A calmodulin-gated calcium channel links pathogen patterns to plant immunity.

Pathogen-associated molecular patterns (PAMPs) activate innate immunity in both animals and plants. Although calcium has long been recognized as an essential signal for PAMP-triggered immunity in plants, the mechanism of PAMP-induced calcium signalling remains unknown1,2. Here we report that calcium nutrient status is critical for calcium-dependent PAMP-triggered immunity in plants. When calcium supply is sufficient, two genes that encode cyclic nucleotide-gated channel (CNGC) proteins, CNGC2 and CNGC4, are essential for PAMP-induced calcium signalling in Arabidopsis3-7. In a reconstitution system, we find that the CNGC2 and CNGC4 proteins together-but neither alone-assemble into a functional calcium channel that is blocked by calmodulin in the resting state. Upon pathogen attack, the channel is phosphorylated and activated by the effector kinase BOTRYTIS-INDUCED KINASE1 (BIK1) of the pattern-recognition receptor complex, and this triggers an increase in the concentration of cytosolic calcium8-10. The CNGC-mediated calcium entry thus provides a critical link between the pattern-recognition receptor complex and calcium-dependent immunity programs in the PAMP-triggered immunity signalling pathway in plants.

Systemic administration of ivabradine, a hyperpolarization-activated cyclic nucleotide-gated channel inhibitor, blocks spontaneous absence seizures.

OBJECTIVE: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to be involved in the generation of absence seizures (ASs), and there is evidence that cortical and thalamic HCN channel dysfunctions may have a proabsence role. Many HCN channel blockers are available, but their role in ASs has been investigated only by localized brain injection or in in vitro model systems due to their limited brain availability. Here, we investigated the effect on ASs of orally administered ivabradine (an HCN channel blocker approved for the treatment of heart failure in humans) following injection of the P-glycoprotein inhibitor elacridar, which is known to increase penetration into the brain of drug substrates for this efflux transporter. The action of ivabradine was also tested following in vivo microinjection into the cortical initiation network (CIN) of the somatosensory cortex and in the thalamic ventrobasal nucleus (VB) as well as on cortical and thalamocortical neurons in brain slices. METHODS: We used electroencephalographic recordings in freely moving Genetic Absence Epilepsy Rats From Strasbourg (GAERSs) to assess the action of oral administration of ivabradine, with and without elacridar, on ASs. Ivabradine was also microinjected into the CIN and VB of GAERSs in vivo and applied to Wistar CIN and GAERS VB slices while recording patch-clamped cortical Layer 5/6 and thalamocortical neurons, respectively. RESULTS: Oral administration of ivabradine markedly and dose-dependently reduced ASs. Ivabradine injection into CIN abolished ASs and elicited small-amplitude 4-7-Hz waves (without spikes), whereas in the VB it was less potent. Moreover, ivabradine applied to GAERS VB and Wistar CIN slices selectively decreased HCN channel-dependent properties of cortical Layer 5/6 pyramidal and thalamocortical neurons, respectively. SIGNIFICANCE: These results provide the first demonstration of the antiabsence action of a systemically administered HCN channel blocker, indicating the potential of this class of drugs as a novel therapeutic avenue for ASs.

Development of correction formula for field potential duration of human induced pluripotent stem cell-derived cardiomyocytes sheets.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used in many studies to assess proarrhythmic risks of chemical compounds. In those studies, field potential durations (FPD) of hiPSC-CMs have been corrected by clinically used Fridericia's and/or Bazett's formulae, however, the rationale for the use of these formulae has not been well established. In the present study, we developed a correction formula for experiments using hiPSC-CMs. First, we analyzed the effect of beating rate on FPD in the hiPSC-CMs sheets with electrical stimuli and a HCN channel inhibitor zatebradine. Next, we examined the relationship between the electrophysiological properties and the expression levels of ion channel genes in the cell sheets. Zatebradine slowed the beating rate and allowed to analyze FPD changes at various pacing cycle lengths. Rate-dependent change in the repolarization period was smaller in the cell sheets than that reported on the human hearts, which can be partly explained by lower gene expression level of hKCNJ2 and hKCNE1. Thus, non-linear equation for correcting FPD in the cell sheet; FPDc = FPD/RR0.22 with RR given in second was obtained, which may make it feasible to assess net repolarization delay by various chemical compounds with a chronotropic action.

Neuronal basis of age-related working memory decline.

Many of the cognitive deficits of normal ageing (forgetfulness, distractibility, inflexibility and impaired executive functions) involve prefrontal cortex (PFC) dysfunction. The PFC guides behaviour and thought using working memory, which are essential functions in the information age. Many PFC neurons hold information in working memory through excitatory networks that can maintain persistent neuronal firing in the absence of external stimulation. This fragile process is highly dependent on the neurochemical environment. For example, elevated cyclic-AMP signalling reduces persistent firing by opening HCN and KCNQ potassium channels. It is not known if molecular changes associated with normal ageing alter the physiological properties of PFC neurons during working memory, as there have been no in vivo recordings, to our knowledge, from PFC neurons of aged monkeys. Here we characterize the first recordings of this kind, revealing a marked loss of PFC persistent firing with advancing age that can be rescued by restoring an optimal neurochemical environment. Recordings showed an age-related decline in the firing rate of DELAY neurons, whereas the firing of CUE neurons remained unchanged with age. The memory-related firing of aged DELAY neurons was partially restored to more youthful levels by inhibiting cAMP signalling, or by blocking HCN or KCNQ channels. These findings reveal the cellular basis of age-related cognitive decline in dorsolateral PFC, and demonstrate that physiological integrity can be rescued by addressing the molecular needs of PFC circuits.

Effect of Selective Heart Rate Slowing in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is associated with significant morbidity and mortality but is currently refractory to therapy. Despite limited evidence, heart rate reduction has been advocated, on the basis of physiological considerations, as a therapeutic strategy in HFpEF. We tested the hypothesis that heart rate reduction improves exercise capacity in HFpEF. METHODS AND RESULTS: We conducted a randomized, crossover study comparing selective heart rate reduction with the If blocker ivabradine at 7.5 mg twice daily versus placebo for 2 weeks each in 22 symptomatic patients with HFpEF who had objective evidence of exercise limitation (peak oxygen consumption at maximal exercise [o2 peak] <80% predicted for age and sex). The result was compared with 22 similarly treated matched asymptomatic hypertensive volunteers. The primary end point was the change in o2 peak. Secondary outcomes included tissue Doppler-derived E/e' at echocardiography, plasma brain natriuretic peptide, and quality-of-life scores. Ivabradine significantly reduced peak heart rate compared with placebo in the HFpEF (107 versus 129 bpm; P<0.0001) and hypertensive (127 versus 145 bpm; P=0.003) cohorts. Ivabradine compared with placebo significantly worsened the change in o2 peak in the HFpEF cohort (-2.1 versus 0.9 mL·kg(-1)·min(-1); P=0.003) and significantly reduced submaximal exercise capacity, as determined by the oxygen uptake efficiency slope. No significant effects on the secondary end points were discernable. CONCLUSION: Our observations bring into question the value of heart rate reduction with ivabradine for improving symptoms in a HFpEF population characterized by exercise limitation. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02354573.

Heart rate and heart failure.

PURPOSE OF REVIEW: Resting heart rate has long been thought to be a risk factor in cardiovascular disease and a prognostic factor in heart failure. ß-Blockers were originally used in heart failure for their heart rate control abilities. However, they also have negative inotropic effects contributing to their overall benefit. The role of isolated heart rate modification is unclear in left ventricular systolic dysfunction. RECENT FINDINGS: Two recent studies looked at the heart rate-lowering effects of the If, or funny current inhibitor ivabradine and its potential role in heart failure therapy. At the doses chosen for the studies, ivabradine is presumed to have only effects on heart rate with no other cardiotropic effects. Thus, the cardiovascular outcome benefits are presumed to be secondary to heart rate modification. SUMMARY: The two recent trials showed both heart rate and cardiovascular events to be significantly lower in the ivabradine-treated group of patients with left ventricular systolic dysfunction and initial heart rate at least 70 beats/min. However, neither of these trials proved causality. Hence, the link between heart rate and improved cardiovascular outcomes still remains muddled.

2,4,6-trichloroanisole is a potent suppressor of olfactory signal transduction.

We investigated the sensitivity of single olfactory receptor cells to 2,4,6-trichloroanisole (TCA), a compound known for causing cork taint in wines. Such off-flavors have been thought to originate from unpleasant odor qualities evoked by contaminants. However, we here show that TCA attenuates olfactory transduction by suppressing cyclic nucleotide-gated channels, without evoking odorant responses. Surprisingly, suppression was observed even at extremely low (i.e., attomolar) TCA concentrations. The high sensitivity to TCA was associated with temporal integration of the suppression effect. We confirmed that potent suppression by TCA and similar compounds was correlated with their lipophilicity, as quantified by the partition coefficient at octanol/water boundary (pH 7.4), suggesting that channel suppression is mediated by a partitioning of TCA into the lipid bilayer of plasma membranes. The rank order of suppression matched human recognition of off-flavors: TCA equivalent to 2,4,6-tribromoanisole, which is much greater than 2,4,6-trichlorophenol. Furthermore, TCA was detected in a wide variety of foods and beverages surveyed for odor losses. Our findings demonstrate a potential molecular mechanism for the reduction of flavor.

High dendritic expression of Ih in the proximity of the axon origin controls the integrative properties of nigral dopamine neurons.

KEY POINTS: The hyperpolarization-activated cation current Ih is expressed in dopamine neurons of the substantia nigra, but the subcellular distribution of the current and its role in synaptic integration remain unknown. We used cell-attached patch recordings to determine the localization profile of Ih along the somatodendritic axis of nigral dopamine neurons in slices from young rats. Ih density is higher in axon-bearing dendrites, in a membrane area close to the axon origin, than in the soma and axon-lacking dendrites. Dual current-clamp recordings revealed a similar contribution of Ih to the waveform of single excitatory postsynaptic potentials throughout the somatodendritic domain. The Ih blocker ZD 7288 increased the temporal summation in all dendrites with a comparable effect in axon- and non-axon dendrites. The strategic position of Ih in the proximity of the axon may influence importantly transitions between pacemaker and bursting activities and consequently the downstream release of dopamine. ABSTRACT: Dendrites of most neurons express voltage-gated ion channels in their membrane. In combination with passive properties, active currents confer to dendrites a high computational potential. The hyperpolarization-activated cation current Ih present in the dendrites of some pyramidal neurons affects their membrane and integration properties, synaptic plasticity and higher functions such as memory. A gradient of increasing h-channel density towards distal dendrites has been found to be responsible for the location independence of excitatory postsynaptic potential (EPSP) waveform and temporal summation in cortical and hippocampal pyramidal cells. However, reports on other cell types revealed that smoother gradients or even linear distributions of Ih can achieve homogeneous temporal summation. Although the existence of a robust, slowly activating Ih current has been repeatedly demonstrated in nigral dopamine neurons, its subcellular distribution and precise role in synaptic integration are unknown. Using cell-attached patch-clamp recordings, we find a higher Ih current density in the axon-bearing dendrite than in the soma or in dendrites without axon in nigral dopamine neurons. Ih is mainly concentrated in the dendritic membrane area surrounding the axon origin and decreases with increasing distances from this site. Single EPSPs and temporal summation are similarly affected by blockade of Ih in axon- and non-axon-bearing dendrites. The presence of Ih close to the axon is pivotal to control the integrative functions and the output signal of dopamine neurons and may consequently influence the downstream coding of movement.

HCN channels--modulators of cardiac and neuronal excitability.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels comprise a family of cation channels activated by hyperpolarized membrane potentials and stimulated by intracellular cyclic nucleotides. The four members of this family, HCN1-4, show distinct biophysical properties which are most evident in the kinetics of activation and deactivation, the sensitivity towards cyclic nucleotides and the modulation by tyrosine phosphorylation. The four isoforms are differentially expressed in various excitable tissues. This review will mainly focus on recent insights into the functional role of the channels apart from their classic role as pacemakers. The importance of HCN channels in the cardiac ventricle and ventricular hypertrophy will be discussed. In addition, their functional significance in the peripheral nervous system and nociception will be examined. The data, which are mainly derived from studies using transgenic mice, suggest that HCN channels contribute significantly to cellular excitability in these tissues. Remarkably, the impact of the channels is clearly more pronounced in pathophysiological states including ventricular hypertrophy as well as neural inflammation and neuropathy suggesting that HCN channels may constitute promising drug targets in the treatment of these conditions. This perspective as well as the current therapeutic use of HCN blockers will also be addressed.

[Evaluation of the Antianginal Efficacy of Ivabradine In Patients With Ischemic Heart Disease Complicated by Heart Failure].

Results of the multicenter open observational program NACHALO (evaluation of therapy in patients with combined coronary artery disease and heart failure as part of daily clinical practice), in which 569 doctors in 58 regions of the Russian Federation participated, were presented. The program included 2751 patients with ischemic heart disease, stable angina complicated by heart failure at the age from 25 to 94 years (mean age 62.6 ± 9.3 years). The follow-up period was 3 months, during which two visits were carried out--after 4 weeks and a final visit--12 weeks after the initiation of therapy. Despite treatment, angina attacks (mean 6.7 6.1 per week), increased blood pressure (average 145.1 ± 20.9/87.2 ± 1.8 mm Hg) and heart rate (average 85.8 ± 9.5 beats/min) were noted in patients before the inclusion in the program. The addition of ivabradine therapy resulted in a significant (p < 0.00001) reduction in heart rate to 73.2 ± 8.7 (at visit 1) and down to 65.2 ± 6.1 beats/min (at visit 2), the number of angina attacks per week to 3.8 (at visit 1) and 1.9 (at visit 2). There were significant (p < 0.00001) positive dynamics of the clinical wellness and the severity of symptoms in patients. Adverse effects that, according to doctors, were associated with the agent, were observed in only 0.15% of cases. Thus, in real clinical practice inhibitor of the if channels ivabradine has been shown to be highly effective in treatment of stable angina in patients with coronary artery disease complicated by heart failure.

[Clinical and Economic Efficacy of Ivabradine in Patients Suffered Myocardial Infarction].

OBJECTIVE: to assess the clinical and cost-effectiveness of the addition to the treatment of patients with coronary artery disease, myocardial infarction (MI). If current blocker ivabradine. MATERIAL AND METHODS: as a basis for pharmacoeconomic research results of the program are taken battleships, in which 1226 patients with MI less than 12 months ago, for 16 weeks received ivabradine in addition to standard therapy. When conducting pharmacoeconomic calculations take into account the direct medical and non-medical costs of treating patients with drugs; the call ambulance crews (CAC); on the patients stay in the hospital at admission. To determine the cost of use of ivabradine used data on daily dosages of the drug presented in the program battleships. In this case, using the retail price of drugs taken from the resource pharmindex.ru (date accessed 6/23/14). When calculating the cost of hospitalization and emergency calls using the values specified in the decree of october 18, 2013 No932 "About the state guarantees the free provision of medical care to citizens for 2014 and the planning period of 2015 and 2016.". RESULTS: according to a study LINCOR adding ivabradine to standard therapy resulted in a significant reduction in the frequency of angina attacks, the need for treatment for CACs and hospitalization. Average total cost of a full 16-week course of therapy with a patient ivabradine 1.87 times lower than with the standard therapy alone. The most obvious benefits to health care institutions: the total cost of emergency calls and hospitalization when added to treatment with ivabradine reduced 20,027.79 to 1,630.45 rubles, le 12.3 times. CONCLUSION: despite the increase in direct costs due to the cost of the drug, the addition to the standard therapy of ivabradine in patients with myocardial infarction, pharmaco is more effective than using only standard therapy, due to a significant reduction in the need for emergency calls and hospitalization.

Neuromodulation of I(h) in layer II medial entorhinal cortex stellate cells: a voltage-clamp study.

Stellate cells in layer II of medial entorhinal cortex (mEC) are endowed with a large hyperpolarization-activated cation current [h current (I(h))]. Recent work using in vivo recordings from awake behaving rodents demonstrate that I(h) plays a significant role in regulating the characteristic spatial periodicity of "grid cells" in mEC. A separate, yet related, line of research demonstrates that grid field spacing changes as a function of behavioral context. To understand the neural mechanism or mechanisms that could be underlying these changes in grid spacing, we have conducted voltage-clamp recordings of I(h) in layer II stellate cells. In particular, we have studied I(h) under the influence of several neuromodulators. The results demonstrate that I(h) amplitude can be both upregulated and downregulated through activation of distinct neuromodulators in mEC. Activation of muscarinic acetylcholine receptors produces a significant decrease in the I(h) tail current and a hyperpolarizing shift in the activation, whereas upregulation of cAMP through application of forskolin produces a significant increase in the I(h) amplitude and a depolarizing shift in I(h) activation curve. In addition, there was evidence of differential modulation of I(h) along the dorsal-ventral axis of mEC. Voltage-clamp protocols were also used to determine whether M current is present in stellate cells. In contrast to CA1 pyramidal neurons, which express M current, the data demonstrate that M current is not present in stellate cells. The results from this study provide key insights into a potential mechanism that could be underlying changes seen in grid field spacing during distinct behavioral contexts.

Contribution of near-threshold currents to intrinsic oscillatory activity in rat medial entorhinal cortex layer II stellate cells.

The temporal lobe is well known for its oscillatory activity associated with exploration, navigation, and learning. Intrinsic membrane potential oscillations (MPOs) and resonance of stellate cells (SCs) in layer II of the entorhinal cortex are thought to contribute to network oscillations and thereby to the encoding of spatial information. Generation of both MPOs and resonance relies on the expression of specific voltage-dependent ion currents such as the hyperpolarization-activated cation current (I(H)), the persistent sodium current (I(NaP)), and the noninactivating muscarine-modulated potassium current (I(M)). However, the differential contributions of these currents remain a matter of debate. We therefore examined how they modify neuronal excitability near threshold and generation of near-threshold MPOs and resonance in vitro. We found that resonance mainly relied on I(H) and was reduced by I(H) blockers and modulated by cAMP and an I(M) enhancer but that neither of the currents exhibited full control over MPOs in these cells. As previously reported, I(H) controlled a theta-frequency component of MPOs such that blockade of I(H) resulted in fewer regular oscillations that retained low-frequency components and high peak amplitude. However, pharmacological inhibition and augmentation of I(M) also affected MPO frequencies and amplitudes. In contrast to other cell types, inhibition of I(NaP) did not result in suppression of MPOs but only in a moderation of their properties. We reproduced the experimentally observed effects in a single-compartment stochastic model of SCs, providing further insight into the interactions between different ionic conductances.

Regulation of epileptiform discharges in rat neocortex by HCN channels.

Hyperpolarization-activated, cyclic nucleotide-gated, nonspecific cation (HCN) channels have a well-characterized role in regulation of cellular excitability and network activity. The role of these channels in control of epileptiform discharges is less thoroughly understood. This is especially pertinent given the altered HCN channel expression in epilepsy. We hypothesized that inhibition of HCN channels would enhance bicuculline-induced epileptiform discharges. Whole cell recordings were obtained from layer (L)2/3 and L5 pyramidal neurons and L1 and L5 GABAergic interneurons. In the presence of bicuculline (10 µM), HCN channel inhibition with ZD 7288 (20 µM) significantly increased the magnitude (defined as area) of evoked epileptiform events in both L2/3 and L5 neurons. We recorded activity associated with epileptiform discharges in L1 and L5 interneurons to test the hypothesis that HCN channels regulate excitatory synaptic inputs differently in interneurons versus pyramidal neurons. HCN channel inhibition increased the magnitude of epileptiform events in both L1 and L5 interneurons. The increased magnitude of epileptiform events in both pyramidal cells and interneurons was due to an increase in network activity, since holding cells at depolarized potentials under voltage-clamp conditions to minimize HCN channel opening did not prevent enhancement in the presence of ZD 7288. In neurons recorded with ZD 7288-containing pipettes, bath application of the noninactivating inward cationic current (Ih) antagonist still produced increases in epileptiform responses. These results show that epileptiform discharges in disinhibited rat neocortex are modulated by HCN channels.

The hyperpolarization-activated non-specific cation current (In ) adjusts the membrane properties, excitability, and activity pattern of the giant cells in the rat dorsal cochlear nucleus.

Giant cells of the cochlear nucleus are thought to integrate multimodal sensory inputs and participate in monaural sound source localization. Our aim was to explore the significance of a hyperpolarization-activated current in determining the activity of giant neurones in slices prepared from 10 to 14-day-old rats. When subjected to hyperpolarizing stimuli, giant cells produced a 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (ZD7288)-sensitive inward current with a reversal potential and half-activation voltage of -36 and -88 mV, respectively. Consequently, the current was identified as the hyperpolarization-activated non-specific cationic current (Ih ). At the resting membrane potential, 3.5% of the maximum Ih conductance was available. Immunohistochemistry experiments suggested that hyperpolarization-activated, cyclic nucleotide-gated, cation non-selective (HCN)1, HCN2, and HCN4 subunits contribute to the assembly of the functional channels. Inhibition of Ih hyperpolarized the membrane by 6 mV and impeded spontaneous firing. The frequencies of spontaneous inhibitory and excitatory postsynaptic currents reaching the giant cell bodies were reduced but no significant change was observed when evoked postsynaptic currents were recorded. Giant cells are affected by biphasic postsynaptic currents consisting of an excitatory and a subsequent inhibitory component. Inhibition of Ih reduced the frequency of these biphasic events by 65% and increased the decay time constants of the inhibitory component. We conclude that Ih adjusts the resting membrane potential, contributes to spontaneous action potential firing, and may participate in the dendritic integration of the synaptic inputs of the giant neurones. Because its amplitude was higher in young than in adult rats, Ih of the giant cells may be especially important during the postnatal maturation of the auditory system.

Heart-rate reduction by If-channel inhibition with ivabradine restores collateral artery growth in hypercholesterolemic atherosclerosis.

AIMS: Collateral arteries protect tissue from ischaemia. Heart rate correlates with vascular events in patients with arterial obstructive disease. Here, we tested the effect of heart-rate reduction (HRR) on collateral artery growth. METHODS AND RESULTS: The I(f)-channel inhibitor ivabradine reduced heart rate by 11% in wild-type and 15% in apolipoprotein E (ApoE)(-/-) mice and restored endothelium-dependent relaxation in aortic rings of ApoE(-/-) mice. Microsphere perfusion and angiographies demonstrated that ivabradine did not change hindlimb perfusion in wild-type mice but improved perfusion in ApoE(-/-) mice from 40.5 ± 15.8-60.2 ± 18.5% ligated/unligated hindlimb. Heart rate reduction (13%) with metoprolol failed to improve endothelial function and perfusion. Protein expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS, and eNOS activity were increased in collateral tissue following ivabradine treatment of ApoE(-/-) mice. Co-treatment with nitric oxide-inhibitor N (G)-nitro-L-arginine methyl ester abolished the effects of ivabradine on arteriogenesis. Following ivabradine, classical inflammatory cytokine expression was lowered in ApoE(-/-) circulating mononuclear cells and in plasma, but unaltered in collateral-containing hindlimb tissue, where numbers of perivascular macrophages also remained unchanged. However, ivabradine reduced expression of anti-arteriogenic cytokines CXCL10and CXCL11 and of smooth muscle cell markers smoothelin and desmin in ApoE(-/-) hindlimb tissue. Endothelial nitric oxide synthase and inflammatory cytokine expression were unchanged in wild-type mice. Ivabradine did not affect cytokine production in HUVECs and THP1 mononuclear cells and had no effect on the membrane potential of HUVECs in patch-clamp experiments. CONCLUSION: Ivabradine-induced HRR stimulates adaptive collateral artery growth. Important contributing mechanisms include improved endothelial function, eNOS activity, and modulation of inflammatory cytokine gene expression.

Rapid loss of dendritic HCN channel expression in hippocampal pyramidal neurons following status epilepticus.

Epilepsy is associated with loss of expression and function of hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels. Previously, we showed that loss of HCN channel-mediated current (I(h)) occurred in the dendrites of CA1 hippocampal pyramidal neurons after pilocarpine-induced status epilepticus (SE), accompanied by loss of HCN1 channel protein expression. However, the precise onset and mechanistic basis of HCN1 channel loss post-SE was unclear, particularly whether it preceded the onset of spontaneous recurrent seizures and could contribute to epileptogenesis or development of the epileptic state. Here, we found that loss of I(h) and HCN1 channel expression began within an hour after SE and involved sequential processes of dendritic HCN1 channel internalization, delayed loss of protein expression, and later downregulation of mRNA expression. We also found that an in vitro SE model reproduced the rapid loss of dendritic I(h), demonstrating that this phenomenon was not specific to in vivo SE. Together, these results show that HCN1 channelopathy begins rapidly and persists after SE, involves both transcriptional and nontranscriptional mechanisms, and may be an early contributor to epileptogenesis.

Molecular mechanism underlying phosphatidylinositol 4,5-bisphosphate-induced inhibition of SpIH channels.

Many ion channels have been shown to be regulated by the membrane signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here, we demonstrate that the binding of PIP(2) to SpIH, a sea urchin hyperpolarization-activated cyclic nucleotide-gated ion channel (HCN), has a dual effect: potentiation and inhibition. The potentiation is observed as a shift in the voltage dependence of activation to more depolarized voltages. The inhibition is observed as a reduction in the currents elicited by the partial agonist cGMP. These two effects were separable and arose from PIP(2) binding to two different regions. Deletion of the C-terminal region of SpIH removed PIP(2)-induced inhibition but not the PIP(2)-induced shift in voltage dependence. Mutating key positively charged amino acids in the C-terminal region adjacent to the membrane selectively disrupted PIP(2)-induced inhibition, suggesting a direct interaction between PIP(2) in the membrane and amino acids in the C-terminal region that stabilizes the closed state relative to the open state in HCN channels.

Role of Ih in the firing pattern of mammalian cold thermoreceptor endings.

Mammalian peripheral cold thermoreceptors respond to cooling of their sensory endings with an increase in firing rate and modification of their discharge pattern. We recently showed that cultured trigeminal cold-sensitive (CS) neurons express a prominent hyperpolarization-activated current (I(h)), mainly carried by HCN1 channels, supporting subthreshold resonance in the soma without participating in the response to acute cooling. However, peripheral pharmacological blockade of I(h), or characterization of HCN1(-/-) mice, reveals a deficit in acute cold detection. Here we investigated the role of I(h) in CS nerve endings, where cold sensory transduction actually takes place. Corneal CS nerve endings in mice show a rhythmic spiking activity at neutral skin temperature that switches to bursting mode when the temperature is lowered. I(h) blockers ZD7288 and ivabradine alter firing patterns of CS nerve endings, lengthening interspike intervals and inducing bursts at neutral skin temperature. We characterized the CS nerve endings from HCN1(-/-) mouse corneas and found that they behave similar to wild type, although with a lower slope in the firing frequency vs. temperature relationship, thus explaining the deficit in cold perception of HCN1(-/-) mice. The firing pattern of nerve endings from HCN1(-/-) mice was also affected by ZD7288, which we attribute to the presence of HCN2 channels in the place of HCN1. Mathematical modeling shows that the firing phenotype of CS nerve endings from HCN1(-/-) mice can be reproduced by replacing HCN1 channels with the slower HCN2 channels rather than by abolishing I(h). We propose that I(h) carried by HCN1 channels helps tune the frequency of the oscillation and the length of bursts underlying regular spiking in cold thermoreceptors, having important implications for neural coding of cold sensation.

Cocaine sensitization inhibits the hyperpolarization-activated cation current Ih and reduces cell size in dopamine neurons of the ventral tegmental area.

The progressive augmentation of motor activity that results from repeated cocaine administration is termed behavioral sensitization. This phenomenon is thought to be a critical component in compulsive drug taking and relapse. Still, the cellular mechanisms that underlie sensitization remain elusive. Cocaine abuse, nonetheless, is known to evoke neuroplastic adaptations in dopamine (DA) neurotransmission originating from the midbrain's ventral tegmental area (VTA). Here, we report that concomitant with the development of locomotor sensitization to cocaine the hyperpolarization-activated cation current (I(h)) amplitude is depressed by ∼40% in VTA DA cells. Such effect did not result from a negative shift in I(h) voltage dependence. Nonstationary fluctuation analysis indicates that this inhibition was caused by an ∼45% reduction in the number of h-channels with no change in their unitary properties. The cocaine-induced I(h) depression was accompanied by a reduction in cell capacitance of similar magnitude (∼33%), leaving h-current density unaltered. Two implications follow from these data. First, I(h) inhibition may contribute to cocaine addiction by increasing bursting probability in DA cells and this effect could be intensified by the decrease in cell capacitance. Second, the cocaine-induced diminution of DA cell capacitance may also lead to reward tolerance promoting drug-seeking behaviors.