Gerlinde Kugler

University of Pennsylvania, Philadelphia, PA, United States

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Publications (9)28.31 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Auxiliary channel subunits regulate membrane expression and modulate current properties of voltage-activated Ca(2+) channels and thus are involved in numerous important cell functions, including muscle contraction. Whereas the importance of the alpha(1S), beta(1a), and gamma Ca(2+) channel subunits in skeletal muscle has been determined by using null-mutant mice, the role of the alpha(2)delta-1 subunit in skeletal muscle is still elusive. We addressed this question by small interfering RNA silencing of alpha(2)delta-1 in reconstituted dysgenic (alpha(1S)-null) myotubes and in BC3H1 skeletal muscle cells. Immunofluorescence labeling of the alpha(1S) and alpha(2)delta-1 subunits and whole cell patch clamp recordings demonstrated that triad targeting and functional expression of the skeletal muscle Ca(2+) channel were not compromised by the depletion of the alpha(2)delta-1 subunit. The amplitudes and voltage dependences of L-type Ca(2+) currents and of the depolarization-induced Ca(2+) transients were identical in control and in alpha(2)delta-1-depleted muscle cells. However, alpha(2)delta-1 depletion significantly accelerated the current kinetics, most likely by the conversion of slowly activating into fast activating Ca(2+) channels. Reverse transcription-PCR analysis indicated that alpha(2)delta-1 is the exclusive isoform expressed in differentiated BC3H1 cells and that depletion of alpha(2)delta-1 was not compensated by the up-regulation of any other alpha(2)delta isoform. Thus, in skeletal muscle the Ca(2+) channel alpha(2)delta-1 subunit functions as a major determinant of the characteristic slow L-type Ca(2+) current kinetics. However, this subunit is not essential for targeting of Ca(2+) channels or for their primary physiological role in activating skeletal muscle excitation-contraction coupling.
    Journal of Biological Chemistry 01/2005; 280(3):2229-37. · 4.65 Impact Factor
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    ABSTRACT: Auxiliary channel subunits regulate membrane expression and modulate current properties of voltage-activated Ca2+ channels and thus are involved in numerous important cell functions, including muscle contraction. Whereas the importance of the α1S, β1a, and γ Ca2+ channel subunits in skeletal muscle has been determined by using null-mutant mice, the role of the α2δ-1 subunit in skeletal muscle is still elusive. We addressed this question by small interfering RNA silencing of α2δ-1 in reconstituted dysgenic (α1S-null) myotubes and in BC3H1 skeletal muscle cells. Immunofluorescence labeling of the α1S and α2δ-1 subunits and whole cell patch clamp recordings demonstrated that triad targeting and functional expression of the skeletal muscle Ca2+ channel were not compromised by the depletion of the α2δ-1 subunit. The amplitudes and voltage dependences of L-type Ca2+ currents and of the depolarization-induced Ca2+ transients were identical in control and in α2δ-1-depleted muscle cells. However, α2δ-1 depletion significantly accelerated the current kinetics, most likely by the conversion of slowly activating into fast activating Ca2+ channels. Reverse transcription-PCR analysis indicated that α2δ-1 is the exclusive isoform expressed in differentiated BC3H1 cells and that depletion of α2δ-1 was not compensated by the up-regulation of any other α2δ isoform. Thus, in skeletal muscle the Ca2+ channel α2δ-1 subunit functions as a major determinant of the characteristic slow L-type Ca2+ current kinetics. However, this subunit is not essential for targeting of Ca2+ channels or for their primary physiological role in activating skeletal muscle excitation-contraction coupling.
    Journal of Biological Chemistry 01/2005; 280(3):2229-2237. · 4.65 Impact Factor
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    ABSTRACT: The plasmalemmal dihydropyridine receptor (DHPR) is the voltage sensor in skeletal muscle excitation-contraction (e-c) coupling. It activates calcium release from the sarcoplasmic reticulum via protein-protein interactions with the ryanodine receptor (RyR). To enable this interaction, DHPRs are arranged in arrays of tetrads opposite RyRs. In the DHPR alpha(1S) subunit, the cytoplasmic loop connecting repeats II and III is a major determinant of skeletal-type e-c coupling. Whether the essential II-III loop sequence (L720-L764) also determines the skeletal-specific arrangement of DHPRs was examined in dysgenic (alpha(1S)-null) myotubes reconstituted with distinct alpha(1) subunit isoforms and II-III loop chimeras. Parallel immunofluorescence and freeze-fracture analysis showed that alpha(1S) and chimeras containing L720-L764, all of which restored skeletal-type e-c coupling, displayed the skeletal arrangement of DHPRs in arrays of tetrads. Conversely, alpha(1C) and those chimeras with a cardiac II-III loop and cardiac e-c coupling properties were targeted into junctional membranes but failed to form tetrads. However, an alpha(1S)-based chimera with the heterologous Musca II-III loop produced tetrads but did not reconstitute skeletal muscle e-c coupling. These findings suggest an inhibitory role in tetrad formation of the cardiac II-III loop and that the organization of DHPRs in tetrads vis-a-vis the RyR is necessary but not sufficient for skeletal-type e-c coupling.
    Molecular Biology of the Cell 01/2005; 15(12):5408-19. · 4.60 Impact Factor
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    ABSTRACT: Interactions of the II-III loop of the voltage-gated Ca(2+) channel alpha(1S) subunit with the Ca(2+) release channel (RyR1) are essential for skeletal-type excitation-contraction (EC) coupling. Here, we characterized the binding site of the monoclonal alpha(1S) antibody mAB 1A and used it to probe the structure of the II-III loop in chimeras with different EC coupling properties. Phage-display epitope mapping of mAB 1A revealed a minimal consensus binding sequence X-P-X-X-D-X-P. Immunofluorescence labeling of (1S), alpha(1C), alpha(1D), and of II-III loop chimeras expressed in dysgenic myotubes established that mAB 1A reacted specifically with amino acids 737-744 in the II-III loop of alpha(1S), which is within the domain (D734-L764) critical for bidirectional coupling with RyR1. Comparing mAB 1A immunoreactivity with known structural and functional properties of II-III loop chimeras in which the non-conserved skeletal residues were systematically mutated to their cardiac counterparts indicated a correlation of mAB 1A immunoreactivity and skeletal-type EC coupling.
    Archives of Biochemistry and Biophysics 08/2004; 427(1):91-100. · 3.37 Impact Factor
  • Journal of Muscle Research and Cell Motility 03/2004; 25(3):239-240. · 1.36 Impact Factor
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    ABSTRACT: Residues Leu720-Leu764 within the II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha1S subunit represent a critical domain for the orthograde excitation-contraction coupling as well as for retrograde DHPR L-current-enhancing coupling with the ryanodine receptor (RyR1). To better understand the molecular mechanism underlying this bidirectional DHPR-RyR1 signaling interaction, we analyzed the critical domain to the single amino acid level. To this end, constructs based on the highly dissimilar housefly DHPR II-III loop in an otherwise skeletal DHPR as an interaction-inert sequence background were expressed in dysgenic (alpha1S-null) myotubes for simultaneous recordings of depolarization-induced intracellular Ca2+ transients (orthograde coupling) and whole-cell Ca2+ currents (retrograde coupling). In the minimal skeletal II-III loop sequence (Asp734-Asp748 required for full bidirectional coupling, eight amino acids heterologous between skeletal and cardiac DHPR were exchanged for the corresponding cardiac residues. Four of these skeletal-specific residues (Ala739, Phe741, Pro742, and Asp744) turned out to be essential for orthograde and two of them (Ala739 and Phe741) for retrograde coupling, indicating that orthograde coupling does not necessarily correlate with retrograde signaling. Secondary structure predictions of the critical domain show that an alpha-helical (cardiac sequence-type) conformation of a cluster of negatively charged residues (Asp744-Glu751 of alpha1S) corresponds with significantly reduced Ca2+ transients. Conversely, a predicted random coil structure (skeletal sequence-type) seems to be prerequisite for the restoration of skeletal-type excitation-contraction coupling. Thus, not only the primary but also the secondary structure of the critical domain is an essential determinant of the tissue-specific mode of EC coupling.
    Journal of Biological Chemistry 03/2004; 279(6):4721-8. · 4.65 Impact Factor
  • Journal of Muscle Research and Cell Motility 02/2004; 25(3):239-40. · 1.36 Impact Factor
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    ABSTRACT: Ca(2+)-induced Ca(2+)-release (CICR)-the mechanism of cardiac excitation-contraction (EC) coupling-also contributes to skeletal muscle contraction; however, its properties are still poorly understood. CICR in skeletal muscle can be induced independently of direct, calcium-independent activation of sarcoplasmic reticulum Ca(2+) release, by reconstituting dysgenic myotubes with the cardiac Ca(2+) channel alpha(1C) (Ca(V)1.2) subunit. Ca(2+) influx through alpha(1C) provides the trigger for opening the sarcoplasmic reticulum Ca(2+) release channels. Here we show that also the Ca(2+) channel alpha(1D) isoform (Ca(V)1.3) can restore cardiac-type EC-coupling. GFP-alpha(1D) expressed in dysgenic myotubes is correctly targeted into the triad junctions and generates action potential-induced Ca(2+) transients with the same efficiency as GFP-alpha(1C) despite threefold smaller Ca(2+) currents. In contrast, GFP-alpha(1A), which generates large currents but is not targeted into triads, rarely restores action potential-induced Ca(2+) transients. Thus, cardiac-type EC-coupling in skeletal myotubes depends primarily on the correct targeting of the voltage-gated Ca(2+) channels and less on their current size. Combined patch-clamp/fluo-4 Ca(2+) recordings revealed that the induction of Ca(2+) transients and their maximal amplitudes are independent of the different current densities of GFP-alpha(1C) and GFP-alpha(1D). These properties of cardiac-type EC-coupling in dysgenic myotubes are consistent with a CICR mechanism under the control of local Ca(2+) gradients in the triad junctions.
    Biophysical Journal 07/2003; 84(6):3816-28. · 3.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ca2+-induced Ca2+-release (CICR)—the mechanism of cardiac excitation-contraction (EC) coupling—also contributes to skeletal muscle contraction; however, its properties are still poorly understood. CICR in skeletal muscle can be induced independently of direct, calcium-independent activation of sarcoplasmic reticulum Ca2+ release, by reconstituting dysgenic myotubes with the cardiac Ca2+ channel α1C (CaV1.2) subunit. Ca2+ influx through α1C provides the trigger for opening the sarcoplasmic reticulum Ca2+ release channels. Here we show that also the Ca2+ channel α1D isoform (CaV1.3) can restore cardiac-type EC-coupling. GFP-α1D expressed in dysgenic myotubes is correctly targeted into the triad junctions and generates action potential-induced Ca2+ transients with the same efficiency as GFP-α1C despite threefold smaller Ca2+ currents. In contrast, GFP-α1A, which generates large currents but is not targeted into triads, rarely restores action potential-induced Ca2+ transients. Thus, cardiac-type EC-coupling in skeletal myotubes depends primarily on the correct targeting of the voltage-gated Ca2+ channels and less on their current size. Combined patch-clamp/fluo-4 Ca2+ recordings revealed that the induction of Ca2+ transients and their maximal amplitudes are independent of the different current densities of GFP-α1C and GFP-α1D. These properties of cardiac-type EC-coupling in dysgenic myotubes are consistent with a CICR mechanism under the control of local Ca2+ gradients in the triad junctions.
    Biophysical Journal - BIOPHYS J. 01/2003; 84(6):3816-3828.

Publication Stats

202 Citations
28.31 Total Impact Points

Institutions

  • 2005
    • University of Pennsylvania
      • Department of Cell and Developmental Biology
      Philadelphia, PA, United States
  • 2004–2005
    • Medizinische Universität Innsbruck
      • Department für Physiologie und Medizinische Physik
      Innsbruck, Tyrol, Austria
  • 2003–2004
    • University of Innsbruck
      • Department of Pharmacology and Toxicology
      Innsbruck, Tyrol, Austria