Abstract
BackgroundVoltage-gated Cav1.2 calcium channels play a crucial role in Ca2+ signaling. The pore-forming α1C subunit is regulated by accessory Cavβ subunits, cytoplasmic proteins of various size encoded by four different genes (Cavβ1 - β4) and expressed in a tissue-specific manner.Methods and ResultsHere we investigated the effect of three major Cavβ types, β1b, β2d and β3, on the structure of Cav1.2 in the plasma membrane of live cells. Total internal reflection fluorescence microscopy showed that the tendency of Cav1.2 to form clusters depends on the type of the Cavβ subunit present. The highest density of Cav1.2 clusters in the plasma membrane and the smallest cluster size were observed with neuronal/cardiac β1b present. Cav1.2 channels containing β3, the predominant Cavβ subunit of vascular smooth muscle cells, were organized in a significantly smaller number of larger clusters. The inter- and intramolecular distances between α1C and Cavβ in the plasma membrane of live cells were measured by three-color FRET microscopy. The results confirm that the proximity of Cav1.2 channels in the plasma membrane depends on the Cavβ type. The presence of different Cavβ subunits does not result in significant differences in the intramolecular distance between the termini of α1C, but significantly affects the distance between the termini of neighbor α1C subunits, which varies from 67 Å with β1b to 79 Å with β3.ConclusionsThus, our results show that the structural organization of Cav1.2 channels in the plasma membrane depends on the type of Cavβ subunits present.
Highlights
Voltage-gated Cav1.2 calcium channels react to membrane depolarization by creating a rapid and transient increase in intracellular free Ca2+ concentration, thereby playing an essential role in initiation of calcium signaling in a wide variety of cells [1]
Conclusions: our results show that the structural organization of Cav1.2 channels in the plasma membrane depends on the type of Cavb subunits present
Using electron microscopy in bird and amphibian cardiac muscle [5,6] and immuno-gold labeling in mammalian ventricular myocytes [7,10] it was shown that Cav1.2 clusters are loosely tethered to ryanodine receptors (RyR) of the sarcoplasmic reticulum
Summary
Voltage-gated Cav1.2 calcium channels react to membrane depolarization by creating a rapid and transient increase in intracellular free Ca2+ concentration, thereby playing an essential role in initiation of calcium signaling in a wide variety of cells [1]. In order to exhibit this function, Cav1.2 calcium channels require association of the pore-forming a1C subunit with accessory Cavb and a2d subunits as well as calmodulin. Calcium channels are clustered rather than evenly distributed along the surface membrane of neurons [2,3,4] and cardiac myocytes [5,6,7]. Association of calcium channels and ryanodine receptors appears to be weaker in cardiac myocytes than in skeletal muscle [11] and may involve different mechanisms of coupling [12], Cav1.2 clustering is essential for excitation-contraction coupling [13,14]. The pore-forming a1C subunit is regulated by accessory Cavb subunits, cytoplasmic proteins of various size encoded by four different genes (Cavb1 - b4) and expressed in a tissue-specific manner
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