Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels as Drug Targets for Neurological Disorders.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that critically modulate neuronal activity. Four HCN subunits (HCN1-4) have been cloned, each having a unique expression profile and distinctive effects on neuronal excitability within the brain. Consistent with this, the expression and function of these subunits are altered in diverse ways in neurological disorders. Here, we review current knowledge on the structure and distribution of the individual HCN channel isoforms, their effects on neuronal activity under physiological conditions, and how their expression and function are altered in neurological disorders, particularly epilepsy, neuropathic pain, and affective disorders. We discuss the suitability of HCN channels as therapeutic targets and how drugs might be strategically designed to specifically act on particular isoforms. We conclude that medicines that target individual HCN isoforms and/or their auxiliary subunit, TRIP8b, may provide valuable means of treating distinct neurological conditions.

Cortical HCN channels: function, trafficking and plasticity.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated potassium ion channels. They are, however, activated by hyperpolarizing potentials and are permeable to cations. Four HCN subunits have been cloned, of which HCN1 and HCN2 subunits are predominantly expressed in the cortex. These subunits are principally located in pyramidal cell dendrites, although they are also found at lower concentrations in the somata of pyramidal neurons as well as other neuron subtypes. HCN channels are actively trafficked to dendrites by binding to the chaperone protein TRIP8b. Somato-dendritic HCN channels in pyramidal neurons modulate spike firing and synaptic potential integration by influencing the membrane resistance and resting membrane potential. Intriguingly, HCN channels are present in certain cortical axons and synaptic terminals too. Here, they regulate synaptic transmission but the underlying mechanisms appear to vary considerably amongst different synaptic terminals. In conclusion, HCN channels are expressed in multiple neuronal subcellular compartments in the cortex, where they have a diverse and complex effect on neuronal excitability.

The LIM homeodomain protein Lhx6 regulates maturation of interneurons and network excitability in the mammalian cortex.

Deletion of LIM homeodomain transcription factor-encoding Lhx6 gene in mice results in defective tangential migration of cortical interneurons and failure of differentiation of the somatostatin (Sst)- and parvalbumin (Pva)-expressing subtypes. Here, we characterize a novel hypomorphic allele of Lhx6 and demonstrate that reduced activity of this locus leads to widespread differentiation defects in Sst(+) interneurons, but relatively minor and localized changes in Pva(+) interneurons. The reduction in the number of Sst-expressing cells was not associated with a loss of interneurons, because the migration and number of Lhx6-expressing interneurons and expression of characteristic molecular markers, such as calretinin or Neuropeptide Y, were not affected in Lhx6 hypomorphic mice. Consistent with a selective deficit in the differentiation of Sst(+) interneurons in the CA1 subfield of the hippocampus, we observed reduced expression of metabotropic Glutamate Receptor 1 in the stratum oriens and characteristic changes in dendritic inhibition, but normal inhibitory input onto the somatic compartment of CA1 pyramidal cells. Moreover, Lhx6 hypomorphs show behavioral, histological, and electroencephalographic signs of recurrent seizure activity, starting from early adulthood. These results demonstrate that Lhx6 plays an important role in the maturation of cortical interneurons and the formation of inhibitory circuits in the mammalian cortex.

TRIP8b-independent trafficking and plasticity of adult cortical presynaptic HCN1 channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are subthreshold activated voltage-gated ion channels. In the cortex, these channels are predominantly expressed in dendrites where they significantly modify dendritic intrinsic excitability as well synaptic potential shapes and integration. HCN channel trafficking to dendrites is regulated by the protein, TRIP8b. Additionally, altered TRIP8b expression may be one mechanism underlying seizure-induced dendritic HCN channel plasticity. HCN channels, though, are also located in certain mature cortical synaptic terminals, where they play a vital role in modulating synaptic transmission. In this study, using electrophysiological recordings as well as electron microscopy we show that presynaptic, but not dendritic, cortical HCN channel expression and function is comparable in adult TRIP8b-null mice and wild-type littermates. We further investigated whether presynaptic HCN channels undergo seizure-dependent plasticity. We found that, like dendritic channels, wild-type presynaptic HCN channel function was persistently decreased following induction of kainic acid-induced seizures. Since TRIP8b does not affect presynaptic HCN subunit trafficking, seizure-dependent plasticity of these cortical HCN channels is not conditional upon TRIP8b. Our results, thus, suggest that the molecular mechanisms underlying HCN subunit targeting, expression and plasticity in adult neurons is compartment selective, providing a means by which pre- and postsynaptic processes that are critically dependent upon HCN channel function may be distinctly influenced.

T-type Ca2+ and persistent Na+ currents synergistically elevate ventral, not dorsal, entorhinal cortical stellate cell excitability.

Dorsal and ventral medial entorhinal cortex (mEC) regions have distinct neural network firing patterns to differentially support functions such as spatial memory. Accordingly, mEC layer II dorsal stellate neurons are less excitable than ventral neurons. This is partly because the densities of inhibitory conductances are higher in dorsal than ventral neurons. Here, we report that T-type Ca2+ currents increase 3-fold along the dorsal-ventral axis in mEC layer II stellate neurons, with twice as much CaV3.2 mRNA in ventral mEC compared with dorsal mEC. Long depolarizing stimuli trigger T-type Ca2+ currents, which interact with persistent Na+ currents to elevate the membrane voltage and spike firing in ventral, not dorsal, neurons. T-type Ca2+ currents themselves prolong excitatory postsynaptic potentials (EPSPs) to enhance their summation and spike coupling in ventral neurons only. These findings indicate that T-type Ca2+ currents critically influence the dorsal-ventral mEC stellate neuron excitability gradient and, thereby, mEC dorsal-ventral circuit activity.

Differential effects of Kv7 (M-) channels on synaptic integration in distinct subcellular compartments of rat hippocampal pyramidal neurons.

The K(V)7/M-current is an important determinant of neuronal excitability and plays a critical role in modulating action potential firing. In this study, using a combination of electrophysiology and computational modelling, we show that these channels selectively influence peri-somatic but not dendritic post-synaptic excitatory synaptic potential (EPSP) integration in CA1 pyramidal cells. K(V)7/M-channels are highly concentrated in axons. However, the competing peptide, ankyrin G binding peptide (ABP) that disrupts axonal K(V)7/M-channel function, had little effect on somatic EPSP integration, suggesting that this effect was due to local somatic channels only. This interpretation was confirmed using computer simulations. Further, in accordance with the biophysical properties of the K(V)7/M-current, the effect of somatic K(V)7/M-channels on synaptic potential summation was dependent upon the neuronal membrane potential. Somatic K(V)7/M-channels thus affect EPSP-spike coupling by altering EPSP integration. Interestingly, disruption of axonal channels enhanced EPSP-spike coupling by lowering the action potential threshold. Hence, somatic and axonal K(V)7/M-channels influence EPSP-spike coupling via different mechanisms. This may be important for their relative contributions to physiological processes such as synaptic plasticity as well as patho-physiological conditions such as epilepsy.

Functional significance of axonal Kv7 channels in hippocampal pyramidal neurons.

Members of the Kv7 family (Kv7.2-Kv7.5) generate a subthreshold K(+) current, the M- current. This regulates the excitability of many peripheral and central neurons. Recent evidence shows that Kv7.2 and Kv7.3 subunits are targeted to the axon initial segment of hippocampal neurons by association with ankyrin G. Further, spontaneous mutations in these subunits that impair axonal targeting cause human neonatal epilepsy. However, the precise functional significance of their axonal location is unknown. Using electrophysiological techniques together with a peptide that selectively disrupts axonal Kv7 targeting (ankyrin G-binding peptide, or ABP) and other pharmacological tools, we show that axonal Kv7 channels are critically and uniquely required for determining the inherent spontaneous firing of hippocampal CA1 pyramids, independently of alterations in synaptic activity. This action was primarily because of modulation of action potential threshold and resting membrane potential (RMP), amplified by control of intrinsic axosomatic membrane properties. Computer simulations verified these data when the axonal Kv7 density was three to five times that at the soma. The increased firing caused by axosomatic Kv7 channel block backpropagated into distal dendrites affecting their activity, despite these structures having fewer functional Kv7 channels. These results indicate that axonal Kv7 channels, by controlling axonal RMP and action potential threshold, are fundamental for regulating the inherent firing properties of CA1 hippocampal neurons.

Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis.

Hyperpolarization-activated cation nonselective 1 (HCN1) plasticity in entorhinal cortical (EC) and hippocampal pyramidal cell dendrites is a salient feature of temporal lobe epilepsy. However, the significance remains undetermined. We demonstrate that adult HCN1 null mice are more susceptible to kainic acid-induced seizures. After termination of these with an anticonvulsant, the mice also developed spontaneous behavioral seizures at a significantly more rapid rate than their wild-type littermates. This greater seizure susceptibility was accompanied by increased spontaneous activity in HCN1(-/-) EC layer III neurons. Dendritic Ih in these neurons was ablated, too. Consequentially, HCN1(-/-) dendrites were more excitable, despite having significantly more hyperpolarized resting membrane potentials (RMPs). In addition, the integration of EPSPs was enhanced considerably such that, at normal RMP, a 50 Hz train of EPSPs produced action potentials in HCN1(-/-) neurons. As a result of this enhanced pyramidal cell excitability, spontaneous EPSC frequency onto HCN1(-/-) neurons was considerably greater than that onto wild types, causing an imbalance between normal excitatory and inhibitory synaptic activity. These results suggest that dendritic HCN channels are likely to play a critical role in regulating cortical pyramidal cell excitability. Furthermore, these findings suggest that the reduction in dendritic HCN1 subunit expression during epileptogenesis is likely to facilitate the disorder.

The subthreshold-active KV7 current regulates neurotransmission by limiting spike-induced Ca2+ influx in hippocampal mossy fiber synaptic terminals.

Little is known about the properties and function of ion channels that affect synaptic terminal-resting properties. One particular subthreshold-active ion channel, the Kv7 potassium channel, is highly localized to axons, but its role in regulating synaptic terminal intrinsic excitability and release is largely unexplored. Using electrophysiological recordings together with computational modeling, we found that the KV7 current was active at rest in adult hippocampal mossy fiber synaptic terminals and enhanced their membrane conductance. The current also restrained action potential-induced Ca2+ influx via N- and P/Q-type Ca2+ channels in boutons. This was associated with a substantial reduction in the spike half-width and afterdepolarization following presynaptic spikes. Further, by constraining spike-induced Ca2+ influx, the presynaptic KV7 current decreased neurotransmission onto CA3 pyramidal neurons and short-term synaptic plasticity at the mossy fiber-CA3 synapse. This is a distinctive mechanism by which KV7 channels influence hippocampal neuronal excitability and synaptic plasticity.

Seizure-induced plasticity of h channels in entorhinal cortical layer III pyramidal neurons.

The entorhinal cortex (EC) provides the predominant excitatory drive to the hippocampal CA1 and subicular neurons in chronic epilepsy. Discerning the mechanisms underlying signal integration within EC neurons is essential for understanding network excitability alterations involving the hippocampus during epilepsy. Twenty-four hours following a single seizure episode when there were no behavioral or electrographic seizures, we found enhanced spontaneous activity still present in the rat EC in vivo and in vitro. The increased excitability was accompanied by a profound reduction in I(h) in EC layer III neurons and a significant decline in HCN1 and HCN2 subunits that encode for h channels. Consequently, dendritic excitability was enhanced, resulting in increased neuronal firing despite hyperpolarized membrane potentials. The loss of I(h) and the increased neuronal excitability persisted for 1 week following seizures. Our results suggest that dendritic I(h) plays an important role in determining the excitability of EC layer III neurons and their associated neural networks.

HCN1 channels reduce the rate of exocytosis from a subset of cortical synaptic terminals.

The hyperpolarization-activated cyclic nucleotide-gated (HCN1) channels are predominantly located in pyramidal cell dendrites within the cortex. Recent evidence suggests these channels also exist pre-synaptically in a subset of synaptic terminals within the mature entorhinal cortex (EC). Inhibition of pre-synaptic HCN channels enhances miniature excitatory post-synaptic currents (mEPSCs) onto EC layer III pyramidal neurons, suggesting that these channels decrease the release of the neurotransmitter, glutamate. Thus, do pre-synaptic HCN channels alter the rate of synaptic vesicle exocytosis and thereby enhance neurotransmitter release? To address this, we imaged the release of FM1-43, a dye that is incorporated into synaptic vesicles, from EC synaptic terminals using two photon microscopy in slices obtained from forebrain specific HCN1 deficient mice, global HCN1 knockouts and their wildtype littermates. This coupled with electrophysiology and pharmacology showed that HCN1 channels restrict the rate of exocytosis from a subset of cortical synaptic terminals within the EC and in this way, constrain non-action potential-dependent and action potential-dependent spontaneous release as well as synchronous, evoked release. Since HCN1 channels also affect post-synaptic potential kinetics and integration, our results indicate that there are diverse ways by which HCN1 channels influence synaptic strength and plasticity.

Recording Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Currents (Ih) in Neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that play a crucial role in many physiological processes such as memory formation and spatial navigation. Alterations in expression and function of HCN channels have also been associated with multiple disorders including epilepsy, neuropathic pain, and anxiety/depression. Interestingly, neuronal HCN currents (Ih) have diverse biophysical properties in different neurons. This is likely to be in part caused by the heterogeneity of the HCN subunits expressed in neurons. This variation in biophysical characteristics is likely to influence how Ih affects neuronal activity. Thus, it is important to record Ih directly from individual neurons. This protocol describes voltage-clamp methods that can be used to record neuronal Ih under whole-cell voltage-clamp conditions, in cell-attached mode, or with outside-out patches. The information obtained using this approach can be used in combination with other techniques such as computational modeling to determine the significance of Ih for neuronal function.

Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Currents in Neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that activate at potentials more negative than -50 mV and are predominantly permeable to Na(+) and K(+) ions. Four HCN subunits (HCN1-4) have been cloned. These subunits have distinct expression patterns and biophysical properties. In addition, cyclic nucleotides as well as multiple intracellular substances including various kinases and phosphatases modulate the expression and function of the subunits. Hence, the characteristics of the current, Ih, are likely to vary among neuronal subtypes. In many neuronal subtypes, Ih is present postsynaptically, where it plays a critical role in setting the resting membrane potential and the membrane resistance. By influencing these intrinsic properties, Ih will affect synaptic potential shapes and summation and thereby affect neuronal excitability. Additionally, Ih can have an effect on resonance properties and intrinsic neuronal oscillations. In some neurons, Ih may also be present presynaptically in axons and synaptic terminals, where it modulates neuronal transmitter release. Hence the effects of Ih on neuronal excitability are complex. It is, however, necessary to fully understand these as Ih has a significant impact on physiological conditions such as learning as well as pathophysiological states such as epilepsy.

Cholinergic afferent stimulation induces axonal function plasticity in adult hippocampal granule cells.

Acetylcholine critically influences hippocampal-dependent learning. Cholinergic fibers innervate hippocampal neuron axons, dendrites, and somata. The effects of acetylcholine on axonal information processing, though, remain unknown. By stimulating cholinergic fibers and making electrophysiological recordings from hippocampal dentate gyrus granule cells, we show that synaptically released acetylcholine preferentially lowered the action potential threshold, enhancing intrinsic excitability and synaptic potential-spike coupling. These effects persisted for at least 30 min after the stimulation paradigm and were due to muscarinic receptor activation. This caused sustained elevation of axonal intracellular Ca(2+) via T-type Ca(2+) channels, as indicated by two-photon imaging. The enhanced Ca(2+) levels inhibited an axonal KV7/M current, decreasing the spike threshold. In support, immunohistochemistry revealed muscarinic M1 receptor, CaV3.2, and KV7.2/7.3 subunit localization in granule cell axons. Since alterations in axonal signaling affect neuronal firing patterns and neurotransmitter release, this is an unreported cellular mechanism by which acetylcholine might, at least partly, enhance cognitive processing.

Recording dendritic ion channel properties and function from cortical neurons.

Dendrites emerging from the cell bodies of neurons receive the majority of synaptic inputs. They possess a plethora of ion channels that are essential for the processing of these synaptic signals. To fully understand how dendritic ion channels influence neuronal information processing, various patch-clamp techniques that allow electrophysiological recordings to be made directly from dendrites have been developed. In this chapter, I describe one such method that is suitable for making electrophysiological recordings from the apical dendrites of hippocampal and cortical pyramidal neurons.

HCN and KV7 (M-) channels as targets for epilepsy treatment.

Voltage-gated ion channels are important determinants of cellular excitability. The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) and KV7 (M-) channels are voltage-gated ion channels. Both channels are activated at sub-threshold potentials and have biophysical properties that mirror each other. KV7 channels inhibit neuronal excitability. Thus, mutations in KV7 channels that are associated with Benign Familial Neonatal Convulsions (BFNC) are likely to be epileptogenic. Mutations in HCN channels have also been associated with idiopathic epilepsies such as GEFS+. In addition, HCN channel expression and function are modulated during symptomatic epilepsies such as temporal lobe epilepsy. It is, though, unclear as to whether the changes in HCN channel expression and function associated with the various forms of epilepsy promote epileptogenesis or are adaptive. In this review, we discuss this as well as the potential for KV7 and HCN channels as drug targets for the treatment of epilepsy. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'.

HCN1 channels: a new therapeutic target for depressive disorders?

The hyperpolarization-activated cyclic nucleotide-gated channels are cation channels that are activated by hyperpolarizing potentials. These channels are concentrated in cortical and hippocampal pyramidal cell dendrites, where they play an important role in determining synaptic input integration and thus neuronal output. These channels have thus been suggested to be involved in physiological processes such as cognition as well as pathophysiological states such as epilepsy. Recent evidence suggests that these channels may also be therapeutic targets for treatment of depressive disorders.

Presynaptic HCN1 channels regulate Cav3.2 activity and neurotransmission at select cortical synapses.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are subthreshold, voltage-gated ion channels that are highly expressed in hippocampal and cortical pyramidal cell dendrites, where they are important for regulating synaptic potential integration and plasticity. We found that HCN1 subunits are also localized to the active zone of mature asymmetric synaptic terminals targeting mouse entorhinal cortical layer III pyramidal neurons. HCN channels inhibited glutamate synaptic release by suppressing the activity of low-threshold voltage-gated T-type (Ca(V)3.2) Ca²(+) channels. Consistent with this, electron microscopy revealed colocalization of presynaptic HCN1 and Ca(V)3.2 subunit. This represents a previously unknown mechanism by which HCN channels regulate synaptic strength and thereby neural information processing and network excitability.

Dendritic ion channel trafficking and plasticity.

Dendritic ion channels are essential for the regulation of intrinsic excitability as well as modulating the shape and integration of synaptic signals. Changes in dendritic channel function have been associated with many forms of synaptic plasticity. Recent evidence suggests that dendritic ion channel modulation and trafficking could contribute to plasticity-induced alterations in neuronal function. In this review we discuss our current knowledge of dendritic ion channel modulation and trafficking and their relationship to cellular and synaptic plasticity. We also consider the implications for neuronal function. We argue that to gain an insight into neuronal information processing it is essential to understand the regulation of dendritic ion channel expression and properties.