The presence of Ca2+-activated Cl− channels (CaCCs) in vascular smooth muscle cells (SMCs) is well established. Their molecular identity is, however, elusive. Two distinct Ca2+-activated Cl− currents (ICl(Ca)) were previously characterized in SMCs. We have shown that the cGMP-dependent ICl(Ca) depends on bestrophin expression, while the “classical” ICl(Ca) is not. Downregulation of bestrophins did not affect arterial contraction but inhibited the rhythmic contractions, vasomotion. In this study, we have used in vivo siRNA transfection of rat mesenteric small arteries to investigate the role of a putative CaCC, TMEM16A. Isometric force, [Ca2+]i, and SMC membrane potential were measured in isolated arterial segments. ICl(Ca) and GTPγS-induced nonselective cation current were measured in isolated SMCs. Downregulation of TMEM16A resulted in inhibition of both the cGMP-dependent ICl(Ca) and the “classical” ICl(Ca) in SMCs. TMEM16A downregulation also reduced expression of bestrophins. TMEM16A downregulation suppressed vasomotion both in vivo and in vitro. Downregulation of TMEM16A reduced agonist (noradrenaline and vasopressin) and K+-induced contractions. In accordance with the depolarizing role of CaCCs, TMEM16A downregulation suppressed agonist-induced depolarization and elevation in [Ca2+]i. Surprisingly, K+-induced depolarization was unchanged but Ca2+ entry was reduced. We suggested that this is due to reduced expression of the L-type Ca2+ channels, as observed at the mRNA level. Thus, the importance of TMEM16A for contraction is, at least in part, independent from membrane potential. This study demonstrates the significance of TMEM16A for two SMCs ICl(Ca) and vascular function and suggests an interaction between TMEM16A and L-type Ca2+ channels.

Matchkov VV, Moeller-Nielsen N, Dam VS, Nourian Z, Boedtkjer DM, Aalkjaer C. The ␣2 isoform of the Na,K-pump is important for intercellular communication, agonist-induced contraction, and EDHF-like response in rat mesenteric arteries. Am J Physiol Heart Circ Physiol 303: H36 -H46, 2012. First published May 4, 2012 doi:10.1152/ajpheart.00673.2011The specific role of different isoforms of the Na,K-pump in the vascular wall is still under debate. We have previously suggested that the ␣2 isoform of the Na,K-pump (␣2), Na ϩ , Ca 2ϩ -exchange (NCX), and connexin43 form a regulatory microdomain in smooth muscle cells (SMCs), which controls intercellular communication and contractile properties of the vascular wall. We have tested this hypothesis by downregulating ␣2 in cultured SMCs and in small arteries with siRNA in vivo. Intercellular communication was assessed by using membrane capacitance measurements. Arteries transfected in vivo were tested for isometric and isobaric force development in vitro; [Ca 2ϩ ]i was measured simultaneously. Cultured rat SMCs were well-coupled electrically, but 10 M ouabain uncoupled them. Downregulation of ␣ 2 reduced electrical coupling between SMCs and made them insensitive to ouabain. Downregulation of ␣2 in small arteries was accompanied with significant reduction in NCX expression. Acetylcholine-induced relaxation was not different between the groups, but the endotheliumdependent hyperpolarizing factor-like component of the response was significantly diminished in ␣2-downregulated arteries. Micromolar ouabain reduced in a concentration-dependent manner the amplitude of norepinephrine (NE)-induced vasomotion. Sixty percent of the ␣ 2-downregulated arteries did not have vasomotion, and vasomotion in the remaining 40% was ouabain insensitive. Although ouabain increased the sensitivity to NE in the control arteries, it had no effect on ␣ 2-downregulated arteries. In the presence of a low NE concentration the ␣ 2-downregulated arteries had higher [Ca 2ϩ ]i and tone. However, the NE EC50 was reduced under isometric conditions, and maximal contraction was reduced under isometric and isobaric conditions. The latter was caused by a reduced Ca 2ϩ -sensitivity. The ␣2-downregulated arteries also had reduced contraction to vasopressin, whereas the contractile response to high K ϩ was not affected. Our results demonstrate the importance of ␣ 2 for intercellular coupling in the vascular wall and its involvement in the regulation of vascular tone. Na ϩ , Ca 2ϩ exchanger; smooth muscle cell synchronization; short interfering RNA; norepinephrine contractility; endothelium-dependent hyperpolarizing factor THE ELECTROGENIC NA,K-PUMP modifies numerous cellular pathways by modulating Na ϩ -coupled transport. The functional significance of the Na,K-pump is dependent upon the isoform. The catalytic ␣-subunit exists in three isoforms (5). These isoforms have different affinities for cardiac glycosides, kinetics, and regulation but show high structural homology and are difficult to distinguish at a fu...

Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain <25% of SQTS cases. Here we identify a missense mutation in the anion exchanger (AE3)-encoding SLC4A3 gene in two unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pHi, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses suggest that an increase in pHi and decrease in [Cl−]i shortened the action potential duration. However, other mechanisms may also play a role. Altered anion transport represents a mechanism for development of arrhythmia and may provide new therapeutic possibilities.

This study demonstrates the importance of bestrophins for synchronization of SMCs and strongly supports our hypothesis for generation of vasomotion.

Under physiological conditions (i.e. with [Formula: see text] present), NBCn1-mediated [Formula: see text] uptake raises VSMC pHi and promotes filopodia, VSMC migration, and hypertrophic inward remodelling. We propose that axial pHi gradients enhance VSMC migration whereas global acidification inhibits VSMC proliferation and media hypertrophy after carotid artery ligation. These findings support a key role of acid-base transport, particularly via NBCn1, for development of occlusive artery disease.