Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor beta 1 subunit transfected with beta 1 cDNA

Beurg, Maryline; Sukhareva, Manana; Strube, Caroline; Powers, Patricia A.; Gregg, Ronald G.; Coronado, Roberto

doi:10.1016/s0006-3495(97)78113-x

Cited by 59 publications

(82 citation statements)

References 40 publications

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“…The ability of Ca v ␤ subunits to promote trafficking of Ca v 1.x and Ca v 2.x channels to the surface membrane has been demonstrated in a variety of studies using techniques ranging from intramembrane charge movement to immunofluorescence assays (3,13,21,27,29). Studies have attributed this effect to the Ca v ␤ subunit binding an ER retention signal localized near the AID in the I-II loop of the Ca 2ϩ channel ␣ 1 -subunit (4).…”

Section: Discussionmentioning

confidence: 99%

Unique modulation of L-type Ca²⁺channels by short auxiliary β_1dsubunit present in cardiac muscle

Cohen

Foell

Balijepalli

et al. 2005

American Journal of Physiology-Heart and Circulatory Physiology

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. Unique modulation of L-type Ca 2ϩ channels by short auxiliary ␤1d subunit present in cardiac muscle. Am J Physiol Heart Circ Physiol 288: H2363-H2374, 2005. First published December 22, 2004; doi:10.1152/ajpheart.00348.2004.-Recent studies have identified a growing diversity of splice variants of auxiliary Ca 2ϩ channel Ca v␤ subunits. The Cav␤1d isoform encodes a putative protein composed of the amino-terminal half of the full-length Cav␤1 isoform and thus lacks the known high-affinity binding site that recognizes the Ca 2ϩ channel ␣1-subunit, the ␣-binding pocket. The present study investigated whether the Ca v␤1d subunit is expressed at the protein level in heart, and whether it exhibits any of the functional properties typical of full-length Cav␤ subunits. On Western blots, an antibody directed against the unique carboxyl terminus of Cav␤1d identified a protein of the predicted molecular mass of 23 kDa from canine and human hearts. Immunocytochemistry and surface-membrane biotinylation experiments in transfected HEK-293 cells revealed that the full-length Cav␤1b subunit promoted membrane trafficking of the pore-forming ␣ 1C (Cav1.2)-subunit to the surface membrane, whereas the Cav␤1d subunit did not. Whole cell patch-clamp analysis of transfected HEK-293 cells demonstrated no effect of coexpression of the Ca v␤1d with the ␣ 1C-subunit compared with the 15-fold larger currents and leftward shift in voltage-dependent activation induced by full-length Ca v␤1b coexpression. In contrast, cell-attached patch single-channel studies demonstrated that coexpression of either Ca v␤1b or Cav␤1d significantly increased mean open probability four-to fivefold relative to the ␣ 1C-channels alone, but only Cav␤1b coexpression increased the number of channels observed per patch. In conclusion, the Ca v␤1d isoform is expressed in heart and can modulate the gating of L-type Ca 2ϩ channels, but it does not promote membrane trafficking of the channel complex. electrophysiology; ion; heart; splice variant CALCIUM CHANNELS ARE FOUND IN many excitable tissues and are intricately involved in important cellular processes including genesis of action potentials, excitation-contraction coupling, and gene expression. Voltage-dependent Ca 2ϩ channels are multisubunit proteins that consist of a pore-forming ␣ 1 -subunit that is associated with auxiliary subunits including the cytoplasmic ␤-subunit, a membrane-associated ␣ 2 ␦-subunit, and a ␥-subunit (10). The ultimate functional properties of Ca 2ϩ channels are determined not only by the pore-forming ␣ 1 -subunits but also by the combination of auxiliary subunits that finely tune the channel properties.

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Section: Discussionmentioning

confidence: 99%

Unique modulation of L-type Ca²⁺channels by short auxiliary β_1dsubunit present in cardiac muscle

Cohen

Foell

Balijepalli

et al. 2005

American Journal of Physiology-Heart and Circulatory Physiology

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“…Outbred Black Swiss mice (Charles River Breeding Laboratories) were used to generate heterozygous muscular dysgenesis gene locus mdg͞ϩ parents. Primary cultures were prepared from enzyme-digested hind limbs of dysgenic (mdg͞mdg) embryos (on embryonic day 18) as described (12). cDNAs were subcloned into the mammalian expression vectors pSG5 (Stratagene) or pCDNA3 (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Intramembrane charge movements and excitation– contraction coupling expressed by two-domain fragments of the Ca ²⁺ channel

Ahern

Arikkath

Vallejo

et al. 2001

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

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To investigate the molecular basis of the voltage sensor that triggers excitation-contraction (EC) coupling, the four-domain pore subunit of the dihydropyridine receptor (DHPR) was cut in the cytoplasmic linker between domains II and III. cDNAs for the I-II domain (␣1S 1-670) and the III-IV domain (␣1S 701-1873) were expressed in dysgenic ␣1S-null myotubes. Coexpression of the two fragments resulted in complete recovery of DHPR intramembrane charge movement and voltage-evoked Ca 2؉ transients. When fragments were expressed separately, EC coupling was not recovered. However, charge movement was detected in the I-II domain expressed alone. Compared with I-II and III-IV together, the charge movement in the I-II domain accounted for about half of the total charge (Q max ‫؍‬ 3 ؎ 0.23 vs. 5.4 ؎ 0.76 fC͞pF, respectively), and the half-activation potential for charge movement was significantly more negative (V 1/2 ‫؍‬ 0.2 ؎ 3.5 vs. 22 ؎ 3.4 mV, respectively). Thus, interactions between the four internal domains of the pore subunit in the assembled DHPR profoundly affect the voltage dependence of intramembrane charge movement. We also tested a two-domain I-II construct of the neuronal ␣1A Ca channels, the opening of the ion-selective pore is coupled to movement of intramembrane charges, the so-called gating charges, buried in transmembrane segments in the channel. The consensus membrane topology of these channels is either six transmembrane segments in each of four subunits (in the case of K ϩ channels) or six transmembrane segments (S1-S6) in each of four internal repeats or domains (I-IV) joined by cytosolic linkers (in the case of Na ϩ and Ca 2ϩ channels) (1). Movement of the gating charges in response to voltage involves a substantial reorientation of transmembrane segments S4 and possibly S3 bearing the gating charges (2). Such a conformational change facilitates the energetically unfavorable opening of the ionselective pore.Skeletal muscle cells use the movement of gating charges in the Ca 2ϩ channel formed by the dihydropyridine receptor (DHPR) to release Ca 2ϩ from the sarcoplasmic reticulum (SR). The coupling mechanism presumably involves voltagedependent gating transitions in the DHPR ␣1S pore subunit, transmission of a conformation change across the transverse tubule͞SR junction, and, ultimately, opening of the ryanodine receptor (RyR1) channel and release of SR-stored Ca 2ϩ (3). The biophysical characteristics of the charge movements that initiate excitation-contraction (EC) coupling have been thoroughly investigated (3, 4). A strong correlation was found between the movement of the so-called suprathreshold charges and the rate of SR Ca 2ϩ release (5). This correlation holds over a wide range of voltages and suggests that muscle charge movements represent surrogate ''gating charges'' for the opening of the RyR1 channel. This suggestion is made explicit in a kinetic model in which opening of the RyR1 channel is coupled with an allosteric mechanism for voltage-dependent transitions in the DHPR (6).The molecular...

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“…␤-null myotubes not only lack the ␤ 1a subunit but also show a severely reduced expression of the ␣ 1S subunit (16). Reconstitution of ␤-null myotubes by transient transfection with ␤ 1a restores Ca 2ϩ currents and EC coupling (17). Thus, ␤ 1a is important for the functional expression of the ␣ 1S subunit in the skeletal muscle.…”

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confidence: 99%

Association of calcium channel α _1S and β _1a subunits is required for the targeting of β _1a but not of α _1S into skeletal muscle triads

Neuhuber

Gerster

Döring

et al. 1998

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

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The skeletal muscle L-type Ca 2؉ channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca 2؉ release from cytoplasmic stores in a process called excitationcontraction coupling. To study the role of ␣ 1S -␤ 1a interactions in the incorporation of the functional channel complex into the triad, ␣ 1S and ␤ 1a [or a ␤ 1a -green f luorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. ␤GFP expressed in dysgenic myotubes that lack the skeletal muscle ␣ 1S subunit was diffusely distributed in the cytoplasm. On coexpression with the ␣ 1S subunit ␤GFP distribution became clustered and colocalized with ␣ 1S immunof luorescence. Based on the colocalization of ␤GFP and ␣ 1S with the ryanodine receptor the clusters were identified as T-tubule͞sarcoplasmic reticulum junctions. Expression of ␣ 1S with and without ␤ 1a restored Ca 2؉ currents and depolarization-induced Ca 2؉ release. The translocation of ␤GFP from the cytoplasm into the junctions failed when ␤GFP was coexpressed with ␣ 1S mutants in which the ␤ interaction domain had been altered (␣ 1S -Y366S) or deleted (␣ 1S -⌬351-380). Although ␣ 1S -Y366S did not associate with ␤GFP it was incorporated into the junctions, and it restored Ca 2؉ currents and depolarization-induced Ca 2؉ release. Thus, ␤ 1a requires the association with the ␤ interaction domain in the I-II cytoplasmic loop of ␣ 1S for its own incorporation into triad junctions, but stable ␣ 1S -␤ 1a association is not necessary for the targeting of ␣ 1S into the triads or for its normal function in Ca 2؉ conductance and excitationcontraction coupling.The skeletal muscle dihydropyridine (DHP) receptor is a L-type Ca 2ϩ channel that functions primarily in the fast activation of Ca 2ϩ release from cytoplasmic stores in a process called excitation-contraction (EC) coupling (1). It is specifically localized in the triad, a junction between the transverse tubules (T-tubules) and the sarcoplasmic reticulum (SR) (2, 3). However, the mechanisms involved in the targeting and organization of the Ca 2ϩ channels in the triad are still elusive (4). The incorporation of the DHP receptor into the junctional T-tubule membrane and the organization of the Ca 2ϩ release channel, also called the ryanodine (RY) receptor, in the SR occur independently of each other (5-7). Thus, targeting and incorporation of DHP and RY receptors into the skeletal muscle triad appear to be intrinsic properties of these channels.The skeletal muscle DHP receptor consists of five subunits (8). The ␣ 1S subunit forms the ion channel and contains the drug binding sites and the molecular domains for interactions with accessory channel subunits and the RY receptor. The ␣ 2 ␦ subunit complex is a disulfide-linked heterodimer that is anchored in the T-tubule membrane by a transmembrane segment in the ␦ subunit. The ␤ 1a subunit is a peripheral membrane protein that associates with the DHP receptor complex via...

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Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor beta 1 subunit transfected with beta 1 cDNA

Cited by 59 publications

References 40 publications

Unique modulation of L-type Ca²⁺channels by short auxiliary β_1dsubunit present in cardiac muscle

Unique modulation of L-type Ca²⁺channels by short auxiliary β_1dsubunit present in cardiac muscle

Intramembrane charge movements and excitation– contraction coupling expressed by two-domain fragments of the Ca ²⁺ channel

Association of calcium channel α _1S and β _1a subunits is required for the targeting of β _1a but not of α _1S into skeletal muscle triads

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Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor beta 1 subunit transfected with beta 1 cDNA

Cited by 59 publications

References 40 publications

Unique modulation of L-type Ca2+channels by short auxiliary β1dsubunit present in cardiac muscle

Unique modulation of L-type Ca2+channels by short auxiliary β1dsubunit present in cardiac muscle

Intramembrane charge movements and excitation– contraction coupling expressed by two-domain fragments of the Ca 2+ channel

Association of calcium channel α 1S and β 1a subunits is required for the targeting of β 1a but not of α 1S into skeletal muscle triads

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Unique modulation of L-type Ca²⁺channels by short auxiliary β_1dsubunit present in cardiac muscle

Unique modulation of L-type Ca²⁺channels by short auxiliary β_1dsubunit present in cardiac muscle

Intramembrane charge movements and excitation– contraction coupling expressed by two-domain fragments of the Ca ²⁺ channel

Association of calcium channel α _1S and β _1a subunits is required for the targeting of β _1a but not of α _1S into skeletal muscle triads