Molecular Determinants of High Affinity Phenylalkylamine Block of l-type Calcium Channels in Transmembrane Segment IIIS6 and the Pore Region of the α1Subunit

Hockerman, Gregory H.; Johnson, Barry D.; Abbott, Mick; Scheuer, Todd; Catterall, William A.

doi:10.1074/jbc.272.30.18759

Cited by 112 publications

(119 citation statements)

References 31 publications

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“…Leucine and valine are both aliphatic, hydrophobic amino acids, but valine has a smaller molecular weight and is less hydrophobic than leucine. However, contrary to previous studies concentrating on segment IIIS6 in the ␣ 1 subunit of L-type Ca 2ϩ channels (11,25,26), our results suggest that the valine in this position actually destabilizes the inactivated state. L1341V and HHT-5421 (HHT-5411 and HHT-5371) (13) shifted the voltage dependence of steady-state inactivation to more positive membrane potentials and inactivated incompletely during the pulse, and a substantial sustained current remained at the end of the depolarization.…”

Section: Discussioncontrasting

confidence: 55%

The Role of Region IVS5 of the Human Cardiac Calcium Channel in Establishing Inactivated Channel Conformation

Bódi¹,

Koch²,

Yamaguchi³

et al. 2002

Journal of Biological Chemistry

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The role of inactivated channel conformation and use dependence for diltiazem, a specific benzothiazepine calcium channel inhibitor, was studied in chimeric constructs and point mutants created in the IVS5 transmembrane segment of the L-type cardiac calcium channel. All mutations, chimeric or point mutations, were restricted to IVS5, while the YAI-containing segment in IVS6, i.e. the primary interaction site with benzothiazepines, remained intact. Slowed inactivation rate and incomplete steady state inactivation, a behavior of some mutants, were accompanied by a reduced or by a complete loss of use-dependent block by diltiazem. L-type Ca 2ϩ channels of cardiac, skeletal, and smooth muscle play a central role in excitation-contraction coupling. It is also believed that these channels may participate in the pathophysiology of some cardiac arrhythmias, hypertension, and angina pectoris (1-4). Increases in [Ca 2ϩ ] i have been linked to a variety of changes in membrane properties that contribute to the changes in excitability, conduction, refractoriness, automaticity, and vascular resistance (1-4). In terms of excitation-contraction coupling, Ca 2ϩ enters the cell during depolarization through voltage-activated calcium channels and triggers contraction but also is responsible for activation of other cellular functions. In the course of sustained depolarization, Ca 2ϩ currents progressively undergo voltage-dependent inactivation with specific kinetics, primarily regulated by the membrane potential.Ca 2ϩ channels are the target for three main classes of drugs that include dihydropyridines (DHP), 1 phenylalkylamines (PAA), and benzothiazepines (BTZ). These molecules bind specifically to different sites of the Ca 2ϩ channel ␣ 1 subunit, and their binding domains are allosterically linked to each other (5-7). Ca 2ϩ channel antagonists, particularly diltiazem and verapamil, block calcium channels in a voltage-and use-dependent manner; thus the binding of the drug facilitates protein conformational changes that alter channel function by interfering with channel gating. These drugs serve as useful tools in dissecting molecular mechanisms of channel inactivation. Several amino acid residues that are involved in Ca 2ϩ antagonist binding, when mutated, change the electrophysiological properties of the channel, such as voltage-dependent inactivation. Single amino acids in segments IIIS6 and IVS6 (5, 8 -11) have been identified as determinants of inactivation. Mutation of key amino acids in these segments alters not only the high affinity drug-binding sites but also the complexity of the kinetic behavior of the channels, consequently changing use-dependent block particularly for the PAAs and BTZs (for review, see Ref. 12).Our previous finding showed that a segment in IVS5 of the human ␣ 1C (Ca v 1.2) subunit is critically involved in inactivation of the channel (13). Consequently, mutants constructed in this region lost the characteristic use-dependent block by PAA and BTZ and recovered from inactivation significantly faster a...

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Section: Discussioncontrasting

confidence: 55%

The Role of Region IVS5 of the Human Cardiac Calcium Channel in Establishing Inactivated Channel Conformation

Bódi¹,

Koch²,

Yamaguchi³

et al. 2002

Journal of Biological Chemistry

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“…These include local anesthetics, class I antiarrhythmics and anticonvulsants that block Na ϩ channels, and dihydropyridines and phenylalkamines that block L-type Ca 2ϩ channels. For both channel types, molecular determinants for high-affinity drug binding have been identified in the S6 regions of domains III and IV by using Ala-scanning mutagenesis (27)(28)(29)(30). Although the drug binding sites are highly complex, there are some similarities with the binding site in HERG.…”

Section: Resultsmentioning

confidence: 99%

A structural basis for drug-induced long QT syndrome

Mitcheson

Chen

Lin

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

864

540

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L ong QT syndrome (LQT) is an abnormality of cardiac muscle repolarization that predisposes affected individuals to a ventricular arrhythmia that can degenerate into ventricular fibrillation and cause sudden death (1). The cellular mechanism of the lengthened QT interval recorded on the body surface electrocardiogram is prolonged ventricular action potentials. Recent genetic discoveries have determined that the molecular mechanism of inherited LQT is mutations in one of several genes [e.g., HERG (2), KCNE2 (3)] that encode ion channel subunits important for normal repolarization of the ventricle. HERG encodes the pore-forming subunits of channels that conduct the rapid delayed rectifier K ϩ current (I Kr ; refs. 4 and 5). LQT also can be acquired as a side effect of treatment with commonly used medications, including some antiarrhythmic, antihistamine, antibiotic, psychoactive, and gastrointestinal prokinetic agents (6, 7). Although drug-induced LQT theoretically could result from reduction of any voltage-gated K ϩ current that contributes to ventricular repolarization, all known drugs with this side effect preferentially block I Kr (1,8,9). It is unclear why so many structurally diverse compounds block HERG channels, but this undesirable side effect now is recognized as a major hurdle in the development of new and safe drugs (9, 10). Previous studies have characterized single residues of HERG that when mutated altered the sensitivity of the channel to block by the methanesulfonanilide antiarrhythmic drug dofetilide. However, these residues are believed to alter drug binding by an allosteric effect related to the loss of inactivation gating (11). A more recent study found that mutation of a Phe located in the S6 domain of HERG decreased block by dofetilide and quinidine (12). However, it is unlikely that a single residue could confer drug sensitivity. Thus, despite the obvious clinical importance of HERG channel block to acquired LQT, the structural basis of the high-affinity drug binding site has not been adequately characterized. Materials and Methods Mutagenesis and Channel Expression in Oocytes.Mutations were introduced into the HERG K ϩ channel (13) by using sitedirected mutagenesis as described previously (14). Complementary RNAs for injection into oocytes were prepared with SP6 Cap-Scribe (Boehringer Mannheim) after linearization of the expression construct with EcoRI. Isolation and maintenance of Xenopus oocytes and cRNA injection were performed as described previously (14-16).Voltage Clamp. The two-microelectrode voltage clamp technique (17) was used to record membrane currents in oocytes 2-4 days after cRNA injection as previously described (18). To attenuate endogenous chloride currents, Cl Ϫ was replaced with Mes (2-[N-morpholino]ethanesulfonic acid) in the external solution that contained 96 mM NaMes͞2 mM KMes͞2 mM CaMes 2 ͞5 mM Hepes͞1 mM MgCl 2 adjusted to pH 7.6 with methane sulfonic acid. In some experiments, KMes was increased to 96 mM with a similar reduction in NaMes.MK-499 was supplied by Me...

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“…Our experimental data do not allow these possibilities to be to distinguished between. Moreover, it is currently not clear if the increase in apparent drug sensitivity with Ca 2ϩ is accomplished by the ion interaction with pore determinants (eg, Glu 1118 and Glu 1419 ; see Hockerman et al 31 ) or, alternatively, with a site that is located in the region of the C-terminus. 22,24,32,33 …”

Section: Role Of the Permeant Ion In Paa Sensitivitymentioning

confidence: 99%

“…The dashed line represents the recovery time course (ϭ15 seconds) observed in Figure 5D. 31 ) or, alternatively, with a site that is located in the region of the C-terminus. 22,24,32,33 Simulation of PAA-Induced Effects on Channel Inactivation…”

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confidence: 99%

On the Role of Ca ²⁺ - and Voltage-Dependent Inactivation in Ca _v 1.2 Sensitivity for the Phenylalkylamine (-)Gallopamil

Sokolov¹,

Timin²,

Hering³

2001

Circulation Research

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Abstract-L-type calcium channels (Ca v 1.m) inactivate in response to elevation of intracellular Ca 2ϩ (Ca 2ϩ -dependent inactivation) and additionally by conformational changes induced by membrane depolarization (fast and slow voltage-dependent inactivation). Molecular determinants of inactivation play an essential role in channel inhibition by phenylalkylamines (PAAs). The relative impacts, however, of Ca 2ϩ -dependent and voltage-dependent inactivation in Ca v 1.2 sensitivity for PAAs remain unknown. In order to analyze the role of the different inactivation processes, we expressed Ca v 1.2 constructs composed of different ␤-subunits (␤ 1a -, ␤ 2a -, or ␤ 3 -subunit) in Xenopus oocytes and estimated their (-)gallopamil sensitivity by means of the two-microelectrode voltage clamp with either Ba 2ϩ or Ca 2ϩ as charge carrier. Ca v 1.2 consisting of the ␤ 2a -subunit displayed the slowest inactivation and the lowest apparent sensitivity for the PAA (-)gallopamil. A significantly higher apparent (-)gallopamil-sensitivity with Ca 2ϩ as charge carrier was observed for all 3 ␤-subunit compositions. The kinetics of Ca 2ϩ -dependent inactivation and slow voltage-dependent inactivation were not affected by drug. The higher sensitivity of the Ca v 1.2 channels for (-)gallopamil with Ca 2ϩ as charge carrier results from slower recovery ( rec,Ca Ϸ15 seconds versus rec,Ba Ϸ3 to 5 seconds) from a PAA-induced channel conformation. We propose a model where (-) antagonists, ie, phenylalkylamines (PAAs), benzothiazepines (ie, diltiazem) and 1,4-dihydropyridines (DHPs). [2][3][4][5] About 20 years ago, several groups demonstrated that Ca v 1.m inhibition by PAAs and diltiazem occurs predominately during membrane depolarizations ("use-dependent" action) when the channels are primarily in the open or inactivated conformation. [2][3][4]6 Since then, numerous studies in myocardial and smooth muscle cells have shown that Ca 2ϩ channel inhibition by these compounds, as well as by the new compound mibefradil, reflect a balance between channel shut-off during membrane depolarization and subsequent recovery at rest. [7][8][9][10][11] Over the last three years there has been substantial progress toward understanding the role of inactivation in Ca 2ϩ channel inhibition by Ca 2ϩ antagonists. Site directed mutagenesis enabled a detailed analysis of the impact of this process in PAA sensitivity. A comparison of the PAA sensitivities of Ca v 2.1 mutants and chimeric channel constructs inactivating at different rates revealed a strong correlation between the rates of fast voltage-dependent inactivation and apparent PAA sensitivities. 11-13 Similar findings were reported for Ca 2ϩ channel block by diltiazem. 11,14 Subsequently, the studies concentrated on the localization of inactivation determinants that are relevant for channel inhibition. Thus, inactivation determinants in the loop between domains I and II and ␤-subunit interaction have been shown to control PAA sensitivity of a Ca v 2.1 mutant 13 as well as the mibefradilsensitivity of wild ...

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Molecular Determinants of High Affinity Phenylalkylamine Block of l-type Calcium Channels in Transmembrane Segment IIIS6 and the Pore Region of the α1Subunit

Cited by 112 publications

References 31 publications

The Role of Region IVS5 of the Human Cardiac Calcium Channel in Establishing Inactivated Channel Conformation

The Role of Region IVS5 of the Human Cardiac Calcium Channel in Establishing Inactivated Channel Conformation

A structural basis for drug-induced long QT syndrome

On the Role of Ca ²⁺ - and Voltage-Dependent Inactivation in Ca _v 1.2 Sensitivity for the Phenylalkylamine (-)Gallopamil

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Molecular Determinants of High Affinity Phenylalkylamine Block of l-type Calcium Channels in Transmembrane Segment IIIS6 and the Pore Region of the α1Subunit

Cited by 112 publications

References 31 publications

The Role of Region IVS5 of the Human Cardiac Calcium Channel in Establishing Inactivated Channel Conformation

The Role of Region IVS5 of the Human Cardiac Calcium Channel in Establishing Inactivated Channel Conformation

A structural basis for drug-induced long QT syndrome

On the Role of Ca 2+ - and Voltage-Dependent Inactivation in Ca v 1.2 Sensitivity for the Phenylalkylamine (-)Gallopamil

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On the Role of Ca ²⁺ - and Voltage-Dependent Inactivation in Ca _v 1.2 Sensitivity for the Phenylalkylamine (-)Gallopamil