Abstract 2331: C-terminal of Cav1.3 L-type Ca Channel is Truncated From the Full Length Protein and is Localized to the Nucleus in Cardiomyocytes
Background: L-type Ca channels encoded by Cav1.2 and Cav1.3 play a major role in activating signaling pathways which control gene expression. The C-terminal fragment of Cav1.2 has been recently shown to translocate to the nucleus and functions as a transcription factor. We therefore, developed an antibody to a unique epitope (1708 –1724) in the C-terminus (C-term Ab) of Cav1.3 and explored for the first time whether Cav1.3 C-terminus is truncated and whether it translocates to the nucleus.
Methods and Results: Immunoblotting of total homogenates from cultured rat neonatal myocytes (RNM) using the C-term Ab showed a 60 –70 kDa band that represents the cleaved C-terminal fragment. Confocal imaging of RNM showed punctuate nuclear as well as nuclear envelope staining (Panel A). Confocal imaging of HEK293 cells transfected with a flag-tagged C-terminus also confirmed the nuclear staining (Panel D). Panels B and E show propidium iodide and DAPI specific nuclear staining. Consistent with these data, prediction software showed that the C-terminus of Cav1.3 contains a conserved nuclear localization signal that promotes its translocation to the nucleus. Electrophsyiological recordings of the truncated Cav1.3 channel expressed in tsA201 cells, showed markedly diminished current amplitude from 5.16±0.36 pA/pF (n=31) to 1.24±0.72 pA/pF (n=7, p<0.05).
Conclusion: The data are first to demonstrate that the C-terminus of Cav1.3 L-type Ca channel is truncated and that it translocates to the nucleus/nuclear envelope. The exact functional role of the cleaved Cav1.3 C-terminus in the nucleus is not known but likely to regulate transcription as demonstrated recently for Cav1.2.