Affinity For Channels Research Articles

Design and synthesis of a novel series of histamine H3 receptor antagonists through a scaffold hopping strategy

Lead compounds 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl-[1,3′]bipyrrolidinyl-1′-yl)-phenyl]-benzamide (1), tetrahydro-pyran-4-carboxylic acid [((2S,3′S)-2-methyl-[1,3′]bipyrrolidinyl-1′-yl)-phenyl]-amide (2), and 3,5-dimethyl-isoxazole-4-carboxylic acid [((2S,3′S)-2-methyl-[1,3′]bipyrrolidinyl-1′-yl)-phenyl]-amide (3) discovered in our laboratory, displayed high histamine H3 receptor (H3R) affinity, good selectivity and weak human Ether-à-go-go-Related Gene (hERG) channel affinity with desirable overall physico-chemical and pharmacokinetic (PK) profiles. Herein, we describe the design and synthesis of a novel series of H3R antagonists utilizing a scaffold hopping strategy. Further structure–activity relationship (SAR) studies of the series culminated in the identification of ((2S,3′S)-2-methyl-[1,3′]bipyrrolidinyl-1′-yl)-naphthalene-2-carboxylic acid (tetrahydro-pyran-4-yl)-amide (4c) and -[4-((2S,3′S)-2-methyl-[1,3′]bipyrrolidinyl-1′-yl)-phenyl]-N-(tetrahydro-pyran-4-yl)-acetamide (4d), which exhibited good H3R affinity in vitro, good selectivity, and desirable PK properties. Compounds 4c and 4d were also assessed in cardiac safety experiments. In particular, the effects of the compounds on action potentials recorded from ventricular myocytes isolated from guinea pigs were used to screen compounds that not only displayed a low affinity towards hERG channel, but also had lower interference with other cardiac ion channels. Compound 4c did not alter the major parameters in this model system at ⩽10μM, and no significant induction of any major haemodynamic effect when intravenously administered at 3mg/kg dose to anaesthetized mongrel dogs. Compound 4c is a new promising lead as orally potent and selective H3R antagonist belonging to a distinct structural class.

Chemical modifications of the N-methyl-laudanosine scaffold point to new directions for SK channels exploration

An asparagine or a histidine are present in a similar position in the outer pore region of SK2 and SK3 channels, respectively. Therefore, this structural difference was targeted in order to develop selective blockers of SK channel subtypes. Following docking investigations, based on theoretical models of truncated SK2 and SK3 channels, the benzyl side chain of N-methyl-laudanosine (NML) was functionalized in order to target this specific amino-acid residues. Chiral butanamide and benzyloxy analogues were prepared, resolved and tested for their affinity for SK2 and SK3 channels. Isoquinolinium (NMIQ) derivatives have a higher affinity for both SK channel subtypes than the corresponding derivative with no functionalized side chain. This trend was observed also for the 1,2,3,4-tetrahydroisoquinoline (THIQ) analogues. A benzyloxy functionalized NML enantiomer has a higher affinity than NML stereoisomers. Otherwise, the conserved affinity of these analogues led to the opportunity to further investigate in terms of possible labeling for in vivo investigations of the role of SK channels.

Structural basis of drugs that increase cardiac inward rectifier Kir2.1 currents

We hypothesize that some drugs, besides flecainide, increase the inward rectifier current (IK1) generated by Kir2.1 homotetramers (IKir2.1) and thus, exhibit pro- and/or antiarrhythmic effects particularly at the ventricular level. To test this hypothesis, we analysed the effects of propafenone, atenolol, dronedarone, and timolol on Kir2.x channels. Currents were recorded with the patch-clamp technique using whole-cell, inside-out, and cell-attached configurations. Propafenone (0.1 nM-1 µM) did not modify either IK1 recorded in human right atrial myocytes or the current generated by homo- or heterotetramers of Kir2.2 and 2.3 channels recorded in transiently transfected Chinese hamster ovary cells. On the other hand, propafenone increased IKir2.1 (EC50 = 12.0 ± 3.0 nM) as a consequence of its interaction with Cys311, an effect which decreased inward rectification of the current. Propafenone significantly increased mean open time and opening frequency at all the voltages tested, resulting in a significant increase of the mean open probability of the channel. Timolol, which interacted with Cys311, was also able to increase IKir2.1. On the contrary, neither atenolol nor dronedarone modified IKir2.1. Molecular modelling of the Kir2.1-drugs interaction allowed identification of the pharmacophore of drugs that increase IKir2.1. Kir2.1 channels exhibit a binding site determined by Cys311 that is responsible for drug-induced IKir2.1 increase. Drug binding decreases channel affinity for polyamines and current rectification, and can be a mechanism of drug-induced pro- and antiarrhythmic effects not considered until now.

1,4-Dihydropyridine derivatives with T-type calcium channel blocking activity attenuate inflammatory and neuropathic pain.

We have recently identified a class of dihydropyridine (DHP) analogues with 30-fold selectivity for T-type over L-type calcium channels that could be attributed to a modification of a key ester moiety. Based on these results, we examined a second series of compounds with similar attributes to determine if they had enhanced affinity for T-type channels. Whole-cell patch clamp experiments in transfected tsA-201 cells were used to screen these DHP derivatives for high affinity and selectivity for Cav3.2 over Cav1.2L-type channels. The effects of the two lead compounds, termed N10 and N12, on Cav3.2 channel activity and gating were characterized in detail. When delivered intrathecally or intraperitoneally, these compounds mediated analgesia in a mouse model of acute inflammatory pain. The best compound from the initial screening, N12, was also able to reverse mechanical hyperalgesia produced by nerve injury. The compounds were ineffective in Cav3.2 null mice. Altogether, our data reveal a novel class of T-type channel blocking DHPs for potential pain therapies.

Structure, molecular modeling, and function of the novel potassium channel blocker urotoxin isolated from the venom of the Australian scorpion Urodacus yaschenkoi.

This communication reports the structural and functional characterization of urotoxin, the first K(+) channel toxin isolated from the venom of the Australian scorpion Urodacus yaschenkoi. It is a basic peptide consisting of 37 amino acids with an amidated C-terminal residue. Urotoxin contains eight cysteines forming four disulfide bridges with sequence similarities resembling the α-potassium channel toxin 6 (α-KTx-6) subfamily of peptides; it was assigned the systematic number of α-KTx-6.21. Urotoxin is a potent blocker of human voltage-gated potassium channel (Kv)1.2 channels, with an IC50 of 160 pM, whereas its affinity for other channels tested was in the nanomolar range (hKv1.1, IC50 = 253 nM; hKv1.3, IC50 = 91 nM; and hKCa3.1, IC50 = 70 nM). The toxin had no effect on hKv1.4, hKv1.5, human ether-à-go-go-related gene type 1 (hERG1), or human ether-à-go-go-like (hELK2) channels. Multiple sequence alignments from the venom gland transcriptome showed the existence of four other new peptides similar to urotoxin. Computer modeling of urotoxin's three-dimensional structure suggests the presence of the α/β-scaffold characteristic of other scorpion toxins, although very likely forming an uncommon disulfide pairing pattern. Using molecular dynamics, a model for the binding of this peptide to human Kv1.2 and hKv1.1 channels is presented, along with the binding of an in silico mutant urotoxin (Lys25Ala) to both channels. Urotoxin enriches our knowledge of K(+) channel toxins and, due to its high affinity for hKv1.2 channels, it may be a good candidate for the development of pharmacologic tools to study the physiologic functions of K(+) channels or related channelopathies and for restoring axonal conduction in demyelinated axons.

Tethering Dimers of Voltage Sensor Toxins can Selectively Amplify their Affinity for Kv Channels

Potent and selective modulators are needed to elucidate the individual roles of ion channel subtypes in physiological systems. Many cystine knot peptides derived from tarantula venom allosterically modulate voltage sensors of ion channels. Although tarantula peptides have varying affinities for different channels, all affect multiple subtypes. Theoretically, a peptide's selectivity between channel types could be amplified by summing binding energies, if a multimeric version of the peptide could bind multiple subunits simultaneously. We have amplified the inherent specificity of a voltage sensor peptide, guangxitoxin-1E (GxTX), by tethering two peptides together to form a dimer coupled by a flexible polyethylene glycol linker. We synthesized GxTX mutants functionalized with artificial amino acids for chemoselective coupling and tethered them with homobifunctional linkers using azide-alkyne cycloaddition chemistry. The GxTX dimers inhibited Kv2.1 with higher affinity than GxTX monomers. Similar to GxTX monomers, these tethered dimers shifted channel opening to positive voltages. Association rates were reduced for the GxTX dimers versus monomers, indicating that increased affinity for Kv2.1 channels was entirely due to the extremely slow dissociation of GxTX dimers. The strong voltage dependence of dimer dissociation was consistent with a two step binding model, where the kinetics of the second GxTX binding domain determines the amplification of affinity by the dimer. This mechanism predicts that dimerization will selectively amplify the affinity for channels with slow toxin dissociation rates. Experimentally, dimerization amplified GxTX specificity for Kv2.1 over other channel subtypes. We conclude that tethered multimers can increase the pharmacological selectivity of voltage sensor modulators.

Modulation of Mouse Rod Photoreceptor Responses by Grb14 Protein

Previous experiments have indicated that growth factor receptor-bound protein 14 (Grb14) may modulate rod photoreceptor cGMP-gated channels by decreasing channel affinity for cGMP; however, the function of Grb14 in rod physiology is not known. In this study, we examined the role of Grb14 by recording electrical responses from rods in which the gene for the Grb14 protein had been deleted. Suction-electrode recordings from single mouse rods showed that responses of dark-adapted Grb14(-/-) mice to brief flashes decayed more rapidly than strain-controlled wild type (WT) rods, with decreased values of both integration time and the exponential time course of decay (τREC). This result is consistent with an increase in channel affinity for cGMP produced by deletion of Grb14. However, Grb14(-/-) mouse rods also showed little change in dark current and a large and significant decrease in the limiting time constant τD, which are not consistent with an effect on channel affinity but seem rather to indicate modulation of the rate of inactivation of cyclic nucleotide phosphodiesterase 6 (PDE6). Grb14 has been reported to translocate from the inner to the outer segment in bright light, but we saw effects on response time course even in dark-adapted rods, although the effects were somewhat greater after rods had been adapted by exposure to bleaching illumination. Our results indicate that the mechanism of Grb14 action may be more complex than previously realized.

Calculation of Cholesterol Binding Affinity for Pentameric Ligand-Gated Ion Channels

Cholesterol has been shown to play a critical role in the function of ion channels and many types of ion channels partition into cholesterol-rich membranes. Several Eukaryotic pentameric ligand-gated ion channels, including the nicotinic Acetylcholine Receptor and the GABA(A) receptor, are highly sensitive to the cholesterol content of reconstitution mixtures or native membranes, but the binding interactions between cholesterol and these receptors are largely uncharacterized. Since the mechanisms for modulation by cholesterol are likely to be at least partially through direct interactions via specific binding, cholesterol molecules may form an essential component of the native structure for some of these receptors.

Removal of Human Ether-à-go-go Related Gene (hERG) K+ Channel Affinity through Rigidity: A Case of Clofilium Analogues

Cardiotoxicity is a side effect that plagues modern drug design and is very often due to the off-target blockade of the human ether-à-go-go related gene (hERG) potassium channel. To better understand the structural determinants of this blockade, we designed and synthesized a series of 40 derivatives of clofilium, a class III antiarrhythmic agent. These were evaluated in radioligand binding and patch-clamp assays to establish structure-affinity relationships (SAR) for this potassium channel. Efforts were especially focused on studying the influence of the structural rigidity and the nature of the linkers composing the clofilium scaffold. It was shown that introducing triple bonds and oxygen atoms in the n-butyl linker of the molecule greatly reduced affinity without significantly modifying the pKa of the essential basic nitrogen. These findings could prove useful in the first stages of drug discovery as a systematic way of reducing the risk of hERG K(+) channel blockade-induced cardiotoxicity.

From venoms to BBB shuttles: Synthesis and blood–brain barrier transport assessment of apamin and a nontoxic analog

Venoms are currently the focus of many drug discovery programs because they contain highly bioactive and selective components. Among them, apamin, a peptide found in bee venom, has received considerable attention because of its affinity for certain potassium channels and also because of its interesting structure and high stability to extreme pH and temperatures. Although apamin has long been claimed to cross the blood-brain barrier (BBB), only a few studies have been performed producing controversial results. In this article, it is shown that not only apamin is indeed able to penetrate the BBB in a cell-based model but also that an analog reported to be nontoxic passes through this barrier. Furthermore, the permeability values obtained, together with some evidence of an active transport mechanism and an amazing stability to serum proteases, make these peptides promising candidates for BBB shuttles.

Structure‐activity relationships of pentamidine‐affected ion channel trafficking and dofetilide mediated rescue

Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied. hERG and K(IR)2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit IKr . Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues. Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and K(IR)2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not K(IR)2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective. Drug-induced trafficking defects can be minimized if certain chemical features are avoided or 'synthesized out'; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS.

Feedback regulation of cone cyclic nucleotide channels by phosphoinositides. Focus on “CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions”

<i>Feedback regulation of cone cyclic nucleotide channels by phosphoinositides</i>. Focus on “CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions”

Propafenone blocks human cardiac Kir2.x channels by decreasing the negative electrostatic charge in the cytoplasmic pore

Human cardiac inward rectifier current (IK1) is generated by Kir2.x channels. Inhibition of IK1 could offer a useful antiarrhythmic strategy against fibrillatory arrhythmias. Therefore, elucidation of Kir2.x channels pharmacology, which still remains elusive, is mandatory. We characterized the electrophysiological and molecular basis of the inhibition produced by the antiarrhythmic propafenone of the current generated by Kir2.x channels (IKir2.x) and the IK1 recorded in human atrial myocytes. Wild type and mutated human Kir2.x channels were transiently transfected in CHO and HEK-293 cells. Macroscopic and single-channel currents were recorded using the patch-clamp technique. At concentrations >1μM propafenone inhibited IKir2.x the order of potency being Kir2.3∼IK1>Kir2.2>Kir2.1 channels. Blockade was irrespective of the extracellular K+ concentration whereas markedly increased when the intracellular K+ concentration was decreased. Propafenone decreased inward rectification since at potentials positive to the K+ equilibrium potential propafenone-induced block decreased in a voltage-dependent manner. Importantly, propafenone favored the occurrence of subconductance levels in Kir2.x channels and decreased phosphatidylinositol 4,5-bisphosphate (PIP2)-channel affinity. Blind docking and site-directed mutagenesis experiments demonstrated that propafenone bound Kir2.x channels at the cytoplasmic domain, close to, but not in the pore itself, the binding site involving two conserved Arg residues (residues 228 and 260 in Kir2.1). Our results suggested that propafenone incorporated into the cytoplasmic domain of the channel in such a way that it decreased the net negative charge sensed by K+ ions and polyamines which, in turn, promotes the appearance of subconductance levels and the decrease of PIP2 affinity of the channels.

Efficient and specific cardiac IK1 inhibition by a new pentamidine analogue

In excitable cells, KIR2.x ion-channel-carried inward rectifier current (IK₁) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific IK1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of IK1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits IK₁ by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific IK₁ inhibitors based on the P structure. We analysed seven pentamidine analogues (PA-1 to PA-7) for IK₁ blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC₅₀ values (12-15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG -44.1 kJ/Mol) than for P (ΔG -31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect INav1.5, ICa-L, IKv4.3, IKv11.1, and IKv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of IK₁ in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM. PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK₁ block.

Why the Drosophila Shaker K+ Channel Is Not a Good Model for Ligand Binding to Voltage-Gated Kv1 Channels

The Drosophila Shaker K(+) channel is the first cloned voltage-gated potassium channel and has, therefore, played an important role in structural and functional studies of those channels. While such a role is well justified for ion permeation, it is not clear whether this also extends to ligand binding. Despite the high degree of homology among Shaker and Kv1 channels, κ-conotoxin PVIIA (κ-PVIIA) binds to Shaker with high affinity but not to Kv1 channels. Here we address this issue by studying binding of κ-PVIIA to Shaker and Kv1 channels using molecular dynamics (MD) simulations. The structures of the channel-toxin complexes are constructed via docking and refinement with MD. The binding mode of each complex is characterized and compared to available mutagenesis data to validate the complex models. The potential of mean force for dissociation of the Shaker-κ-PVIIA complex is calculated from umbrella sampling MD simulations, and the corresponding binding free energy is determined, which provides further validation of the complex structure. Comparison of the Shaker and Kv1 complex models shows that a few mutations in the turret and extended regions are sufficient to abolish the observed sensitivity of Shaker to κ-PVIIA. This study demonstrates that Shaker is not always a good model for Kv1 channels for ligand binding. It also provides insights into the binding of the toxin to potassium channels that will be useful for improving affinity and selectivity properties of Kv1 channels.

Dissecting Gating Rules of GIRK Channels: Role of PIP2 in Ethanol-Dependent Activation

G protein-gated inwardly rectifying potassium (GIRK) channels are implicated in alcohol abuse and addiction. We discovered a discrete alcohol-binding pocket in the channel mediating ethanol-dependent activation. Here, we investigated the role of G proteins and PIP2 in ethanol-dependent gating. We engineered GIRK2 with single, modifiable cysteine at L257 in alcohol pocket and found that alcohol-like methanthiosulfonate (MTS) reagents activate GIRK2-L257C, similar to ethanol-dependent activation. We assessed role of G proteins in alcohol activation by either increasing levels of Gβγ (+Gβ1γ2) or decreasing Gβγ through chelation with membrane bound Phosducin (+mPhos). Neither Gβ1γ2 nor mPhos altered the rate of MTS-HE-mediated GIRK2-L257C activation. For comparison, we examined GIRK2-L344C, a key site for Gβγ activation that is inhibited by MTS modification. In contrast to L257C, rate of MTS modification showed a clear dependence on Gβγ levels. These results suggest that alcohol activation of GIRK channel is independent of G proteins.To investigate the role of PIP2, we used voltage-sensitive phosphatase DR-VSP to transiently deplete PIP2 in the membrane. Activation of DR-VSP completely reversed MTS-HE activated current of GIRK2-L257C. Furthermore, MTS-HE treatment significantly slowed the rate of GIRK2-L257C current inhibition following DR-VSP activation, suggesting an increase in apparent affinity for PIP2 and GIRK2-L257C channels modified by MTS-HE. Lastly, we examined the role of PIP2 on alcohol-dependent activation of wild-type GIRK2. Addition of propanol significantly slowed the rate of wild-type GIRK2 current inhibition following PIP2 depletion. Taken together, these data demonstrate that alcohol-dependent activation of GIRK involves an increase in apparent affinity for PIP2, with little influence of Gβγ subunits. The fundamental dichotomy between alcohol and Gβγ arises from distinct gating mechanisms converging on PIP2-dependent opening, revealing novel pathways for antagonizing alcohol's effects on ion channels.

Probability Based hERG Blocker Classifiers.

The US Food and Drug Administration (FDA) require in vitro human ether-a-go-go related (hERG) ion channel affinity tests for all drug candidates prior to clinical trials. In this study, probabilistic-based methods were employed to develop prediction models on hERG inhibition prediction, which are different from traditional QSAR models that are mainly based on supervised 'hard point' (HP) classification approaches giving 'yes/no' answers. The obtained models can 'ascertain' whether or not a given set of compounds can block hERG ion channels. The results presented indicate that the proposed probabilistic-based method can be a valuable tool for ranking compounds with respect to their potential cardio-toxicity and will be promising for other toxic property predictions.

Phase II Therapeutic Trial of Mexiletine in Non-Dystrophic Myotonia: Secondary Outcomes Show Improvement in Symptoms and Signs of Myotonia (S55.005)

Objective: To determine if mexiletine is effective therapy for myotonia in patients with Non-Dystrophic Myotonia (NDM). Background NDM results from rare mutations of muscle sodium and chloride channels. Patients experience delayed muscle relaxation following contraction and functionally limiting stiffness and pain. Mexiletine, a class 1B antiarrhythmic medication with a high affinity for muscle sodium channels, may reduce myotonia in NDM. Design/Methods: We conducted a randomized, double-blind, placebo controlled, cross over trial of mexiletine 200 mg three times daily in 59 patients with NDM at 7 centers in 4 countries. Each treatment period was 4 weeks, separated by a 1 week wash-out. The primary outcome was patient reported stiffness on an interactive voice response diary (IVR). Secondary endpoints included changes in pain, weakness, and tiredness on the IVR, SF-36, INQoL, clinical myotonia assessment, electrophysiological short and long exercise testing, quantitative grip myotonia, and needle EMG. Results: Last year we reported mexiletine provides major benefit for all symptoms of myotonia on the IVR and SF-36 quality of life measures, without study-related serious adverse events. Here we report mexiletine improves symptoms and signs of myotonia: (1) For almost all subdomains on the INQoL, most notably in muscle locking and perceived treatment effects; (2) Reduces time to open the eyes (-0.75 seconds, p=0.0008) and fist (-1.32 seconds, p Conclusions: Mexiletine at a dose of 200 mg three times daily is effective therapy for symptoms and signs of myotonia in patients with NDM. Supported by: FDA Orphan Products Division grant# RO1 FD003454 and the NIH National Institute of Neurological Disorders and Stroke grant# U54 NS059065-05S1. Disclosure: Dr. Statland has nothing to disclose. Dr. Salajegheh has nothing to disclose. Dr. Bundy has nothing to disclose. Dr. Wang has nothing to disclose. Dr. Raja Rayan has nothing to disclose. Dr. Trivedi has nothing to disclose. Dr. Sansone has nothing to disclose. Dr. Venance has received personal compensation for activities with Genzyme Corporation. Dr. Ciafaloni has nothing to disclose. Dr. Matthews has nothing to disclose. Dr. Meola has nothing to disclose. Dr. Zanolini has nothing to disclose. Dr. Ciocca has nothing to disclose. Dr. Herbelin has nothing to disclose. Dr. Griggs has nothing to disclose. Dr. Barohn has received personal compensation for activities with Talecris Biotherapeutics, Genzyme Corporation and Speakers Bureau. Dr. Barohn has received research support from Alexion Pharmaceuticals. Dr. Hanna has nothing to disclose. The Consortium for Clinical Investigation of Neurologic Channelopathies has nothing to disclose.

Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of CaV2.1 voltage-gated calcium channels

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of CaV2.1 voltage-gated Ca2+ channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human CaV2.1α1 subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK‐293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant CaV2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca2+ channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.

Hg1, Novel Peptide Inhibitor Specific for Kv1.3 Channels from First Scorpion Kunitz-type Potassium Channel Toxin Family

The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nM for recombinant LmKTT-1a, 160 nM for LmKTT-1b, 124 nM for LmKTT-1c, 136 nM for BmKTT-1, 420 nM for BmKTT-2, 760 nM for BmKTT-3, and 107 nM for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ~50-80% of Kv1.3 channel currents at a concentration of 1 μM. The exception was rBmKTT-3, which had weak activity. The IC(50) values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ~129.7, 371.3, and 6.2 nM, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.