues relevant to two important ion channel anesthetic targets, HCN and K 2P ., (© 2018 Elsevier Inc. All rights reserved.)

Neuropathic pain arises from injury to the nervous system. Conditions associated with neuropathic pain are diverse, and lesions and/or pathological changes in the central nervous system (CNS) or peripheral nervous system (PNS) can frequently, but not always, be identified. It is difficult to treat, with patients often on multiple, different classes of medications, all with appreciable adverse side effect profiles. Consequently, there is a pressing need for the development of new medications. The development of such therapeutics is predicated on a clear understanding of the relevant molecular and cellular processes that contribute to the development, and maintenance, of the neuropathic pain state. One proposed mechanism thought to contribute to the ontogeny of neuropathic pain is altered expression, trafficking, and functioning of ion channels expressed by primary sensory neurons. Here, we will focus on three voltage-gated ion channel families, CaV, HCN, and NaV, first reviewing the preclinical data and then the human data where it exists., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na+-K+ permeable IH pacemaker current. As with other voltage-gated members of the 6-transmembrane KV channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6 albeit this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg2+ which blocks the open channel pore and blunts the inhibitory effect of outward K+ flux. Here, we show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg2+ block. A combination of experimental and simulation studies demonstrates that agonist acceleration of block is mediated via acceleration of the blocking reaction itself rather than as a secondary consequence of the cAMP enhancement of channel opening. These results suggest that the activation status of the gating ring and the open state of the pore are not coupled in an obligate manner (as required by the often invoked Monod-Wyman-Changeux allosteric model) but couple more loosely (as envisioned in a modular model of protein activation). Importantly, the emergence of second messenger sensitivity of open channel rectification suggests that loose coupling may have an unexpected consequence: it may endow these erstwhile "slow" channels with an ability to exert voltage and ligand-modulated control over cellular excitability on the fastest of physiologically relevant time scales.

Chronic pain after peripheral nerve injury is associated with afferent hyperexcitability and upregulation of hyperpolarization-activated, cyclic nucleotide-regulated (HCN)-mediated IH pacemaker currents in sensory neurons. HCN channels thus constitute an attractive target for treating chronic pain. HCN channels are ubiquitously expressed; analgesics targeting HCN1-rich cells in the peripheral nervous system must spare the cardiac pacemaker current (carried mostly by HCN2 and HCN4) and the central nervous system (where all four isoforms are expressed). The alkylphenol general anesthetic propofol (2,6-di-iso-propylphenol) selectively inhibits HCN1 channels versus HCN2-HCN4 and exhibits a modest pharmacokinetic preference for the periphery. Consequently, we hypothesized that propofol, and congeners, should be antihyperalgesic. Alkyl-substituted propofol analogs have different rank-order potencies with respect to HCN1 inhibition, GABA(A) receptor (GABA(A)-R) potentiation, and general anesthesia. Thus, 2,6- and 2,4-di-tertbutylphenol (2,6- and 2,4-DTBP, respectively) are more potent HCN1 antagonists than propofol, whereas 2,6- and 2,4-di-sec-butylphenol (2,6- and 2,4-DSBP, respectively) are less potent. In contrast, DSBPs, but not DTBPs, enhance GABA(A)-R function and are general anesthetics. 2,6-DTBP retained propofol's selectivity for HCN1 over HCN2-HCN4. In a peripheral nerve ligation model of neuropathic pain, 2,6-DTBP and subhypnotic propofol are antihyperalgesic. The findings are consistent with these alkylphenols exerting analgesia via non-GABA(A)-R targets and suggest that antagonism of central HCN1 channels may be of limited importance to general anesthesia. Alkylphenols are hydrophobic, and thus potential modifiers of lipid bilayers, but their effects on HCN channels are due to direct drug-channel interactions because they have little bilayer-modifying effect at therapeutic concentrations. The alkylphenol antihyperalgesic target may be HCN1 channels in the damaged peripheral nervous system.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate a pacemaking current, I(h), which regulates neuronal excitability and oscillatory activity in the brain. Although all four HCN isoforms are expressed in the brain, the functional contribution of HCN3 is unknown. Using immunohistochemistry, confocal microscopy, and whole-cell patch-clamp recording techniques, we investigated HCN3 function in thalamic intergeniculate leaflet (IGL) neurons, as HCN3 is reportedly preferentially expressed in these cells. We observed that I(h) recorded from IGL, but not ventral geniculate nucleus, neurons in HCN2(+/+) mice and rats activated slowly and were cAMP insensitive, which are hallmarks of HCN3 channels. We also observed strong immunolabeling for HCN3, with no labeling for HCN1 and HCN4, and only very weak labeling for HCN2. Deletion of HCN2 did not alter I(h) characteristics in mouse IGL neurons. These data together indicate that the HCN3 channel isoform generated I(h) in IGL neurons. Intracellular phosphatidylinositol-4,5-bisphosphate (PIP(2)) shifted I(h) activation to more depolarized potentials and accelerated activation kinetics. Upregulation of HCN3 function by PIP(2) augmented low-threshold burst firing and spontaneous oscillations; conversely, depletion of PIP(2) or pharmacologic block of I(h) resulted in a profound inhibition of excitability. The results indicate that functional expression of HCN3 channels in IGL neurons is crucial for intrinsic excitability and rhythmic burst firing, and PIP(2) serves as a powerful modulator of I(h)-dependent properties via an effect on HCN3 channel gating. Since the IGL is a major input to the suprachiasmatic nucleus, regulation of pacemaking function by PIP(2) in the IGL may influence sleep and circadian rhythms.

Investigation of the mechanistic bases and physiological importance of cAMP regulation of HCN channels has exploited an arginine to glutamate mutation in the nucleotide-binding fold, an approach critically dependent on the mutation selectively lowering the channel's nucleotide affinity. In apparent conflict with this, in intact Xenopus oocytes, HCN and HCN-RE channels exhibit qualitatively and quantitatively distinct responses to the tyrosine kinase inhibitor, genistein -- the estrogenic isoflavonoid strongly depolarizes the activation mid-point of HCN1-R538E, but not HCN1 channels (+9.8 mV + or - 0.9 versus +2.2 mV + or - 0.6) and hyperpolarizes gating of HCN2 (-4.8 mV + or - 1.0) but depolarizes gating of HCN2-R591E (+13.2 mV + or - 2.1). However, excised patch recording, X-ray crystallography and modeling reveal that this is not due to either a fundamental effect of the mutation on channel gating per se or of genistein acting as a mutation-sensitive partial agonist at the cAMP site. Rather, we find that genistein equivalently moves both HCN and HCN-RE channels closer to the open state (rendering the channels inherently easier to open but at a cost of decreasing the coupling energy of cAMP) and that the anomaly reflects a balance of these energetic effects with the isoform-specific inhibition of activation by the nucleotide gating ring and relief of this by endogenous cAMP. These findings have specific implications with regard to findings based on HCN-RE channels and kinase antagonists and general implications with respect to interpretation of drug effects in mutant channel backgrounds.

We explored the structural basis of voltage sensing in the HCN1 hyperpolarization-activated cyclic nucleotide-gated cation channel by examining the relative orientation of the voltage sensor and pore domains. The opening of channels engineered to contain single cysteine residues at the extracellular ends of the voltage-sensing S4 (V246C) and pore-forming S5 (C303) domains is inhibited by formation of disulfide or cysteine:Cd(2+) bonds. As Cd(2+) coordination is promoted by depolarization, the S4-S5 interaction occurs preferentially in the closed state. The failure of oxidation to catalyze dimer formation, as assayed by Western blotting, indicates the V246C:C303 interaction occurs within a subunit. Intriguingly, a similar interaction has been observed in depolarization-activated Shaker voltage-dependent potassium (Kv) channels at depolarized potentials but such an intrasubunit interaction is inconsistent with the X-ray crystal structure of Kv1.2, wherein S4 approaches S5 of an adjacent subunit. These findings suggest channels of opposite voltage-sensing polarity adopt a conserved S4-S5 orientation in the depolarized state that is distinct from that trapped upon crystallization.

I(H) pacemaker channels carry a mixed monovalent cation current that, under physiological ion gradients, reverses at approximately -34 mV, reflecting a 4:1 selectivity for K over Na. However, I(H) channels display anomalous behavior with respect to permeant ions such that (a) open channels do not exhibit the outward rectification anticipated assuming independence; (b) gating and selectivity are sensitive to the identity and concentrations of externally presented permeant ions; (c) the channels' ability to carry an inward Na current requires the presence of external K even though K is a minor charge carrier at negative voltages. Here we show that open HCN channels (the hyperpolarization-activated, cyclic nucleotide sensitive pore forming subunits of I(H)) undergo a fast, voltage-dependent block by intracellular Mg in a manner that suggests the ion binds close to, or within, the selectivity filter. Eliminating internal divalent ion block reveals that (a) the K dependence of conduction is mediated via K occupancy of site(s) within the pore and that asymmetrical occupancy and/or coupling of these sites to flux further shapes ion flow, and (b) the kinetics of equilibration between K-vacant and K-occupied states of the pore (10-20 micros or faster) is close to the ion transit time when the pore is occupied by K alone ( approximately 0.5-3 micros), a finding that indicates that either ion:ion repulsion involving Na is adequate to support flux (albeit at a rate below our detection threshold) and/or the pore undergoes rapid, permeant ion-sensitive equilibration between nonconducting and conducting configurations. Biophysically, further exploration of the Mg site and of interactions of Na and K within the pore will tell us much about the architecture and operation of this unusual pore. Physiologically, these results suggest ways in which "slow" pacemaker channels may contribute dynamically to the shaping of fast processes such as Na-K or Ca action potentials.

Activation of native IH pacemaker channels and channels formed on heterologous expression of some isoforms of their pore forming HCN (hyperpolarization-activated, cyclic nucleotide-regulated) subunits is inhibited by the intravenous general anaesthetic propofol (2,6-diisopropylphenol). Here, we show that inhibition of homomeric HCN1 channels is mediated through anaesthetic association with the membrane embedded channel core, a domain that is highly conserved between this isoform and the relatively insensitive HCN2 and 4 subunits. Decoupling of HCN channel gating from cAMP and internal protons reveals that changes in these second messengers are neither necessary nor sufficient to account for propofol's actions. Modelling of the equilibrium and kinetic behaviour of HCN1 channels in the absence and presence of anaesthetic reveals that (1) gating is best described by models wherein closed and open states communicate via a voltage-independent reaction with no significant equilibrium occupancy of a deactivated open state at non-permissive voltages, and (2) propofol modifies gating by preferentially associating with closed-resting and closed-activated states but a low affinity interaction with the activated open state shapes the effect of the drug under physiological conditions. Our findings illuminate the mechanism of HCN channel gating and provide a framework that will facilitate development of propofol derivates that have altered pharmacological properties and therapeutic potentials.

Hyperpolarization-activated pacemaker currents (I(H)) contribute to the subthreshold properties of excitable cells and thereby influence behaviors such as synaptic integration and the appearance and frequency of intrinsic rhythmic activity. Accordingly, modulation of I(H) contributes to cellular plasticity. Although I(H) activation is regulated by a plethora of neurotransmitters, including some that act via phospholipase C (PLC), the only second messengers known to alter I(H) voltage dependence are cAMP, internal protons (H+(I)s), and phosphatidylinositol-4,5-phosphate. Here, we show that 4beta-phorbol-12-myristate-13-acetate (4betaPMA), a stereoselective C-1 diacylglycerol-binding site agonist, enhances voltage-dependent opening of wild-type and cAMP/H+(I)-uncoupled hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels, but does not alter gating of the plant hyperpolarization-activated channel, KAT1. Pharmacological analysis indicates that 4betaPMA exerts its effects on HCN gating via sequential activation of PKC and diacylglycerol kinase (DGK) coupled with upregulation of MAPK (mitogen-activated protein kinase) and phospholipase A2 (PLA2), but its action is independent of phosphoinositide kinase 3 (PI3K) and PI4K. Demonstration that both phosphatidic acid and arachidonic acid (AA) directly facilitate HCN gating suggests that these metabolites may serve as the messengers downstream of DGK and PLA2, respectively. 4BetaPMA-mediated suppression of the maximal HCN current likely arises from channel interaction with AA coupled with an enhanced membrane retrieval triggered by the same pathways that modulate channel gating. These results indicate that regulation of excitable cell behavior by neurotransmitter-mediated modulation of I(H) may be exerted via changes in three signaling lipids in addition to the allosteric actions of cAMP and H+(I)s.

Propofol (2,6-diisopropylphenol) is a widely used intravenous general anesthetic, which has been reported to produce bradycardia in patients at concentrations associated with profound sedation and loss of consciousness. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels conduct a monovalent cationic current I(h) (also known as I(q) or I(f)) that contributes to autorhythmicity in both the brain and heart. Here we studied the effects of propofol on recombinant HCN1, HCN2, and HCN4 channels and found that the drug inhibits and slows activation of all three channels at clinically relevant concentrations. In oocyte expression studies, HCN1 channel activation was most sensitive to slowing by propofol (EC(50) values of 5.6 +/- 1.0 microM for fast component and 31.5 +/- 7.5 microM for slow component). HCN1 channels also showed a marked propofol-induced hyperpolarizing shift in the voltage dependence of activation (EC(50) of 6.7 +/- 1.0 microM) and accelerated deactivation (EC(50) of 4.5 +/- 0.9 microM). Furthermore, propofol reduced heart rate in an isolated guinea pig heart preparation over the same range of concentrations. These data suggest that propofol modulation of HCN channel gating is an important molecular mechanism that can contribute to the depression of central nervous system function and also lead to bradyarrhythmias in patients receiving propofol during surgical anesthesia.

Hyperpolarization-activated cation channels of the HCN gene family contribute to spontaneous rhythmic activity in both heart and brain. All four family members contain both a core transmembrane segment domain, homologous to the S1-S6 regions of voltage-gated K+ channels, and a carboxy-terminal 120 amino-acid cyclic nucleotide-binding domain (CNBD) motif. Homologous CNBDs are responsible for the direct activation of cyclic nucleotide-gated channels and for modulation of the HERG voltage-gated K+ channel--important for visual and olfactory signalling and for cardiac repolarization, respectively. The direct binding of cyclic AMP to the cytoplasmic site on HCN channels permits the channels to open more rapidly and completely after repolarization of the action potential, thereby accelerating rhythmogenesis. However, the mechanism by which cAMP binding modulates HCN channel gating and the basis for functional differences between HCN isoforms remain unknown. Here we demonstrate by constructing truncation mutants that the CNBD inhibits activation of the core transmembrane domain. cAMP binding relieves this inhibition. Differences in activation gating and extent of cAMP modulation between the HCN1 and HCN2 isoforms result largely from differences in the efficacy of CNBD inhibition.

The hyperpolarization-activated cation current (termed I(h), I(q), or I(f)) was recently shown to be encoded by a new family of genes, named HCN for hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective. When expressed in heterologous cells, each HCN isoform generates channels with distinct activation kinetics, mirroring the range of biophysical properties of native I(h) currents recorded in different classes of neurons. To determine whether the functional diversity of I(h) currents is attributable to different patterns of HCN gene expression, we determined the mRNA distribution across different regions of the mouse CNS of the three mouse HCN genes that are prominently expressed there (mHCN1, 2 and 4). We observe distinct patterns of distribution for each of the three genes. Whereas mHCN2 shows a widespread expression throughout the CNS, the expression of mHCN1 and mHCN4 is more limited, and generally complementary. mHCN1 is primarily expressed within neurons of the neocortex, hippocampus, and cerebellar cortex, but also in selected nuclei of the brainstem. mHCN4 is most highly expressed within neurons of the medial habenula, thalamus, and olfactory bulb, but also in distinct neuronal populations of the basal ganglia. Based on a comparison of mRNA expression with an electrophysiological characterization of native I(h) currents in hippocampal and thalamic neurons, our data support the idea that the functional heterogeneity of I(h) channels is attributable, in part, to differential isoform expression. Moreover, in some neurons, specific functional roles can be proposed for I(h) channels with defined subunit composition.

The molecular basis of the hyperpolarization-activated cation channels that underlie the anomalous rectifying current variously termed Ih, Iq, or I(f) is discussed. On the basis of the expression patterns and biophysical properties of the newly cloned HCN ion channels, an initial attempt at defining the identity and subunit composition of channels underlying native Ih is undertaken. By comparing the sequences of HCN channels to other members of the K channel superfamily, we discuss how channel opening may be coupled to membrane hyperpolarization and to direct binding of cyclic nucleotide. Finally, we consider some of the questions in cardiovascular physiology and neurobiology that can be addressed as a result of the demonstration that Ih is encoded by the HCN gene family.

Cyclic nucleotide-gated ion channels are composed of four pore-forming subunits. Binding of cyclic nucleotide to a site in the intracellular carboxyl terminus of each subunit leads to channel activation. Since there are four subunits, four binding events are possible. In this study, we investigate the effects of individual binding events on activation by studying channels containing one, two, three, or four functional binding sites. The binding of a single ligand significantly increases opening, although four ligands are required for full activation. The data are inconsistent with models in which the four subunits activate in a single concerted step (Monod-Wyman-Changeux model) or in four independent steps (Hodgkin-Huxley model). Instead, the four subunits may associate and activate as two independent dimers.

The generation of pacemaker activity in heart and brain is mediated by hyperpolarization-activated cation channels that are directly regulated by cyclic nucleotides. We previously cloned a novel member of the voltage-gated K channel family from mouse brain (mBCNG-1) that contained a carboxy-terminal cyclic nucleotide-binding domain (Santoro et al., 1997) and hence proposed it to be a candidate gene for pacemaker channels. Heterologous expression of mBCNG-1 demonstrates that it does indeed code for a channel with properties indistinguishable from pacemaker channels in brain and similar to those in heart. Three additional mouse genes and two human genes closely related to mBCNG-1 display unique patterns of mRNA expression in different tissues, including brain and heart, demonstrating that these channels constitute a widely expressed gene family.

Activation of cyclic nucleotide-gated channels is thought to involve two distinct steps: a recognition event in which a ligand binds to the channel and a conformational change that both opens the channel and increases the affinity of the channel for an agonist. Sequence similarity with the cyclic nucleotide-binding sites of cAMP- and cGMP-dependent protein kinases and the bacterial catabolite activating protein (CAP) suggests that the channel ligand binding site consists of a beta-roll and three alpha-helices. Recent evidence has demonstrated that the third (or C) alpha-helix moves relative to the agonist upon channel activation, forming additional favorable contacts with the purine ring. Here we ask if channel activation also involves structural changes in the beta-roll by investigating the contribution of a conserved arginine residue that, in CAP and the kinases, forms an important ionic interaction with the cyclized phosphate of the bound ligand. Mutations that conserve, neutralize, or reverse the charge on this arginine decreased the apparent affinity for ligand over four orders of magnitude but had little effect on the ability of bound ligand to open the channel. These data indicate that the cyclized phosphate of the nucleotide approaches to within 2-4 A of the arginine, forming a favorable ionic bond that is largely unaltered upon activation. Thus, the binding site appears to be polarized into two distinct structural and functional domains: the beta-roll stabilizes the ligand in a state-independent manner, whereas the C-helix selectively stabilizes the ligand in the open state of the channel. It is likely that these distinct contributions of the nucleotide/C-helix and nucleotide/beta-roll interactions may also be a general feature of the mechanism of activation of other cyclic nucleotide-binding proteins.

Despite recent advances in the identification of ligand-binding and voltage-sensing regions of ion channels, the domains that couple such regions to channel opening have not been identified. Moreover, it is uncertain whether ligand binding or depolarization are obligatory steps that must precede channel opening (according to linear reaction schemes) or whether they act to stabilize the channel in an open state that can exist independently of ligand binding or depolarization (according to cyclic allosteric models). By comparing ligand-independent and ligand-dependent channel openings, we now show that retinal and olfactory cyclic-nucleotide-gated channels are activated by a cyclic allosteric mechanism. We further show that an amino-terminal domain, distinct from the pore and ligand-binding motifs, participates in the allosteric gating transition, accounting for differences in the free energy of gating of the two channels. The allosteric transition provides an important mechanism for tuning the physiological response of ligand-binding proteins, such as cyclic-nucleotide-gated channels, to different biological signals.

The effects of four K+-channel inhibitors on synaptosomal free Ca2+ concentrations and 86Rb+ fluxes are analysed. 4-Aminopyridine, alpha-dendrotoxin, charybdotoxin, and tetraethylammonium all increase the free Ca2+ concentration, although their potencies differ widely. In each case, the elevation in free Ca2+ concentration is reversed by the subsequent addition of tetrodotoxin. The transient 86Rb+ efflux from preequilibrated synaptosomes induced with high concentrations of veratridine is partially inhibited by 4-aminopyridine and alpha-dendrotoxin. In contrast, when 4-aminopyridine or alpha-dendrotoxin is added to polarized synaptosomes, and enhanced 86Rb+ flux is seen, both for uptake and for efflux with no change in the total 86Rb+/K+ content of the synaptosomes and with only a slight time-averaged plasma membrane depolarization (6.4 and 3.3 mV, respectively). The enhancements of flux by 4-aminopyridine or alpha-dendrotoxin are sensitive to ouabain and/or to tetrodotoxin. Furthermore, these flux changes show the same concentration dependencies as the blocked component of veratridine-stimulated 86Rb+ efflux, the elevation of free Ca2+ concentration, and the facilitation of glutamate exocytosis that are elicited by 4-aminopyridine or alpha-dendrotoxin. It is concluded that these findings support the proposal of spontaneous, repetitive firing of synaptosomes evoked by K+-channel inhibitors and that the enhanced 86Rb+ flux is a consequence of the activity of 4-aminopyridine- and alpha-dendrotoxin-insensitive K+ channels during these action potentials.

Cyclic nucleotide-gated (CNG) ion channels are multimeric structures containing at least two subunits. However, the total number of subunits per functional channel is unknown. To determine the subunit stoichiometry of CNG ion channels, we have coexpressed the 30 pS conductance bovine retinal channel (RET) with an 85 pS conductance chimeric retinal channel containing the catfish olfactory channel P region (RO133). When RO133 and RET monomers are coexpressed, channels with four distinct intermediate conductances are observed. Dimer constructs reveal that two of these conductance levels arise from channels with the same subunit composition (2 RO133:2 RET) but distinct subunit order (like subunits adjacent to each other versus like subunits across from each other). Thus, the data demonstrate that cyclic nucleotide-gated ion channels are tetrameric like the related voltage-gated potassium ion channels; the order of subunits affects the conductance of the channel; and the channel has 4-fold symmetry in which four asymmetric subunits assemble head to tail around a central axis.

Studies on the activation of ligand- and voltage-gated ion channels have identified regions involved in both ligand binding and voltage sensing, but relatively little is known about how such domains are coupled to channel opening. Here we investigate the structural basis for the activation of cyclic-nucleotide-gated channels, which are directly opened by cytoplasmic cyclic nucleotides but are structurally homologous to voltage-gated channels. By constructing chimaeras between cyclic-nucleotide-gated channels cloned from bovine retinal photoreceptors and catfish olfactory neurons, we identify two distinct domains that are important for ligand binding and channel gating. A putative alpha-helix in the carboxy-terminal binding domain determines the selectivity of the channel for activation by cGMP relative to cAMP. A domain in the amino-terminal region determines the ease with which channels open and thus influences agonist efficacy. We propose that channel opening is coupled to an allosteric conformational change in the binding site which enhances agonist binding. Thus, cyclic nucleotides activate the channel by binding tightly to the open state and stabilizing it.

Due to the small size of most nerve terminals, the ion channels which underlie presynaptic currents are usually inaccessible to investigation by conventional electrophysiological techniques. Here we describe a simple method for obtaining single-channel recordings from synaptic plasma membranes that does not require exposure of the native membranes to exogenous lipids or fusogens. To illustrate the method, we have recorded single-channel activity from rat cerebrocortical synaptosomal membranes. Under conditions designed to isolate calcium-independent currents, we describe three channel types that are most commonly observed.

Ion permeation through membrane channels is thought to be governed by a narrow region of the channel pore termed the selectivity filter, which has been proposed to discriminate among ions by both specific binding and molecular sieving, as determined by pore diameter. Recent evidence suggests that a conserved domain (known as H5, P or SS1-SS2) in voltage-gated potassium, sodium and calcium channels contributes to the lining of the pore. Here we investigate whether the H5 domain determines pore diameter and examine the role of pore diameter in controlling ion permeation. These studies rely on differences in single channel conductance, ion selectivity and apparent pore diameter between cyclic nucleotide-gated channels cloned from bovine retina and catfish olfactory neurons. Using chimaeric retinal-olfactory channels, we find that the H5 domain determines these differences in permeation properties, providing structural evidence that the cyclic nucleotide-gated channels are indeed members of the voltage-gated channel family. Moreover, these results show directly that the H5 domain helps form the selectivity filter and that molecular sieving is important in controlling ion permeation.

Three polypeptides (?, ? and ?) homologous to ?-dendrotoxin, an inhibitor of certain voltage-activated K(+) channels, were found to elevate the cytosolic free concentration of calcium ([Ca(2+)](c)) in isolated central nerve terminals. Relative to ?-dendrotoxin (EC(50) ? 2.1 nM), the ?-, ?- and ?-toxins were 790-, 214- and 5.7-fold less effective; no additivity was apparent in the toxins' effects on [Ca(2+)](c). Each toxin antagonized the high affinity binding of (125)I-labelled ?- and ?-dendrotoxin to synaptosomes but with different potencies. The mutual interaction of ?- and ?-dendrotoxin with the acceptor appeared complex, the inhibition curves being noticeably extended. For the inhibition of binding of ?- and ?-dendrotoxin, the respective K(i)'s (nM) observed for ?, ?, ? and ? toxins were 0.78, 50, 99, 8, and 2.3, 95, 61, 0.53. Apparently, interactions of ?- and ?-, but not ?- or ?-dendrotoxin with the acceptor are closely coupled to an inhibition of K(+) channel(s) as observed indirectly by elevation of [Ca(2+)](c).

Nanomolar concentrations of charybdotoxin or dendrotoxin increase the cytoplasmic free Ca2+ concentration in isolated central nerve terminals. The effects of the two toxins, normally considered to be blockers of K+ channels controlled by voltage in a Ca2+-sensitive or -insensitive manner, respectively, show only marginal additivity. Apamin, and inhibitor of low conductance Ca2+-activated K+ channels, was without effect in either the absence or presence of dendrotoxin. The effect of charybdotoxin on polarized, isolated central nerve terminals seems to be mediated largely by a block of K+ channels sensitive to dendrotoxin. Apparently, these voltage-operated K+ channels make a more significant contribution to maintaining the polarized potential of synaptosomes than do those activated by Ca2+.

The release of endogenous glutamate from guinea-pig cerebrocortical synaptosomes evoked by dendrotoxin, beta-bungarotoxin, and 4-aminopyridine is compared. Dendrotoxin and 4-aminopyridine cause Ca2+-dependent release, representing a partial depletion of the KCl-releasable transmitter pool. The decrease in the plasma membrane potential caused by 4-aminopyridine or dendrotoxin and the evoked release of glutamate from a transmitter pool accord with the inhibitory action of these agents on certain K+ conductances. In contrast, the massive release of glutamate evoked by beta-bungarotoxin is produced in the presence of Ca2+ but not of Sr2+, a result consistent with a generalised permeabilisation of synaptosomal plasma membranes. Although dendrotoxin inhibits the binding of beta-bungarotoxin and the resultant synaptosomal lysis, demonstration of a direct effect of beta-bungarotoxin binding per se on K+ permeability is impractical owing to its phospholipase A2 activity.

The mechanisms by which an elevated KCl level and the K+-channel inhibitor 4-aminopyridine induce release of transmitter glutamate from guinea-pig cerebral cortical synaptosomes are contrasted. KCl at 30 mM caused an initial spike in the cytosolic free Ca2+ concentration ([Ca2+]c), followed by a partial recovery to a plateau 112 +/- 13 nM above the polarized control. The Ca2+-dependent release of endogenous glutamate, determined by continuous fluorimetry, was largely complete by 3 min, by which time 1.70 +/- 0.35 nmol/mg was released. [Ca2+]c elevation and glutamate release were both insensitive to tetrodotoxin. KCl-induced elevation in [Ca2+]c could be observed in both low-Na+ medium and in the presence of low concentrations of veratridine. 4-Aminopyridine at 1 mM increased [Ca2+]c by 143 +/- 18 nM to a plateau similar to that following 30 mM KCl. The initial rate of increase in [Ca2+]c following 4-aminopyridine administration was slower than that following 30 mM KCl, and a transient spike was less apparent. Consistent with this, the 4-aminopyridine-induced net uptake of 45Ca2+ is much lower than that following an elevated KCl level. 4-Aminopyridine induced the Ca2+-dependent release of glutamate, although with somewhat slower kinetics than that for KCl. The measured release was 0.81 nmol of glutamate/mg in the first 3 min of 4-aminopyridine action. In contrast to KCl, glutamate release and the increase in [Ca2+]c with 4-aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na+ channels. Basal [Ca2+]c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

The predicted presence in perfused rat hearts of a rapidly metabolized but hitherto unrecognized form of adenosine phosphate has been confirmed by specific radioactive labelling. The properties of the purified compound suggest that it is a heteropolymer of a small organic acid, phosphate and purine nucleoside in the proportions 1:4:1.