Generation and Characterization of a Polyclonal Antibody Specific for the Extracellular Loop of CaV2.1 Voltage-Gated Ca2+ Channels

Abstract

Multiple mutations in human CACNA1A, the gene encoding the pore-forming CaV2.1 subunit of P/Q-type VGCCs, have been associated with many neurological diseases including familial hemiplegic migraine type 1 (FHM-1), episodic ataxia type 2 (EA2) and epilepsy. The effects of many CACNA1A mutations on the biophysical properties of the P/Q-type channels have been extensively studied. However, questions remain as to whether and how these mutations affect CaV2.1 protein expression and/or the plasma membrane trafficking. To develop tools in exploring the mechanisms underlying CaV2.1 trafficking, we inserted a 15 amino acids epitope (PQ-I) in the IS5-S6 loop of human CaV2.1 into the hepatitis B virus core (HBc) protein. The virus-like particles self-assembled from the HBc-PQ-I fusion protein was used to immunize mice to obtain antisera. The PQ-I antibody specifically recognized human CaV2.1 subunits expressed in HEK293 cells but not CaV1.2, CaV2.2 or CaV2.3. Next, we expressed EGFP-tagged wild-type or mutant CaV2.1 subunits in HEK cells and examined CaV2.1-immunoreactivity (CaV2.1-ir) in transfected, non-permeabilized cells with PQ-I antibody. EGFP signals were observed in both the plasma membrane and intracellular compartments. In contrast, CaV2.1-ir was restricted to the cell surface. Our data showed that missense FHM-1 mutations did not affect the expression or the plasma membrane trafficking of CaV2.1 subunits. On the contrary, the nonsense EA2 mutation Y1854X resulted in a significant reduction of both CaV2.1 protein expression as well as its efficiency of trafficking to the plasma membrane. This may account for the diminished current through Y1854X mutant channels. Furthermore, the PQ-I antibody did not attenuate P/Q-type currents, nor did it affect the effect of P/Q-channel blocker-agatoxin-IVA, suggesting that it can be used to track CaV2.1 trafficking in vivo without perturbing its physiological functions.