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In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2&#948-2 and α2&#948-3 with mutated metal iondependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density. [ABSTRACT FROM AUTHOR]

L-type Ca2+ currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca2+ signal, which regulates the contraction force. The auxiliary Ca2+ channel sub-units α2δ-1 and β2 are important regulators of membrane expression and current properties of the cardiac Ca2+ channel (Cav1.2). However, their role in cardiac excitation—contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the α2δ-1 subunit in a muscle expression system with simulation of AP5 and Ca2+ transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with Cav1.2 (GFP-α-1c) recapitulates key properties of cardiac excitation—contraction coupling. Concomitant depletion of the α2δ-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-α1c subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However. α2δ-1 depletion shifted the voltage dependence of Ca2+ current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca2+ concentration during each contraction. Thus, the Ca2+ channel α2δ-1 subunit is not essential for normal Ca2+ channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of α2δ-1 function is predicted to severely perturb normal heart function. [ABSTRACT FROM AUTHOR]

Homozygous zebrafish of the mutant relaxed (redts25) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarization-induced but not caffeine-induced Ca²+ transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the α1s subunit is normal, whereas the β1a subunit harbors a single point mutation resulting in a premature stop. Quantitative RT-PCR revealed that the mutated gene is transcribed, but Western blot analysis and immunocytochemistry demonstrated the complete loss of the β1a protein in mutant muscle. Thus, the immotile zebrafish relaxed is a β1a-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the α1s subunit in the absence of β1a. Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an ≈2-fold reduction in cluster size with a normal density of DHPR particles within the clusters. Most importantly, DHPR particles in the junctional membranes of the immotile zebrafish mutant relaxed entirely lacked the normal arrangement in arrays of tetrads. Thus, our data indicate that the lack of the β1a subunit does not prevent triad targeting of the DHPR α1s subunit but precludes the skeletal muscle-specific arrangement of DHPR particles opposite the ryanodine receptor (RyR1). This defect properly explains the complete deficiency of skeletal muscle excitation-contraction coupling in β1-null model organisms. [ABSTRACT FROM AUTHOR]

In nerve cells the genes encoding for α 2 δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α 2 δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α 2 δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α 2 δ isoforms as synaptic organizers is highly redundant, as each individual α 2 δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α 2 δ-2 and α 2 δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α 2 δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α 2 δ implicates α 2 δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α 2 δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Homozygous zebrafish of the mutant relaxed (red(ts25)) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarization-induced but not caffeine-induced Ca(2+) transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the alpha(1S) subunit is normal, whereas the beta(1a) subunit harbors a single point mutation resulting in a premature stop. Quantitative RT-PCR revealed that the mutated gene is transcribed, but Western blot analysis and immunocytochemistry demonstrated the complete loss of the beta(1a) protein in mutant muscle. Thus, the immotile zebrafish relaxed is a beta(1a)-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the alpha(1S) subunit in the absence of beta(1a). Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an approximately 2-fold reduction in cluster size with a normal density of DHPR particles within the clusters. Most importantly, DHPR particles in the junctional membranes of the immotile zebrafish mutant relaxed entirely lacked the normal arrangement in arrays of tetrads. Thus, our data indicate that the lack of the beta(1a) subunit does not prevent triad targeting of the DHPR alpha(1S) subunit but precludes the skeletal muscle-specific arrangement of DHPR particles opposite the ryanodine receptor (RyR1). This defect properly explains the complete deficiency of skeletal muscle excitation-contraction coupling in beta(1)-null model organisms.