Troponin T3 Regulates Calcium Channel Beta1a Subunit Nuclear Translocation in Skeletal Muscle

Abstract

Voltage-dependent calcium channel (VDCC) beta subunits (CaVβ) are critical for CaVα subunit membrane expression and gating properties. While CaVβ interacts with a variety of other molecular partners, nonchannel functions of skeletal muscle CaVβ1a have not been reported. In a yeast two hybrid (Y2H) screening of the mouse tibialis anterior (TA) muscle cDNA library, troponin T3 (TNNT3), specific to fast skeletal muscle, was identified using full-length CaVβ1a as the bait. Further studies revealed that TNNT3, but not the slow skeletal muscle TNNT1, co-immunoprecipitated with CaVβ1a in the mouse skeletal muscle cell line C2C12 and mouse skeletal muscle. Consistently, both CaVβ1a and TNNT3 were highly expressed in mouse fast, but not slow, skeletal muscle. Surprisingly, when DsRed-tagged TNNT3 and YFP tagged CaVβ1a (CaVβ1a -YFP) were transiently co-expressed in C2C12 cells or the mouse flexor digitorum brevis (FDB) muscle, both showed a punctuate distribution pattern in the cytoplasm and nucleus. In comparison, CaVβ1a-YFP alone localized uniformly in the cytoplasm. Further truncation analysis revealed that TNNT3 C-terminus (aa 161-244) localized exclusively in FDB nuclei and enriched CaVβ1a -YFP accordingly. In contrast, the TNNT3 middle region (aa 55-160) localized only in the cytoplasm, while the N-terminus (aa 1-54) was found in both cytoplasm and nucleus. Note that the N-terminus could also recruit CaVβ1a-YFP in the nucleus. Y2H assay verified that the TNNT3 C-terminus has the strongest interaction with CaVβ1a. Immunoblotting with an antibody targeting the TNNT3 N-terminus detected an increased TNNT3 fragment in the aging mouse skeletal muscle. We conclude that TNNT3 interacts with CaVβ1a and regulates its nuclear translocation mainly through its TNNT3 C- or N-terminus. These findings strongly support a transcription-regulation function for CaVβ1a and TNNT3.