Splice Variant Specific Modulation of Ca _V 1.2 Calcium Channel by Galectin-1 Regulates Arterial Constriction

Abstract

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 ⌬9* 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. Key Words: Ca V 1.2 Ⅲ galectin-1 Ⅲ vascular smooth muscle cell Ⅲ arterial constriction T he Ca V 1.2 L-type calcium channels (LTCCs) are the primary conduits for Ca 2ϩ influx that lead to initiation of numerous physiological processes, including muscle excitation-contraction coupling, neurotransmitter release, gene expression, and hormone secretion. Under optimal conditions, Ca 2ϩ influx through the activated Ca V 1.2 channels in the vascular smooth muscle cells (VSMCs) tightly influences the contractility of small arteries and arterioles, and maintains the myogenic tone of resistance vessels. 1,2 However, if this process goes awry during the pathogenesis of hypertension, an increase in Ca 2ϩ influx may contribute to the development of an abnormal vascular tone and result in elevated peripheral vascular resistance. 3,4 Of the 4 subunits (␣ 1 , ␤, ␣2/␦, and ␥) that composed the calcium channels, the Ca V ␣ 1 subunit not only forms the aqueous pore but also possesses the voltage sensor, gating apparatus and sites for channel regulation by second messengers, drugs, and toxins. 5,6 Within the intracellular linker region between homologous domains I and II of ␣ 1 subunit (I-II loop) is the ␣ 1 -subunit interaction domain (AID), a site where the auxiliary Ca V ␤ subunit binds to traffic ␣ 1 subunit to cell membrane. 7 Moreover, alternative splicing at the Ca V 1.2 I-II loop produces a subpopulation of Ca V 1.2 channel splice variant that contain exon 9*, which is important for vasotone maintenance in blood vessels ( Figure 1A). 8 -11 To date, we and others have identified and characterized 3 predominant Ca V 1.2 splice combinations that are found in arterial smooth muscles, namely, Ca V 1.2 77WT (1-8-⌬9*-33), Ca V 1.2 SM (1-8-9*-⌬33),...

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Emerging Role of Ca_V1.2 Channels in Proliferation and Migration in Distinct Cancer Cell Lines

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