The IVS6 segment of the L-type calcium channel is critical for the action of dihydropyridines and phenylalkylamines.

Schuster, Anja; Lacinová, Ľubica; Klugbauer, Norbert; Ito, Hiroyuki; Birnbaumer, Lutz; Hofmann, Franz

doi:10.1002/j.1460-2075.1996.tb00592.x

Cited by 115 publications

(122 citation statements)

References 25 publications

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“…Kass and colleagues found that the DHP receptor site lies within the lipid bilayer approximately 11-14 Å from the extracellular surface of the membrane (3). These conclusions are consistent with mutagenic studies where individual amino acid residues located on transmembrane segments IIIS5, IIIS6 and IVS6 were found to be critical for DHP binding and activity (4)(5)(6)(7)(8).…”

supporting

confidence: 90%

Calcicludine Binding to the Outer Pore of L-type Calcium Channels Is Allosterically Coupled to Dihydropyridine Binding

Wang

Peterson

2007

Biochemistry

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How dihydropyridines modulate L-type voltage-gated Ca 2+ channels is not known. Dihydropyridines bind cooperatively with Ca 2+ binding to the selectivity filter, suggesting that they alter channel activity by promoting structural rearrangements in the pore. We used radioligand binding and patchclamp electrophysiology to demonstrate that calcicludine, a toxin from the venom of the green mamba snake, binds in the outer vestibule of the pore and, like Ca 2+ , is a positive modulator of dihydropyridine binding. Data were fit using an allosteric scheme where dissociation constants for dihydropyridine and calcicludine binding, K DHP and K CaC , are linked via the coupling factor, α. Nine acidic amino acids located within the S5-Pore-helix segment of repeat III were sequentially changed to alanine in groups of three resulting in the mutant channels, Mut-A, Mut-B and Mut-C. Mut-A, whose substitutions are proximal to IIIS5, exhibits a 4.5-fold reduction in dihydropyridine binding and is insensitive to calcicludine binding. Block of Mut-A currents by calcicludine is indistinguishable from wild-type, indicating that K CaC is unchanged and that the coupling between dihydropyridine and calcicludine binding (i.e., α) is disrupted. Mut-B and Mut-C possess K DHP values that resemble wild-type. Mut-C, the most C-terminal of the mutant channels, is insensitive to calcicludine binding and block. K CaC values for the Mut-C single mutants, E1122A, D1127A and D1129A, increase from 0.3 (Wild-type) to 1.14, 2.00 and 20.5 μM, respectively. Together, these findings suggest that dihydropyridine antagonist and calcicludine binding to L-type Ca 2+ channels promote similar structural changes in the pore that stabilize the channel in a nonconducting, blocked state.The flow of Ca 2+ ions through voltage-gated Ca 2+ channels drives a variety of cellular processes including excitation-contraction coupling, neurotransmitter release and gene expression. Voltage activated Ca 2+ channels are heteromultimeric complexes consisting of α 1 , β, α 2 /δ and sometimes γ subunits. The pore-forming α 1 subunit contains all of the structural determinants required for voltage-dependent gating, drug binding and ion permeation. The membrane topology of the α 1 subunit consists of four homologous repeats (I, II, III, IV), each consisting of six transmembrane segments (S1-S6). Each of the four S5/S6 connecting segments contains a highly conserved negatively charged glutamate residue that together form a binding site for Ca 2+ ions called the selectivity filter. The selectivity filter is the narrowest region in the pore and is the site that enables the channel to conduct Ca 2+ ions under physiological conditions where Na + is in excess (1). † This work was supported by research grants from the American Heart Association (0230298N) and the National Institutes of Health (RO1 HL074143) to B.Z.P. Important clues regarding the molecular basis for DHP action came from the finding that DHP binding to a site that lies outside the permeation pathway is cooperative w...

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supporting

confidence: 90%

Calcicludine Binding to the Outer Pore of L-type Calcium Channels Is Allosterically Coupled to Dihydropyridine Binding

Wang

Peterson

2007

Biochemistry

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“…This property was responsible for the inhibition of cardiac contraction by 1 M nisoldipine, since at depolarized membrane potentials which are necessary for channel opening the affinity for nisoldipine increased significantly. The affinity of the Ca v 1.2 channel for DHPs is lowered 100-fold by mutation of Tyr-1485, Met-1486, and Ile-1493 in the IVS6 segment (39) and is lost upon mutation of Thr-1061 in the IIIS5 segment (see Refs. 3,5,and 20).…”

Section: Resultsmentioning

confidence: 99%

Functional Embryonic Cardiomyocytes after Disruption of the L-type α1C (Ca 1.2) Calcium Channel Gene in the Mouse

Seisenberger¹,

Specht²,

Welling³

et al. 2000

Journal of Biological Chemistry

252

207

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“…Mutagenesis was performed using the QuikChangeXL kit (Stratagene) according to the manufacturer's instructions. This DHP-insensitive mutant contained mutations at the critical residues Thr1066 in IIIS5 [4,14,15], as well as Phe 1463, Met1464, and Iso1471 in IVS6 [3][4][5]. These mutations have previously been shown to decrease the affinity of α 1c for dihydropyridines by over 100-fold, without significant effects on the biophysical properties of the channel [3].…”

Section: Experimental Procedures α 1c-d-plasmid Preparationmentioning

confidence: 99%

“…The largest component is the pore-forming α 1 subunit (∼220 kDa), the critical determinant of most functional properties of the channel, including voltage-dependent gating, Ca 2+ permeability, Ca 2+ -dependent inactivation [1,2], and inhibition by the three principal classes of Ca 2+ channel antagonists [3][4][5].…”

Section: Introductionmentioning

confidence: 99%

Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

Ganesan

O’Rourke

Maack

et al. 2005

Biochemical and Biophysical Research Communications

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The α 1c subunit of the cardiac L-type Ca 2+ channel, which contains the channel pore, voltage-and Ca 2+ -dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca 2+ channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length α 1c gene cassettes into the adenovirus backbone. Wildtype (α 1c-wt ) and mutant (α 1c-D-) Ca 2+ channel adenoviruses were constructed. The α 1c-D-contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both α 1c-wt and α 1c-D-expressed robustly in A549 cells (peak L-type Ca 2+ current (I CaL ) at 0 mV: α 1c-wt −9.94 ± 1.00 pA/pF, n = 9; α 1c-D-−10.30 pA/pF, n = 12). I CaL carried by α 1c-D-was markedly less sensitive to nitrendipine (IC 50 17.1 µM) than α 1c-wt (IC 50 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 µM nitrendipine blocked only 51 ± 5% (n = 9) of I CaL in α 1c-D--expressing myocytes, in comparison to 86 ± 8% (n = 9) of I CaL in control myocytes. Moreover, in 20 µM nitrendipine, calcium transients could still be evoked in α 1c-D--transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca 2+ release. These α 1c adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitationcontraction coupling and L-type Ca 2+ channel regulation in the native myocyte background. KeywordsDihydropyridine; Excitation-contraction coupling; Gene transfer; Adenovirus; Ion channelThe cardiac L-type Ca 2+ channel (Ca v 1.2a) is a multisubunit complex of five proteins located on the surface membrane of the cardiac myocyte. The largest component is the pore-forming α 1 subunit (∼220 kDa), the critical determinant of most functional properties of the channel, including voltage-dependent gating, Ca 2+ permeability, Ca 2+ -dependent inactivation [1,2], and inhibition by the three principal classes of Ca 2+ channel antagonists [3][4][5].

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The IVS6 segment of the L-type calcium channel is critical for the action of dihydropyridines and phenylalkylamines.

Cited by 115 publications

References 25 publications

Calcicludine Binding to the Outer Pore of L-type Calcium Channels Is Allosterically Coupled to Dihydropyridine Binding

Calcicludine Binding to the Outer Pore of L-type Calcium Channels Is Allosterically Coupled to Dihydropyridine Binding

Functional Embryonic Cardiomyocytes after Disruption of the L-type α1C (Ca 1.2) Calcium Channel Gene in the Mouse

Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

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