Figure 7.

CaBP4 interacts with and modulates Ca_v1.4. (a) Affinity chromatography of purified recombinant mCaBP4 on Ca1.4_vα1 column. The eluted fractions were probed with anti-CaBP4. Lanes 1–4, elution with 3 mM EGTA; lane 5, final elution with 3 mM EGTA; lanes 6–8: further elution with 0.1 M glycine buffer, pH 2.1. (b) Colocalization of CaBP4-DsRed2 with Ca_v1.4-GFP in HEK293 cells. Confocal images of HEK293 cells transfected with mCaBP4-DsRed2 and Ca_v1.4–GFP (left three panels) or transfected with mCaBP4-DsRed2 alone (right panel). The yellow color indicates colocalization of the overexpressed fusion proteins. Scale bars, 12.5 μ) m. (c) Modulation of Ca_v1.4 activation by CaBP4 in transfected HEK293T cells. Whole-cell Ca²⁺ currents recorded in cells transfected with Ca_v1.4 subunits (α₁ 1.4, β_2A, α₂δ) alone (left) or cotransfected with CaBP4 (right). Shown are representative traces of Ca²⁺ currents evoked by 50-ms steps from a holding voltage of −80 mV to various test voltages. Extracellular recording solution contained 20 mM Ca²⁺ and intracellular solution contained 5 mM EGTA. CaBP4 enhanced activation of Ca²⁺ currents at negative voltages as indicated. (d) Current-voltage relationship from cells transfected with Ca_v1.4 alone (○) or cotransfected with CaBP4 (•). For each test voltage (V_m), I/Imax represents the current amplitude measured at 45 ms normalized to the maximal current amplitude obtained in the series (mean ± s.e.m.; n = 7–10).