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.
Highlights
Excitation-contraction (EC)1 coupling in skeletal muscle is understood as a protein-protein or “mechanical” interaction of two distinct Ca2ϩ channels, the voltage-gated L-type Ca2ϩ channel or dihydropyridine receptor (DHPR) and the Ca2ϩ release channel or ryanodine receptor (RyR1) in the sarcoplasmic reticulum (1, 2; reviewed in Refs. 3 and 4)
Are Required for the Bidirectional Signaling Interaction with RyR1—In previous studies, residues Leu720-Leu764 of the rabbit skeletal muscle DHPR ␣1S II-III loop were demonstrated to be essential for the full restoration of skeletal-type EC coupling
To better understand the molecular mechanism underlying the direct skeletal DHPR-RyR1 interaction, we identified the minimum sequence within ␣1S residues Leu720-Leu764 required for the bidirectional coupling
Summary
Construction of DHPR Chimeras—The design of the DHPR II-III loop chimeras used in the present study was based on the sequence of chimeras GFP-SkLM and GFP-SkLMS45 [15]. GFP-SkLMC31: GFP-SkLMS31 and ␣1C were used as the respective cDNA templates to introduce the M/C transition (nt M2211/C2593) as well as a ClaI* RE site (nt C2685) to generate the corresponding MfeI-ClaI* PCR fragment (nt M2024 –C2685). Point mutants D734S, A739P, D740E, F741T, P742T, D744E, D745E, E746D: Base exchanges from skeletal to the corresponding cardiac coding triplets within the skeletal sequence portion (nt 2200 – 2244) of clone GFP-SkLMS15C16 were introduced by SOE-PCR to yield the respective MfeI-ClaI* PCR fusion fragments (nt M2024 –C2685). For the recordings of immobilization-resistant intramembrane charge movements (gating currents), ionic Ca2ϩ currents were blocked by the addition of 0.5 mM Cd2ϩ and 0.1 mM La3ϩ to the extracellular recording solution, and depolarizing steps of the prepulse protocol were reduced to 50 ms. Data were analyzed using Clampfit® 8.0 (Axon Instruments, Foster City, CA) and SigmaPlot® 6.0 (SPSS Science, Chicago, IL) software
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