Fig. 2.

The Ca_Vβ₃ subunit is cleaved by Ca²⁺-dependent proteases. A, Recombinant Ca_Vβ₃ ΔP1–2 protein is more stable than the wild-type Ca_Vβ₃ to endogenous Ca²⁺-dependent proteases. Lanes 1 and 2 show the proteolytic breakdown of wild-type Ca_Vβ₃ (β₃) heterologously expressed in HEK- 293 cells. 200 μg of microsomes were incubated with Ca²⁺ (750 μM) at 30°C for 20 min in absence or presence of EDTA (1.5 mM). No proteolytic degradation was observed (lanes 3–4) when the PEST-like regions were deleted (ΔP1–2). B, Coomassie-stained SDS-PAGE in a 10% resolving gel using Laemmli buffer system. Lane 1, control; lanes 2–7, microsomes from HEK- 293 cells expressing the wild-type Ca_Vβ₃ incubated for 20 min at 30°C with Ca²⁺, EDTA or calpeptin or exposed to increasing concentrations of μ-calpain. C, Degradation of the Ca_Vβ₃ subunits in the presence of Ca²⁺ and a calpain protease inhibitor. Ca_Vβ₃ subunits were incubated at 30°C for 20 min with 750 μM Ca²⁺, 180 nM of calpeptin or 1.5 mM EDTA. The full-length (β₃) or the double PEST deletion mutant (ΔP1–2) are indicated. D, ΔPEST mutant Ca_Vβ₃ proteins were incubated for 20 min at 30°C with Ca²⁺, EDTA or calpeptin as indicated above or exposed to two increasing doses of μ-calpain as listed (in the presence of 750 μM Ca²⁺). In each case, one representative of at least two independent degradation experiments of wild-type and mutant Ca_Vβ₃ proteins is presented.