Construction Of Functional N-type Ca2+ Channels (Cav2.2) With Accessible External Epitope Tags Suitable For Live Cell Labeling

Abstract

Recent studies of voltage-gated Ca2+ channels indicate that mechanisms other than alterations in gating contribute to modulation. For example, internalization of receptor/channel complexes (e.g., ORL1) or formation of non-conducting species (e.g., RGK proteins), potentially contribute to CaV2.2 function. An essential technique for investigating these phenomena is the ability to specifically label fully functional CaV2.2 α-subunits (CACNA1B) in the plasma membrane of living cells. Towards this end, we attempted a systematic insertion of a hemagglutinin (HA) epitope tag into each of the 12 predicted extracellular loops of the rat CaV2.2 α-subunit. The first round of mutagenesis resulted in the addition of sites to 1E1 (domain 1, extracellular loop 1), 1E2, 2E2, 3E1, 3E2 and 4E3. A sequence coding for a short flexible linker plus two contiguous copies of the hemagglutinin epitope was ligated into each site. The resulting constructs were electroporated into HEK293 cells along with plasmids encoding Ca2+ channel β2a and α2δ subunits, and the K+ channel IRK1. Robust “rim type” immunofluorescent labeling (in living cells) was detected for 4 of 6 clones (1E2, 3E1, 3E2 and 4E3). Expression of these constructs in HEK293 cells produced channels with electrophysiological properties similar to wildtype as determined by whole-cell patch-clamp with 10 mM Ca2+ as the charge carrier. Average peak currents were (in nA) -5.4±0.7 for the control and -3.8±0.7, -1.2±0.2, -3.1±0.7 and -4.0±0.7 for 1E2, 3E1, 3E2 and 4E3, respectively. The IV curves and individual current trajectories for each clone were superficially similar to the wildtype. These clones should provide powerful tools for the study of trafficking and modulatory mechanisms of CaV2.2. Additionally, the tagging strategy may be applicable to additional members of the CaV and NaV families of voltage-gated channels.