Bestrophin-3 (Vitelliform Macular Dystrophy 2–Like 3 Protein) Is Essential for the cGMP-Dependent Calcium-Activated Chloride Conductance in Vascular Smooth Muscle Cells

Abstract

Abstract-Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca 2ϩ -activated Cl Ϫ currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca 2ϩ -activated Cl Ϫ current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a "classic" (cGMP-independent) Ca 2ϩ -activated Cl Ϫ current and to have characteristics distinct from those previously known for Ca 2ϩ -activated Cl Ϫ currents. Here, we suggest that a bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca 2ϩ -activated Cl Ϫ current based on similarities between the membrane currents produced by heterologous expressions of bestrophins and the cGMP-dependent Ca 2ϩ -activated Cl Ϫ current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca 2ϩ -activated Cl Ϫ current and bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca 2ϩ -activated Cl Ϫ current, whereas the magnitude of the classic Ca 2ϩ -activated Cl Ϫ current was not affected. Ϫ channel, which results in depolarization in vascular smooth muscle. Furthermore, the current is of similar magnitude as "classic" Ca 2ϩ -activated Cl Ϫ currents in most vascular beds and even larger in some vascular smooth muscles. 3 It is, therefore, highly desirable to know the molecular structure of the channel responsible for this current because it is likely to play an important role in smooth muscle function.Although their biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) are well established, 4 -6 the molecular identity of Ca 2ϩ -sensitive Cl Ϫ channels is still controversial. 7 Recently, the gene responsible for vitelliform macular dystrophy 8 and its homologs that code for bestrophin proteins have been suggested as candidates. 9,10 Four bestrophin family members in the mammalian genome and many homologues in genomes of invertebrates and even prokaryotes have been identified. 11-13 Two different nomenclatures for mammalian bestrophins were previously devel- The majority of suggestions that bestrophins function as Cl Ϫ channels are based on the findings that expression of the gene in different cell types leads to the appearance of a Cl Ϫ conductance 9,10 and that mutations or chemical modifications of the predicted channel pore change this conductance. [15][16][17][18] Although downregulation by small interfering (si)RNA in recent studies demonstrated a direct association between the endogenous Cl Ϫ current in epithelial cell culture and Best-1 expression, 19 -21 the exact role of the bestrophins in native tissues remains questionable...

Expression, localization, and functional properties of Bestrophin 3 channel isolated from mouse heart

“…This study of mBest3 expression and cellular location in cardiac myocytes, together with biophysical properties as a calcium-dependent chloride channel, may provide clues to its function. A recent study by Matchkov et al (25) assigned a role for Best3 as a cGMP-dependent Ca 2ϩ -sensitive Cl Ϫ current with unique characteristics in certain smooth muscle cells. To propose a similar physiological role for Best3 in the heart is difficult because a cGMP-dependent, Ca 2ϩ -sensitive Cl Ϫ conductance has not been reported in this tissue.…”

Activation of Ca2+‐activated Cl− channel ANO1 by localized Ca2+ signals

“…2004, 2005), which possibly depends on bestrophins (Matchkov et al . 2008; Bulley & Jaggar, 2014). Several excellent recent reviews discuss the roles and regulation of CaCCs in smooth muscles (Sanders et al .…”

Simply the Best?

“…In this issue of Circulation Research, Matchkov et al present compelling evidence that best-3 expression is essential for the generation of calcium-sensitive cGMP-dependent chloride channels in rat mesenteric artery. 1 Chloride channels are enigmatic beasts. Numerous phenotypes exist, as determined by their mode of activation, channel kinetics, and pore properties, but the molecular identity has only really been identified for the voltagedependent Cl Ϫ channels (CLCs) and the cAMP-dependent, cystic fibrosis transmembrane regulator (CFTR) channels.…”

“…3 In this issue of Circulation Research, Matchkov et al suggest that we can add 1 more Cl Ϫ current to the list of identified species. 1 Perversely, the calcium-sensitive cGMP-dependent Cl current, which is the focus of the work by Matchkov et al, is among the newest additions to the Cl Ϫ current family. This article completes a body of work by this group that begins and ends with Circulation Research.…”

“…1 This was based on bioinformatics studies that revealed that the C terminus of best-3 proteins contains cGMP phosphorylation motifs (see the online data supplement of the article 1 ), comparison of the best-3 expression profile and the I ClcGMP distribution profile, consolidated by the use of interference RNA. Thus, abundant best-3 message was detected by RT-PCR in rat aorta and mesenteric artery, as well as A7r5 cultured myocytes, in which robust I ClcGMP was recorded but was less abundant in pulmonary artery myocytes, in which I ClcGMP is negligible.…”