Cardiac-type EC-Coupling in Dysgenic Myotubes Restored with Ca(2+) Channel Subunit Isoforms α(1C) and α(1D) Does not Correlate with Current Density

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 α(1C) (Ca(V)1.2) subunit. Ca(2+) influx through α(1C) provides the trigger for opening the sarcoplasmic reticulum Ca(2+) release channels. Here we show that also the Ca(2+) channel α(1D) isoform (Ca(V)1.3) can restore cardiac-type EC-coupling. GFP-α(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-α(1C) despite threefold smaller Ca(2+) currents. In contrast, GFP-α(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-α(1C) and GFP-α(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

The short-EMBU in East-Germany and Sweden: A cross-national factorial validity extension

The factorial stability and reliability of the 23-item s(hort)-EMBU previously demonstrated to be satisfactory in samples of students from Greece, Guatemala. Hungary and Italy, were extended with 791 students from East-Germany and Sweden. Previous findings on factorial validity, internal reliability and correlations among scales were replicated. The 23-item form thus continues to be recommended as a reliable functional equivalent to the early 81-item EMBU, when the clinical and/or research context does nor adequately permit application of time-consuming test batteries

A CaV1.1 Ca2+ Channel Splice Variant with High Conductance and Voltage-Sensitivity Alters EC Coupling in Developing Skeletal Muscle

The Ca2+ channel α1S subunit (CaV1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca2+ release from internal stores and conducts a slowly activating Ca2+ current. However, this Ca2+ current is not essential for skeletal muscle EC coupling. Here, we identified a CaV1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged α1S subunit and expressed it in dysgenic (α1S-null) myotubes. GFP-α1SΔ29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca2+ currents through GFP-α1SΔ29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca2+ influx contributed substantially to the depolarization-induced Ca2+ transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary α2δ-1 subunit. Thus, characterizing the CaV1.1Δ29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca2+ channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle

Functional Interaction of Ca(V) Channel Isoforms with Ryanodine Receptors Studied in Dysgenic Myotubes

The L-type Ca(2+) channels Ca(V)1.1 (α(1S)) and Ca(V)1.2 (α(1C)) share properties of targeting but differ by their mode of coupling to ryanodine receptors in muscle cells. The brain isoform Ca(V)2.1 (α(1A)) lacks ryanodine receptor targeting. We studied these three isoforms in myotubes of the α(1S)-deficient skeletal muscle cell line GLT under voltage-clamp conditions and estimated the flux of Ca(2+) (Ca(2+) input flux) resulting from Ca(2+) entry and release. Surprisingly, amplitude and kinetics of the input flux were similar for α(1C) and α(1A) despite a previously reported strong difference in responsiveness to extracellular stimulation. The kinetic flux characteristics of α(1C) and α(1A) resembled those in α(1S)-expressing cells but the contribution of Ca(2+) entry was much larger. α(1C) but not α(1A)-expressing cells revealed a distinct transient flux component sensitive to sarcoplasmic reticulum depletion by 30 μM cyclopiazonic acid and 10 mM caffeine. This component likely results from synchronized Ca(2+)-induced Ca(2+) release that is absent in α(1A)-expressing myotubes. In cells expressing an α(1A)-derivative (α(1)Aas(1592-clip)) containing the putative targeting sequence of α(1S), a similar transient component was noticeable. Yet, it was considerably smaller than in α(1C), indicating that the local Ca(2+) entry produced by the chimera is less effective in triggering Ca(2+) release despite similar global Ca(2+) inward current density

The proximal C-terminus of α1C subunits is necessary for junctional membrane targeting of cardiac L-type calcium channels

In cardiac myocytes, LTCCs (L-type calcium channels) form a functional signalling complex with ryanodine receptors at the JM (junctional membrane). Although the specific localization of LTCCs to the JM is critical for excitation-contraction coupling. their targeting mechanism is unclear. Transient transfection of GFP (green fluorescent protein)-alpha(1S) or GFP-alpha(1C) but not P/Q-type calcium channel alpha(1A), in dysgenic (alpha(1S)-null) GLT myotubes results in correct targeting of these LTCCs to the JMs and restoration of action-potential-induced Ca2+ transients. To identify the sequences of alpha(1C) responsible for JM targeting, we generated a range of alpha(1C)-alpha(1A) chimaeras, deletion mutants and alanine substitution mutants and studied their targeting properties in GLT myotubes. The results revealed that amino acids L-1681 QAGLRTL(1688) and P(1693)EIRRAIS(1700), predicted to form two adjacent alpha-helices in the proximal C-terminus, are necessary for the JM targeting of alpha(1C). The efficiency of restoration of action-potential-induced Ca2+ transients in GLT myotubes was significantly decreased by mutations in the targeting motif. JM targeting was not disrupted by the distal C-terminus of alpha(1C) which binds to the second alpha-helix. Therefore we have identified a new structural motif in the C-terminus of alpha(1C) that mediates the targeting of cardiac LTCCs to JMs independently of the interaction between proximal and distal C-termini of alpha(1C).ArticleBIOCHEMICAL JOURNAL. 448:221-231 (2012)journal articl