Magic™ Anti-CAV2 (Phospho S23) polyclonal antibody

Insulin and glucagon are the major hormones of the islets of Langerhans that are stored and released from the B- and A-cells, respectively. Both hormones are secreted when the intracellular cytosolic Ca2+ concentration ([Ca2+](i)) increases. The [Ca2+](i) is modulated by mutual inhibition and activation of different voltage-gated ion channels. The precise interplay of these ion channels in either glucagon or insulin release is unknown, owing in part to the difficulties in distinguishing A- from B-cells in electrophysiological experiments. We have established a single-cell RT-PCR method to identify A- and B-cells from the mouse. A combination of PCR, RT-PCR, electrophysiology and pharmacology enabled us to characterize the different sodium and calcium channels in mouse islet cells. In both A- and B-cells, 60% of the inward calcium current (I-Ca) is carried by L-type calcium channels. In B-cells, the predominant calcium channel is Ca(V)1.2, whereas Ca(V)1.2 and Ca(V)1.3 were identified in A-cells. These results were confirmed by using mice carrying A- or B-cell-specific inactivation of the Ca(V)1.2 gene. In B-cells, the remaining I-Ca flows in equal amounts through Ca(V)2.1, Ca(V)2.2 and CaCa(V)2.3. In A-cells, 30 and 15% Of ICa is due to Ca(V)2.3 and Ca(V)2.1 activity, respectively, whereas CaV2.2 current was not found in these cells. Low-voltage-activated T-type calcium channels could not be identified in A- and B-cells. Instead, two TTX-sensitive sodium currents were found: an early inactivating and a residual current. The residual current was only recovered in a subpopulation of B-cells. A putative genetic background for these currents is Na(V)1. 7.

High voltage-gated calcium channels consist of a pore-forming subunit (alpha(1)) and three nonhomologous subunits (alpha(2)/delta,beta, and gamma). Although it is well established that the beta-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the alpha(1)-subunit remains controversial. Here, we address this issue by examining the effect of purified beta(2)a protein on Ca(V)1.2 and Ca(V)2.3 channels expressed in Xenopus oocytes. The beta(2a)-subunit binds to the alpha(1)-interaction domain ( AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of CaV1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the alpha(1)-beta interaction through the AID site can take place at the plasma membrane. Furthermore, injection of beta(2a) protein inhibited inactivation of CaV2.3 channels and converted fast inactivating Ca(V)2.3/beta(1b) channels to slow inactivating channels. Inhibition of inactivation required larger concentration of beta(2a) in oocytes expressing Ca(V)2.3/beta(1b) channels than expressing CaV2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the alpha(1)-beta interaction is reversible in intact cells and defines calcium channels beta-subunits as regulatory proteins rather than stoichiometric subunits.