Figure 3. Effect of γ₇ and C-terminally truncated γ₇(1-217) on Ca_V2.2 mRNA stability.

A, Effect of γ₇ on Ca_V2.2 mRNA degradation rate. Constructs were expressed in Xenopus oocytes either without (■) or with γ₇ (Δ) or with γ₇(1-217) (□). In the absence of a γ₇ construct, an equivalent amount of a similar sized transcript, a non-functional K⁺ channel Kir-AAA cDNA was used. After 24 h (T₀), actinomycin D (50 μg/ml) was added to the medium, and the Ca_V2.2 mRNA levels measured at the times shown after this. The numbers of determinations from individual oocytes are between 6 and 12 for each data point; * P<0.05 compared to Ca_V2.2 (Student’s t test). The lines are apparent linear fits using errors as weight.

B, Bar chart showing the % of Ca_V2.2, α₂δ-2 and KCC1 mRNA present at time t after actinomycin D addition at time T₀. For Ca_V2.2 mRNA turnover, t = 9 h. Bars 1 - 3 are control (black bar, n = 11); + γ₇, (white bar, n = 7) and +γ₇(1-217) (hatched bar, n = 8). Bars 4 -7 show the % of α₂δ-2 mRNA and KCC1 mRNA present 9 and 24 h respectively after actinomycin D addition for the control condition (black bar, n = 8 and 9), + γ₇ (white bar, n = 9 and 9). These times were chosen as the % of mRNA remaining in control conditions was similar for all mRNA species. * P<0.05, compared to control, Student’s t test.

C, Degradation rate for endogenous Ca_V2.2 in the γ₇-CFP PC12 cell line (○, n = 3), compared to control pcDNA3.1-transfected PC12 cell line (■, n = 5), following differentiation with NGF. The Ca_V2.2 mRNA level is expressed as % of time 0 (T₀), when actinomycin D was added. The half-life for endogenous Ca_V2.2 mRNA was 19.5 h in the pcDNA3.1 transfected PC12 cell line. In γ₇-transfected PC12 cells the half-life was 9.1 h (** P<0.01, Student’s t test). Insert: Reduction of endogenous Ca_V2.2 mRNA level in γ₇-CFP PC12 cell line, compared to control pcDNA3.1-transfected PC12 cell line. *** P<0.001 vs control.