Splice variant specific modulation of CaV1.2 calcium channel by galectin-1 regulates arterial constriction.

RATIONALE: Ca(V)1.2 channels are essential for excitation-contraction coupling in the cardiovascular system, and alternative splicing optimizes its role. Galectin-1 (Gal-1) has been reported to regulate vascular smooth muscle cell (VSMC) function and play a role in pulmonary hypertension. We have identified Gal-1 multiple times in yeast 2-hybrid assays using the Ca(V)1.2 I-II loop as bait. OBJECTIVE: Our hypothesis is that Gal-1 interacts directly with Ca(V)1.2 channel at the I-II loop to affect arterial constriction. METHODS AND RESULTS: Unexpectedly, Gal-1 was found to selectively bind to the I-II loop only in the absence of alternatively spliced exon 9*. We found that the current densities of Ca(V)1.2(Δ9*) channels were significantly inhibited as a result of decreased functional surface expression due to the binding of Gal-1 at the export signal located on the C-terminus of exon 9. Moreover, the suppression of Gal-1 expression by siRNA in rat A7r5 and isolated VSMCs produced the opposite effect of increased I(Ca,L). The physiological significance of Gal-1 mediated splice variant-specific inhibition of Ca(V)1.2 channels was demonstrated in organ bath culture where rat MAs were reversibly permeabilized with Gal-1 siRNA and the arterial wall exhibited increased K(+)-induced constriction. CONCLUSION: The above data indicated that Gal-1 regulates I(Ca,L) via decreasing the functional surface expression of Ca(V)1.2 channels in a splice variant selective manner and such a mechanism may play a role in modulating vascular constriction.

The CtBP2 co-repressor is regulated by NADH-dependent dimerization and possesses a novel N-terminal repression domain.

The C-terminal binding protein 2 (CtBP2) is a 48 kDa phosphoprotein reported to function as a co- repressor for a growing list of transcriptional repressors. It was recently demonstrated that CtBP is a dimeric NAD+-regulated d-isomer-specific 2-hydroxy acid dehydrogenase. However, the specific substrate(s) of CtBP enzymatic activity and the relationship of this activity to its co-repression function remain unknown. The ability of a human CtBP to bind and serve as a co-repressor of E1A has been shown to be regulated by nuclear NADH levels. Here we extend the functional characterization of CtBP by demonstrating that amino acid substitutions at Gly189 in the conserved NAD+-binding fold both abrogate the ability of CtBP2 to homodimerize and are associated with a dramatic loss of co-repressor activity. Consistent with the known enzymatic activity of CtBP2, mutations at Arg272 in the substrate-binding domain and at His321 in the catalytic domain result in significant loss of CtBP2 transcriptional co-repressor activity. High resolution serial C-terminal deletion analysis of CtBP2 also revealed a novel N-terminal repression domain that is distinct from its dehydrogenase domain. Our results suggest a model in which CtBP2 co-repressor function is regulated, at least in part, through the effect of NADH on CtBP2 homodimerization.