No abstract

Background and purpose: Inhibition of HERG channels prolongs the ventricular action potential and the QT interval with the risk of torsade de pointes arrhythmias and sudden cardiac death. Many drugs induce greater inhibition of HERG channels when the cell membrane is depolarized frequently. The dependence of inhibition on the pulsing rate may yield different IC 50 values at different frequencies and thus affect the quantification of HERG channel block. We systematically compared the kinetics of HERG channel inhibition and recovery from block by 8 blockers at different frequencies. Experimental approach: HERG channels were expressed heterologously in Xenopus oocytes and currents were measured with the two-electrode voltage clamp technique. Key results: Frequency-dependent block was observed for amiodarone, cisapride, droperidol and haloperidol (group 1) whereas bepridil, domperidone, E-4031 and terfenadine (group 2) induced similar pulse-dependent block at all frequencies. With the group 1 compounds, HERG channels recovered from block in the presence of drug (recovery being voltagedependent). No substantial recovery from block was observed with the second group of compounds. Washing out of bepridil, domperidone, E-4031 and terfenadine was substantially augmented by frequent pulsing. Mutation D540K in the HERG channel (which exhibits reopening at negative voltages) facilitated recovery from block by these compounds at À140 mV. Conclusion and implications: Drug molecules dissociate at different rates from open and closed HERG channels ('usedependent' dissociation). Our data suggest that apparently 'trapped' drugs (group 2) dissociated from the open channel state whereas group 1 compounds dissociated from open and resting states.

The entry of Ca 2ϩ through voltage-gated Ca 2ϩ channels has direct effects on muscle contraction, release of hormones and neurotransmitters, hearing, vision, gene expression, and other important physiological functions (2). The pore-forming ␣ 1 -subunits of voltage-gated Ca 2ϩ channels are composed of four homologous domains formed by six transmembrane segments (S1-S6) that are linked together on a single polypeptide (3). A membrane depolarization initiates channel openings (activation) and closures (inactivation). These events can be considered a multistep process consisting of a conformational change in the voltage sensor, a transmission of the signal to the pore region, the opening of the pore, and channel closure due to inactivation. The voltage-sensing machinery is formed by multiple charged amino acids located in segment S4 and adjacent structures of each domain (4). A large number of amino acids involved in Ca 2ϩ channel inactivation have been identified and several molecular mechanisms for this process have been proposed (for reviews see Refs. 5-7).The molecular mechanism of the voltage-dependent pore opening of Ca 2ϩ channels, however, is less studied and largely unknown. The first attempt to localize the structural elements in Ca 2ϩ channel ␣ 1 -subunits that are involved in channel activation was made by Tanabe et al. (8) who constructed chimeric channels in which sequence stretches of a slow activating ("skeletal muscle-like") Ca V 1.1 ␣ 1 -subunit were replaced by sequences from a fast activating ("cardiac-like") Ca V 1.2 ␣ 1 -subunit. The chimeras activated slowly if repeat I of the Ca V 1.2 ␣ 1 -subunit was replaced by the Ca V 1.1 ␣ 1 -sequence. In a later study, replacement of domains I, II, and III of the low voltage and fast activating Ca V 3.1 ␣ 1 -subunit with the corresponding domains of the high voltage-activated Ca V 1.2 ␣ 1 -subunit resulted in a high voltage-activated channel (9). An important role of domains I and III but not II and IV on midpoint voltage and time constants of activation was reported by Garcia et al. (10) who mutated the arginines in the S4 segments of all four domains of a chimeric channel to neutral or negative amino acids. The removal of prolines that are conserved in segments IS4 and IIIS4 of voltage-gated Ca 2ϩ channels resulted in shortening of channel open time, whereas introduction of extra prolines to corresponding positions of IIS4 and IVS4 lengthened the channel open time (11).Our present study was initiated by the recent finding that a novel retinal disorder is caused by a point mutation (I745T) in segment IIS6 of the Ca V 1.4 ␣ 1 -subunit that shifts the voltage dependence of Ca V 1.4 channel activation by approximately Ϫ30 mV (1, 12). As Ca V 1.4 channels express only at low density in mammalian cell lines (13) we have decided to study the functional roles of this residue and neighboring residues in segment IIS6 by introducing and characterizing mutations in the homologous Ca V 1.2 channel. Our findings demonstrate that residue Ile-781 and three neigh...

Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K + current (I Kr ) and are essential for the repolarization of the cardiac action potential. hERG1 inhibition by structurally diverse drugs may lead to life threatening arrhythmia. Putative binding determinants of hERG1 channel blockers include T623, S624 and V625 on the pore helix, and residues G648, Y652 and F656, located on segment S6. We and others have previously hypothesized that additional binding determinants may be located on helix S5, which is in close contact with the S6 segments. In order to test this hypothesis, we performed a detailed investigation combining ionic current measurements with two-microelectrode voltage clamp and molecular modeling techniques. We identified a novel aromatic high affinity binding determinant for blockers located in helix S5, F557, which is equally potent as Y652. Modeling supports a direct interaction with the outer pore helix.Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K + current (I Kr ) and are essential for regulating the duration of the plateau phase of the cardiac action potential 1,2 . Inherited loss-of-function mutations in hERG1 can lead to life threatening torsades de pointes (TdP) arrhythmia 3 , while gain-of-function mutations are associated with short QT syndrome 4 . Most frequently, TdP arrhythmia is an acquired disorder, resulting from "off-target" block of this channel by structurally diverse drugs including antiarrhythmics, antihistamines, antipsychotics and antibiotics 5 . Since this inhibition can lead to sudden cardiac death, several pharmaceuticals such as cisapride or terfenadine were withdrawn from the market, or had their use severely restricted 6,7 . Recently, the Cardiac Safety Research Consortium (CSRC) and the Food and Drug Administration (FDA) proposed a new cardiac safety paradigm labelled "Comprehensive In Vitro Proarrhythmia Assay" (CiPA). The new CiPA guidelines emphasize the importance of studying pharmacological effects of drugs on three different ion channel types including hERG, Nav1.5 and Cav1.2, which proposed to play an important role in shaping the ventricular action potential 8 . hERG1 blockers might also have beneficial therapeutic potential. During routine preclinical screening for hERG1, new modulators, so-called activators, have been identified. These modulators may have the potential of shortening the action potential duration 9 . Thus, they might be beneficial for the treatment of inherited long QT syndrome.Great efforts have been directed toward a better understanding of the molecular and structural mechanisms of hERG1 channel drug interactions, including in vivo, in vitro and in silico approaches (for a recent reviews see Durdagi, S. et al. 10 and Vandenberg, J. et al. 11 ). Substantial progress has been made by identifying amino acids essential for drug block. The majority of hERG inhibitors are interacting with the pore module. This homo tetrameric module consists of an outer S5 helix, a...

GABA A receptors composed of ␣ 1 , ␤ 2 , ␥ 1 subunits are expressed in only a few areas of the brain and thus represent interesting drug targets. The pharmacological properties of this receptor subtype, however, are largely unknown. In the present study, we expressed ␣ 1 ␤ 2 ␥ 1 -GABA A receptors in Xenopus laevis oocytes and analyzed their modulation by 21 ligands from 12 structural classes making use of the two-microelectrode voltage-clamp method and a fast perfusion system. Modulation of GABA-induced chloride currents (I GABA ) was studied at GABA concentrations eliciting 5 to 10% of the maximal response. Triazolam, clotiazepam, midazolam, 2-(4-methoxyphenyl)-2, 3,5,6,7,8,9,10-octahydro-cyclohepta-(b) (CGS 9896), diazepam, zolpidem, and bretazenil at 1 M concentrations were able to significantly (Ͼ20%) enhance I GABA in ␣ 1 ␤ 2 ␥ 1 receptors. Methyl-6,7-dimethoxy-4-ethyl-␤-carboline-3-carboxylate, 3-methyl-6-[3-trifluoromethyl-phenyl]-1,2,4-triazolo [4,3-b]pyridazine (Cl 218,872), clobazam, flumazenil,, and 6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)phenylmethanone (Ru 32698) (1 M each) had no significant effect, and flunitrazepam and 2-phenyl-4-(4-ethyl-piperidinyl)-quinoline (PK 8165) inhibited I GABA . The most potent compounds triazolam, clotiazepam, midazolam, and CGS 20625 were investigated in more detail on ␣ 1 ␤ 2 ␥ 1 and ␣ 1 ␤ 2 ␥ 2S receptors. The potency and efficiency of these compounds for modulating I GABA was smaller for ␣ 1 ␤ 2 ␥ 1 than for ␣ 1 ␤ 2 ␥ 2S receptors, and their effects on ␣ 1 ␤ 2 ␥ 1 could not be blocked by flumazenil. CGS 20625 displayed the highest efficiency by enhancing at 100 M I GABA (␣ 1 ␤ 2 ␥ 2 ) by 775 Ϯ 17% versus 526 Ϯ 14% I GABA (␣ 1 ␤ 2 ␥ 1 ) and 157 Ϯ 17% I GABA (␣ 1 ␤ 2 ) (p Ͻ 0.05). These data provide new insight into the pharmacological properties of GABA A receptors containing ␥ 1 subunits and may aid in the design of specific ligands for this receptor subtype.GABA is the principal inhibitory neurotransmitter in the mammalian brain. It mediates fast synaptic inhibition by interaction with the GABA A receptor. GABA A receptors are ligandgated ion channels that are modulated by a large number of clinically relevant drugs such as benzodiazepines (BZs), barbiturates, neurosteroids, and anesthetics (Sieghart, 1995). They are assembled from individual subunits forming a pentameric structure. Nineteen isoforms of mammalian GABA A receptor subunits have been cloned: ␣ 1-6 , ␤ 1-3 , ␥ 1-3 , ␦, ⑀, p, 1-3 , and (Barnard et al., 1998;Simon et al., 2004). The major receptor subtype of the GABA A receptor in adults consists of ␣ 1 , ␤ 2 , and ␥ 2 subunits, and the most likely stoichiometry is two ␣ subunits, two ␤ subunits, and one ␥ subunit (Sieghart and Sperk, 2002). CGS 20625, 2-(4-methoxyphenyl)-2,3,5,6,7,8,9,10-octahydro-cyclohepta-(b) ABBREVIATIONS: