Purified calcium channels have three allosterically coupled drug receptors

Striessnig, Jörg; Göll, A.; Moosburger, Kurt; Glossmann, Hartmut

doi:10.1016/0014-5793(86)80327-1

Cited by 71 publications

(37 citation statements)

References 24 publications

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“…This allosteric interaction was easily observed at 37°C but could be demonstrated also at lower temperatures. Similar results have been observed previously with the skeletal muscle membrane-bound receptor [36, 411, the cardiac muscle membrane-bound and solubilized receptor [17, 181 and a receptor purified from microsomes of guinea pig skeletal muscle [23]. The similarity of the properties of the membrane-bound and purified receptor suggests that the allosteric, temperaturedependent characteristics of the binding of various calcium channel blockers to this receptor are mainly a property of the channel protein and not determined by other membrane constituents.…”

Section: Discussionsupporting

confidence: 90%

“…A similar high density value has been reported by Curtis and Catterall [20]. The specific densities of the receptors purified by different techniques were 0.8 nmol/mg [22] and 0.5 nmol/mg in the absence [23] and 1.5 nmol/mg in the presence of diltiazem [23]. This comparison suggests therefore that the receptor used in this study was at least as pure as those used in other studies.…”

Section: Discussionsupporting

confidence: 89%

“…The arrow in C indicates the position of thyroglobulin (19.2 S). Fraction size was 2 ml in A, 1.5 ml in B and 2.8 ml in C muscle microsomes [23]. At 18°C the density of the purified rabbit skeletal muscle receptor was about half that measured at 4°C.…”

Section: N-mentioning

confidence: 94%

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Purification of a functional receptor for calcium‐channel blockers from rabbit skeletal‐muscle microsomes

Flockerzi

Oeken

Hofmann

1986

European Journal of Biochemistry

101

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The dihydropyridine receptor was purified from rabbit skeletal muscle microsomes in the presence of [3H]nitrendipine plus diltiazem or [3H](+)PN 200-1 10 to an apparent density of 1.5 -2 nmol binding sites/mg protein. Sodium dodecyl sulfate gel electrophoresis in the absence of reducing agents yielded three peptide bands of 142, 56 and 30 kDa in a relative ratio of 11 : 1 : 1.3, whereas in the presence of 40 mM dithiothreitol bands of 142, 122, 56, 31, 26 and 22 kDa were obtained in a relative ratio of 5.5:2.2: 1 :0.9: 14:0.09. This gel pattern was observed regardless of whether the receptor was purified as a complex with nitrendipine plus diltiazem or with (+)PN 200-1 10. CAMP-dependent protein kinase phosphorylated preferentially the 142-kDa band up to a stoichiometry of 0.82 f 0.07 (15) mol phosphate/mol peptide. The 56-kDa band was phosphorylated only in substoichiometric amounts. These results suggest that the purified dihydropyridine receptor retains the basic properties of the membranebound receptor and contains separate sites for at least dihydropyridines and phenylalkylamines.Most excitable cells contain two types of voltage-regulated calcium channels, namely a transient (t) and a long-lasting (1) channel [l -41. The opening of the cardiac muscle 1-type depends on membrane depolarization to less then -35 mV and is facilitated by CAMP-dependent phosphorylation of the channel [5-91. Both types of calcium channels are present in skeletal muscle [4]. The skeletal muscle 1-type channel shares many electrophysiological properties with the cardiac muscle 1-type channel, although these channels are not identical [lo]. Apparently, the skeletal muscle 1-type channel is affected by CAMP-dependent phosphorylation [ll]. The conductance of -1 10,isopropy1-4-(2,1,3-benzoxadia-dine-3-carboxylate; PhMeS02F, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; WGA, wheat germ agglutinin; I&, concentration required to inhibit 50% of a maximal effect. the 1-type but not the t-type calcium channel is blocked by a heterogenous group of organic compounds which include the dihydropyridines nitrendipine and PN 200-1 10, the phenylalkylamines verapamil and desmethoxyverapamil and the benzothiazepine diltiazem. The tritiated congeners of these compounds bind to specific sites in skeletal and cardiac muscle membranes 112 -191. The high-affinity binding sites for these compounds interact allosterically with each other, suggesting that the membrane-receptor complex contains distinct sites for dihydropyridines, phenylalkylamines and presumably diltiazem [12-15, 17, 181. The high-affinity sites have been considered to represent the voltage-operated calcium channel, although until recently a direct proof has not been presented for this suggestion.Purification of the 1-type calcium channel has not been achieved from cardiac muscle since this muscle contains a very low density of this channel [17, 181. In contrast, t-tubular membranes of skeletal muscle have a high density of binding sites for dihydropyridines [13,15] and have b...

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Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 89%

See 1 more Smart Citation

Purification of a functional receptor for calcium‐channel blockers from rabbit skeletal‐muscle microsomes

Flockerzi

Oeken

Hofmann

1986

European Journal of Biochemistry

101

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“…Western Blot Analysis-Partially purified SCG membranes from wild-type and ␤3-Tg 3 mice were prepared (18). For protein analysis of the heart, we purified membrane protein from the entire heart.…”

Section: Methodsmentioning

confidence: 99%

Modified Sympathetic Nerve System Activity with Overexpression of the Voltage-dependent Calcium Channel β3 Subunit

Murakami

Ohba

et al. 2008

Journal of Biological Chemistry

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N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory ␤3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a ␤3-overexpressing transgenic (␤3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of ␤3-null and CaV2.2 (␣1B)-null mice. The ␤3-Tg mice showed increased expression of the ␤3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The ␤3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the ␤3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the ␤3-Tg mice, whereas ␤3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the ␤3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of ␤3-Tg mice revealed increased calcium channel currents, especially N-and L-type currents. These results identify a determining role for the ␤3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.Voltage-dependent Ca 2ϩ channels (VDCCs) 2 are present in excitable tissues. VDCCs mediate the influx of Ca 2ϩ in response to membrane depolarization and regulate many fundamental functions, including muscle contraction, neurotransmitter release, and gene transcription (1). The VDCC ␣1 subunit families are classified into three main groups (CaV1.1-4, CaV2.1-3, and CaV3.1-3) based on their physiological properties and sequence similarities (2). VDCCs are heteromultimeric proteins, composed of four subunits (␣1, ␣2/␦, ␤, and ␥). The ␣1 subunit serves as the channel pore, voltage sensor, and the binding site for various channel modulators (1, 3). The ␤ subunit, which is hydrophilic and has no transmembrane domain, is the most important auxiliary subunit in the VDCC complex, with four different genes having been identified. The ␤ subunits have distant homology with the Src homology region 3 and guanylate kinase module, which interact with the ␣1 subunit (4).Several important roles of the ␤ subunit in channel properties, such as multifold increases in peak calcium current density and acceleration of inactivation kinetics, have been demonstrated using heterologous cDNA co-expression systems (5, ...

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“…11 The protein concentrations were determined by the Bradford assay. Aliquots of homogenate (100 μg) from each rat were resolved by 6% SDSpolyacrylamide gel electrophoresis.…”

Section: Western Blot Analysismentioning

confidence: 99%

Molecular and Electrical Remodeling of L- and T-Type Ca2+ Channels in Rat Right Atrium With Monocrotaline-Induced Pulmonary Hypertension

Koyama

Ono

Watanabe

et al. 2009

Circ J

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t is well known that fibrillating or hemodynamically overloaded atria are subject to cellular electrical remodeling, which is characterized by shortening of the action potential 1 and often contributes to the occurrence and persistence of atrial fibrillation (AF). AF and chronic obstructive pulmonary disease (COPD) frequently coexist and complicate treatment of both clinical conditions. 2,3 COPD patients are susceptible to secondary pulmonary hypertension (PH), and the resulting hemodynamic changes, and in addition hypoxia, hypercapnia, acid-base disorders, disturbed sympathovagal balance, and medication can lead to the development of AF. 2,3 Recent studies demonstrated that the densities of the L-type calcium (Ca 2+ ) current (ICaL), transient outward current (Ito), and voltage-dependent Na + current (INa) are decreased in experimental animal models of atrial tachycardia-related AF. [4][5][6] In contrast to tachycardia-induced ionic remodeling, several clinical and experimental studies have indicated a decrease, no change, or even an increase in the ICaL density in chronic heart failure (CHF), 1,7,8 depending on the experimental conditions. Nonetheless, it is generally agreed that a possible change in ICaL is a key factor in electrical remodeling of cardiac myocytes in CHF and/or hypertrophy. In addition, the T-type Ca 2+ channel is involved in electrical remodeling during CHF or hypertrophy. The T-type Ca 2+ channel is normally expressed in embryonic and neonatal myocytes, and in the conducting system of the adult heart, 9 and in rats it is re-expressed in pathologic conditions such as ventricular hypertrophy and post-myocardial infarction. 10 In the present study, the electrical properties of ICaL and ICaT were characterized in rat right atrial cells with monocrotaline (MCT)-induced PH, and the possible causes are discussed in relation to changes in the expression and function of Ca 2+ channel subunits. Methods AnimalsThe Animal Ethics Committee of Akita University School of Medicine, Japan, approved the study protocol. Four-week-old Wistar rats (CLEA Japan, Tokyo, Japan), weighing 140-150 g each, were treated with 500 mg MCT (Crotaline; Sigma, St Louis, MO, USA) to produce PH. A single dose of MCT 60 mg/kg was injected subcutaneously in the interscapular region. Measurement of Hemodynamic Parameters and Assessment of Right Ventricular (RV) HypertrophyRV pressure and heart rate were measured, using a pressure transducer connected to a monitor (DynaScope 5100E; Fukuda Denshi, Tokyo, Japan). Right atrial (RA) and RV hypertrophy were evaluated by measuring the ratio of the RA weight to body weight (BW), the ratio of the RV free wall weight to BW, and the ratio of the RV weight to the left ventricular free wall plus septum (LV + IVS) weight. (Received June 19, 2008; revised manuscript received September 8, 2008; accepted September 24, 2008; released online December 26, 2008) Departments of Cardiology, *Physiology, Akita University School of Medicine, Akita, Japan Mailing address: Hiroshi Ito, Department of Ca...

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Purified calcium channels have three allosterically coupled drug receptors

Cited by 71 publications

References 24 publications

Purification of a functional receptor for calcium‐channel blockers from rabbit skeletal‐muscle microsomes

Purification of a functional receptor for calcium‐channel blockers from rabbit skeletal‐muscle microsomes

Modified Sympathetic Nerve System Activity with Overexpression of the Voltage-dependent Calcium Channel β3 Subunit

Molecular and Electrical Remodeling of L- and T-Type Ca2+ Channels in Rat Right Atrium With Monocrotaline-Induced Pulmonary Hypertension

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