IMPACT OF HCN CHANNEL MUTATION ON EPILEPSY

YANAMALA VIJAY RAJ05/01/2023

Outline

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Epilepsy HCN channel K channel Signal transduction pathways Interactions

Hyper-polarization activated cyclic nucleotide (HCN) Non selective channel Brian and heart Oscillatory rhythm HCN channels have a major role in controlling

neuronal excitability, dendritic integration of synaptic potentials, synaptic transmission, and rhythmic oscillatory activity in individual neurons and neuronal networks.

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Structure

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Distribution

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Reference HCN channels : Function and clinical implications Eduardo E. Benarroch, MD

Functions

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Reference HCN channels : Function and clinical implications Eduardo E. Benarroch, MD

HCN role in epilepsy Abnormal regulation of HCN expression or function has been

implicated in epilepsy.

However, the role of HCN dysregulation in epilepsy is complex. Studies in experimental models indicate that both upregulation and downregulation of HCN channels can be associated with seizures;

Different HCN types may contribute to different extents to focal or generalized epilepsy syndromes; and the role of HCN channels strongly depends on cellular localization and physiologic context.05/01/2023

Complications The number of surface-expressed HCN channels influences the

magnitude and properties of Ih.

Mechanisms controlling the number of surface HCN channels act at different time-scales.

At a relatively long time scale (hours to days), transcriptional regulation determines the total amount of HCN channel protein in the cell.

This regulation is isoform-specific, acts via specific transcription factors, and depends on network activity.

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Transcription factors altering HCN NRSF also known as REST, repressor

element 1-silencing transcription factor). repressor element 1-silencing

transcription factor), which binds to neuron restrictive silencer elements (NRSE).

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Post-translational modifications of HCN HCN channels are heavily glycosylated in the mammalian brain

and glycosylation influences both the total number of HCN channels in the membrane and their heteromerization.

Modulation of HCN channel surface expression at shorter time-scales can occur via local regulation of channel membrane insertion, internalization and recycling.

Importantly, the dynamics of HCN channel trafficking and surface expression are activity-dependent, with implications to neuroplasticity and disease.

TRIP8b (an auxiliary protein that interacts directly with HCN channels) can either up- or down-regulate HCN channel surface expression.

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The targeting of HCN channels to distinct sub-cellular domains influences their location dependent roles in regulation of excitability.

Distinct distribution patterns of HCN channels exist in dendrites of specific cell types and brain regions, as well as during development.

Whereas ample information exists on the distribution of HCN channels, little is known of the molecular mechanisms that underlie their targeting.

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Reporting of HCN role in epilepsy

Enhanced levels of HCN1 channel expression and dendritic localization were found in granule cells of the dentate gyrus.

Recent work has identified a mutation in the HCN2 gene and augmentation of Ih in patients with genetic epilepsy with febrile seizures plus (GEFS).

Deletion of the HCN1 gene in mice results in increased excitability and seizure susceptibility, and reduction or deletion of the HCN2 isoform leads to spontaneous ‘absence’ seizures.

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In accord with the diverse regulatory mechanisms and versatile functions of Ih in the normal brain, the dysregulation of Ih and HCN channels in epilepsy is dynamic and intricate.

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REFERENCE: Jasper's Basic Mechanisms of the Epilepsies. 4th edition, Noebels JL, Avoli M, Rogawski MA, et al., editors. Bethesda (MD): National Center for Biotechnology Information (US); 2012.

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REFERENCE: Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th edition, Noebels JL, Avoli M, Rogawski MA, et al., editors. Bethesda (MD): National Center for Biotechnology Information (US); 2012.

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Do Seizures Cause Neuronal Death in the Human Hippocampus?Luisa L Rocha, MD PhD, Maria-Leonor Lopez-Meraz, PhD, Jerome Niquet PhD and Claude G Wasterlain MD,

Based on Indirect Markers of Neuronal Injury, it is conformed that repetitive seizures caused neuronal death.

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“CypD” The Key to the Death Door: Shaik M. Fayaz, Yanamala V. Raj and Rajanikant G. Krishnamurthy: School of Biotechnology, Coordinator, DBT - Centre for Bioinformatics, National Institute of Technology Calicut, Calicut - 673601, India.

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Genotype–phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor of K v7.2 potassium channel subunits Francesco Micelia, Maria Virginia Soldovierib, Paolo Ambrosinob, Vincenzo Barresea, Michele Migliorec, Maria Roberta Ciliod,e,1, and Maurizio Taglialatela

1. Mutations in Kv7.2 gene encoding for voltage dependent potassium channel cause neonatal epilepsies.

2. Kv7.2 channels fall under delayed rectifiers category.3. Mutations reported in S4 domain

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Potassium channels

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Pore visualization

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Domains

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Mutations1. R213W2. R213Q Mutations markedly destabilized open state causing decrease in channel voltage sensitivity.

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Functional changes of mutation These functional changes were

1. More pronounced for channels incorporating R213Q- than R213W-carrying KV7.2 subunits.

2. (ii) proportional to the number of mutant subunits incorporated; 3. (iii) fully restored by the neuronal Kv7 activator retigabin.

NOTE: Both mutations increased firing, but R213Q has more dramatically functional changes compared to others.

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Anticonvulsant therapy Kv7 openers as a targeted anticonvulsant therapy to improve

developmental outcome in neonates with KV7.2 encephalopathy

CLINICAL IDENTIFICATIONS:1. seizures with psychomotor retardation, 2. suppression-burst pattern at EEG, 3. and distinct neuroradiological features

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Threshold voltages for current activation were-50mV in Kv7.2−10 mV in Kv7.2 R6W channels0 mV in Kv7.2 R6Q channels.

The half activation potential (V1/2) was −23 mV in Kv7.2 channels; this value was right-shifted by i. 58 mV R6W mutant ii. 68 mV R6Q mutant

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Deactivation kinetics Deactivation kinetics of KV7.2 R6W & KV7.2 R6Q channels

were faster than KV7.2 channels.

At the end of a −20 mV step, a significant fraction of KV7.2 channels appeared to still be in the open state; by contrast, more complete deactivation was observed for KV7.2 R6W and KV7.2 R6Q channels, the latter being fully closed at the end of the voltage step.

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Functional and Pharmacological Properties of KV7.2 and/or KV7.3 Heteromeric Channels Incorporating Mutant KV7.2 R6W and R6Q Subunits.

To evaluate the possible functional changes caused by KV7.2 R6W and KV7.2 R6Q mutant subunits in Heteromeric assembly with KV7.2 and/or KV7.3 subunits.

CHO cells were transfected with KV7.2+KV7.3 cDNAs at a 1:1 ratio to mimic the genetic balance of normal individuals, and with KV7.2+KV7.2 R6W+KV7.3 or KV7.2+KV7.2 R6Q+KV7.3 (each at 0.5:0.5:1 ratio) to mimic the genetic balance of individuals who carried the mutant KV7.2 alleles in heterozygosity.

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Incorporation of KV7.3 subunits into KV7.2+KV7.3, heteromeric channels generated currents with larger size and a reduced sensitivity to the pore blocker tetraethylammonium (TEA) than KV7.2 homomers.

Compared with KV7.2+KV7.3-transfected cells, cells transfected with KV7.2+KV7.2 R6W+KV7.3 or KV7.2+KV7.2 R6Q+KV7.3 cDNAs generated K+ currents of identical size and TEA sensitivity, suggesting that these mutations failed to interfere with heteromeric subunit assembly.

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HCN hyperpolarization-activated cation channels inhibit EPSPs by interactions with M-type K+ channels Meena S George, L F Abbott & Steven A Siegelbaum

Characterization of HCN channels on dendritic processing of subthreshold excitatory postsynaptic potentials (EPSPs).

The HCN channels generated an excitatory inward current (Ih) that exerted a direct depolarizing effect on the peak voltage of weak EPSPs, but produced a paradoxical hyperpolarizing effect on the peak voltage of stronger, but still subthreshold EPSPs.

Its found that the inhibitory action of (Ih) was caused by its interaction with the delayed-rectifier M-type K current.05/01/2023

M current  M current is a type of non-inactivating potassium

current first discovered in bullfrog sympathetic ganglion cells.

The M-channel is important in raising the threshold for firing an action potential. It is unique because it is open at rest and even more likely to be open during depolarization.

The M-channel is a PIP2-regulated ion channel.05/01/2023

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PIP2 role in rundown of HCN The voltage dependence of activation of the HCN

hyperpolarization-activated cation channels is shifted in inside-out patches by -40 to -60 mV relatively.

Role of phosphatidylinositol 4,5-bisphosphate PIP(2) in HCN channel rundown, as hydrolysis of PIP(2) by lipid phosphatases is thought to underlie rundown of several other channels.

We find that bath application of exogenous PIP(2) reverses the effect of rundown, producing a large depolarizing shift in HCN2 activation.

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Coordinated signal integration a t the M-typepotassium channel upon muscarinic stimulationAnastasia Kosenko1, Seungwoo Kang1, Ida M Smith1, Derek L Greene1, Lorene K Langeberg2, John D Scott2and Naoto Hoshi1,*1Department of Pharmacology, University of California, Irvine, CA, USA and 2Department of Pharmacology, Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA

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NEUROSCIENCEThird Edition

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Regulation of gating and rundown of HCN hyperpolarization-activated channels by exogenous and endogenous PIP2.Pian P1, Bucchi A, Robinson RB, Siegelbaum SA.Author information1Center for Neurobiology and Behavior, Columbia University Medical Center, New York, NY 10032, USA.

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The Cyclic AMP PathwayPaolo Sassone-CorsiCenter for Epigenetics and Metabolism, School of Medicine, University of California, Irvine, California 92697Correspondence: psc@uci.edu

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cAMP Activated G protein bind to PLC. PLC cleaves PIP2 into IP3 and DAG. IP3 triggers calcium release from endo-plastic

reticulum, and it ultimately regulates cAMP. DAG in a chain of reaction too increase cyclic

nucleotide activity.

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Dendride channels Voltage gated sodium and calcium

channels amplify synaptic potential. Voltage gated calcium activated

potassium channel inhibit synaptic potential.

HCN channel has both inhibitory and excitatory action on synaptic potential.

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HCN role Unlike most voltage-gated channels, HCN channels activate with

hyperpolarization and deactivate with depolarization.

Their mixed permeability to K+ and Na+ ions results in a reversal potential ( Eh) of approximately –30 mV, causing these channels to generate anexcitatory inward current ( Ih) at subthreshold potentials.

These biophysical properties underlie the role of (Ih) as a pacemaker current in cardiac myocytes and thala-mocortical relay neurons, in which activation of Ih following action potential repolarization generates a depolarizing current that drives spontaneous, rhythmic firing.

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HCN channels are not spontaneously active, Ih contributes 5 to 10-mV

depolarizing influence on the resting membrane potential (RMP) and increases the resting membrane conductance (that is, it lowers the input resistance).

Conversely from the fact that Ih provides a depolarizing current at subthreshold potentials, results from several studies have indicated that it has a paradoxical inhibitory effect on the ability of an EPSP to trigger an action potential.

Thus, enhancement of Ih by the anticonvulsant lamotrigine, application of dopamine or induction of long-term potentiation decreases excitability and spike firing.

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Conversely, downregulation of Ih via genetic deletion of HCN1, pharmacological blockade using cesium or the organic antagonist ZD7288 following induction of long-term depression or seizures increases EPSP amplitude, temporal summation and spike firing.

The inhibitory effects of Ih , by which we mean the inhibition that is seen when Ih is enhanced, have generally been attributed to its ability to increase the resting membrane conductance.

This so-called ‘shunting effect’ on the excitatory postsynaptic current decreases the amplitude of an EPSP with EPSP amplitude being defined as the difference between the peak voltage of an EPSP (V peak) and the resting potential.

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The Yin and Yang of the H-Channel and Its Role in Epilepsy: Nicholas P. Poolos, M.D., Ph.D

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However, the effect of an EPSP depends not on its amplitude, but on the voltage reached at its peak, which determines whether an EPSP is supra-threshold.

Notably, Ih exerts two opposing influences on V peak: its shunting effect decreases EPSP peak voltage and its direct depolarizing effect increases V peak.

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By computational it is proved that HCN has more excitatory role than inhibitory role.

Depolarizing effect greater than shunting effect.

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In principle, this effect of Ih on resting potential and resting inactivation could also explain how Ih suppresses the firing of Na+ action potentials.

But it remains unclear whether Ih can exert an inhibitory effect on Vpeak for subthreshold EPSPs.

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Results

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Ih suppresses the firing of Na+ action potentials.

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Ih shifts membrane potential to more positive side, with weak EPSP.

Ih shifts membrane potential to more negative side, with strong EPSP.

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Conclusion Dual influence of Ih on EPSPs in CA1 pyramidal

neurons. Ih is purely excitatory when it is the sole active

conductance. Ih can inhibit EPSPs by interactions with a K+

current. Ih interacts with the M-type K+ current to inhibit

EPSPs. Inhibitory effects of Ih are prevented by M-current

blockade.

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Molecular mechanism ofcAMP modulation of HCN pacemaker channels

All four family members contain both a core transmembrane segment domain, homologous to the S1 to S6 regions of voltage-gated K+ channels, and a carboxy-terminal 120 amino-acid cyclic nucleotide-binding domain (CNBD) motif.

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.

Reference: Molecular mechanism of cAMP modulation of HCN pacemaker channels Brian J. Wainger*, Matthew DeGennaro², Bina Santoro*, Steven A. Siegelbaum*²³ & Gareth R. Tibbs³§ * Center for Neurobiology and Behavior; ² Howard Hughes Medical Institute; ³ Department of Pharmacology; and § Department of Anesthesiology, Columbia University, New York, NY 10032, USA

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Activation kinetics It is demonstrated 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.

HCN1 channels activate more rapidly on hyperpolarization than HCN2 channels; additionally, HCN1 channels turn on at voltages of about 20 mV more positive than HCN2 channels.

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Mean tail current I/V curves:Normalized peak inward tail-current amplitude as a function of voltage during preceding hyperpolarization for HCN1 (top panel, circles) and HCN2 (bottom panel, squares) channels in absence (open symbols) or) of cAMP presence.

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HCN1 vs HCN2V1/2 of HCN1 = -110.6V1/2 of HCN2 = -129.6HCN2 activation was about 15-fold slower than HCN1 activation.

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HCN1 vs HCN2 wrt cAMP V1/2 of HCN1shifted by +5.8 V1/2 of HCN2 shifted by +17.3

cAMP accelerated HCN2 by 3.5 fold cAMP accelerated HCN1 by 1.5 fold

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