Figure 2. Stac interacts with the channel carboxy-terminus.
(A) Live-cell FRET 2-hybrid assay shows high-affinity interaction between CFP-tagged stac3 with YFP-tethered holo-CaV1.3 channels in the presence of auxiliary β2A and α2δ subunits. (B) Cartoon shows FRET pairs, CFP-stac3 with YFP-CI, YFP-PCI, and YFP-IQ of CaV1.3. (C) FRET-binding curves show robust binding of stac3 to both the CI and PCI segment while binding to IQ is weaker. (D) Bar graph summarizes the relative association constant, Ka,EFF, of stac2 binding to major channel intracellular domains. (E–F) Transferring CaV1.3S CI to CaV2.3 (CaV2.3/1.3 CI) unveils latent stac2-mediated suppression of CDI. Format as in Figure 1A – B.
Figure 2—figure supplement 1. Systematic FRET 2-hybrid scan of major intracellular loops of CaV1.3 with stac.
(A) Schematic depicts design of YFP-tagged CaV1.3 intracellular loop constructs (NT, I-II loop, II-III loop, III-IV loop, CI region, PCI region, IQ domain). The exact sequence of the N- and C-termini of each peptide as well as locations on the CaV1.3 α subunit are denoted. (B–E) FRET 2-hybrid binding curves for stac3 interaction with various channel intracellular loops (black symbol and fit). Each symbol denotes FRET measurements from a single cell. Stac3 binds very weakly to the amino terminal (NT) peptide (B) that includes the CaM-binding segment, NSCaTE. Both the I-II (C) and the II-III loop peptides (D) also showed little or no FRET binding. By contrast, the III-IV loop peptide bound appreciably to stac3 (E). (F) Transfer of CI module of CaV1.3 onto CaV2.3 confers stac modulation. Format as in Figure 1—figure supplement 1A. In the absence of stac2, CaV2.3/1.3 CI exhibits robust CDI triggered by local Ca2+ isolated using 10 mM BAPTA (middle subpanel). Stac2 abolishes CDI of CaV2.3/1.3 CI (right subpanel).