Molecular Origin of the L-Type Ca2+ Current of Skeletal Muscle Myotubes Selectively Deficient in Dihydropyridine Receptor β1a Subunit

Abstract

The origin of Ibetanull, the Ca2+ current of myotubes from mice lacking the skeletal dihydropyridine receptor (DHPR) beta1a subunit, was investigated. The density of Ibetanull was similar to that of Idys, the Ca2+ current of myotubes from dysgenic mice lacking the skeletal DHPR alpha1S subunit (-0.6 +/- 0.1 and -0.7 +/- 0.1 pA/pF, respectively). However, Ibetanull activated at significantly more positive potentials. The midpoints of the GCa-V curves were 16.3 +/- 1.1 mV and 11.7 +/- 1.0 mV for Ibetanull and Idys, respectively. Ibetanull activated significantly more slowly than Idys. At +30 mV, the activation time constant for Ibetanull was 26 +/- 3 ms, and that for Idys was 7 +/- 1 ms. The unitary current of normal L-type and beta1-null Ca2+ channels estimated from the mean variance relationship at +20 mV in 10 mM external Ca2+ was 22 +/- 4 fA and 43 +/- 7 fA, respectively. Both values were significantly smaller than the single-channel current estimated for dysgenic Ca2+ channels, which was 84 +/- 9 fA under the same conditions. Ibetanull and Idys have different gating and permeation characteristics, suggesting that the bulk of the DHPR alpha1 subunits underlying these currents are different. Ibetanull is suggested to originate primarily from Ca2+ channels with a DHPR alpha1S subunit. Dysgenic Ca2+ channels may be a minor component of this current. The expression of DHPR alpha1S in beta1-null myotubes and its absence in dysgenic myotubes was confirmed by immunofluorescence labeling of cells.

Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor β1-null Zebrafish Relaxed Is an Exclusive Function of the β1a Subunit

“…Because quantification of the ␣ 1S protein expression in the mouse myotube-typical peripheral couplings was never performed, a clear differentiation between non-functional ␣ 1S expression and functional ␣ 1S expression in the membrane (charge movement) was not feasible. Thus, the ␤ 1a -induced facilitation of ␣ 1S charge movement was not detected in the mouse system and consequently experimental attempts on the ultrastructural level were not pushed forward (14,17,18). Due to the lack of these essential informations, the data of a large series of ␤-expression experiments (24, 25, 49 -52) were in general interpreted in a way that domains of the DHPR ␤ 1a subunit, similar to elements present in the ␣ 1S subunit, might be directly involved in activation of RyR1 channels (26).…”

“…␤ subunits of voltage-gated Ca 2ϩ channels were repeatedly shown to be responsible for the facilitation of ␣ 1 membrane insertion and to be potent modulators of ␣ 1 current kinetics and voltage dependence (15,16). Whether the loss of EC coupling in ␤ 1 -null mice was caused by decreased DHPR membrane expression or by the lack of a putative specific contribution of the ␤ subunit to the skeletal muscle EC coupling apparatus (17,18) was not clearly resolved. Recently, other ␤-functions were identified in skeletal muscle using the ␤ 1 -null mutant zebrafish relaxed (19,20).…”

The β 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle

“…Using molecular biology, protein biochemistry, and immunocytochemistry techniques, we show that the zebrafish mutant relaxed lacks the DHPR ␤ 1a subunit. In contrast to previous studies (8,11), we can use this novel model organism to demonstrate that the ␣ 1S is correctly targeted into skeletal muscle triad junctions in the absence of ␤ 1a . However, DHPRs lacked the skeletal muscle-specific arrangement in tetrads and displayed substantially reduced charge movement.…”

“…So, how does the lack of the ␤ 1a protein affect expression and function of the DHPR in skeletal muscle? Based on extensive coexpression experiments in heterologous systems (31,32) and on results from the ␤ 1a -null mouse (8,11), a lack of ␣ 1S triad expression would have been expected. However, immunofluorescence analysis of the fully differentiated zebrafish myotubes clearly demonstrated that ␤ 1a deficient cells are able to correctly target ␣ 1S into the triads (Fig.…”

“…Myotubes from ␤ 1a -null mice show reduced L-type Ca 2ϩ currents, charge movements, and 1,4-dihydropyridine binding (10). The staining intensity for ␣ 1S in immunocytochemistry was initially described to be below detectability (8), but later it was found to be significantly higher than that of dysgenic myotubes (11). Therefore, it remained unclear whether the loss of EC coupling in ␤ 1a -null myotubes was caused by decreased membrane expression of DHPRs, or by the lack of a specific contribution of the ␤ subunit to the EC coupling mechanism (10,12).…”