Cl⁻ channels in smooth muscle cells

Abstract

In smooth muscle cells (SMCs), the intracellular chloride ion (Cl−) concentration is high due to accumulation by Cl−/HCO3− exchange and Na+–K+–Cl− cotransportation. The equilibrium potential for Cl− (ECl) is more positive than physiological membrane potentials (Em), with Cl− efflux inducing membrane depolarization. Early studies used electrophysiology and nonspecific antagonists to study the physiological relevance of Cl− channels in SMCs. More recent reports have incorporated molecular biological approaches to identify and determine the functional significance of several different Cl− channels. Both "classic" and cGMP-dependent calcium (Ca2+)-activated (ClCa) channels and volume-sensitive Cl− channels are present, with TMEM16A/ANO1, bestrophins, and ClC-3, respectively, proposed as molecular candidates for these channels. The cystic fibrosis transmembrane conductance regulator (CFTR) has also been described in SMCs. This review will focus on discussing recent progress made in identifying each of these Cl− channels in SMCs, their physiological functions, and contribution to diseases that modify contraction, apoptosis, and cell proliferation.

Fig 1. Predicted membrane topologies of Cl⁻ channels described in vascular SMCs

TMEM16A/ANO1 was adapted from [144], although an alternative membrane topology has been suggested [144]. Bestrophin modified from ref [79], ClC-3 from ref [29] and CFTR from ref[152].

Fig 2. Original electrophysiological recordings of recombinant TMEM16A/ANO1 and SMC Cl_Ca currents

Whole-cell currents of TMEM16A-expressing HEK-293 cells in different free [Ca²⁺]_i [106]. Reproduced with permission, from Scudieri P, Sondo E, Caci E, Ravazzolo R, Galietta LJV, (2013), (Biochem J), (452), (443–455). © the Biochemical Society. Whole-cell recordings of Cl⁻ currents in cerebral artery SMCs with 200 nM and 1 μM free [Ca²⁺]_i (adapted from ref [117]).

Fig 3. Recombinant bestrophin-3 and SMC cGMP-dependent Cl_Ca currents

Whole-cell mBest3 currents expressed in COS-7 cells at a [Ca²⁺]_i of 500 nM [88]. Adapted with permission from O’Driscoll KE, Hatton WJ, Burkin, HR, Leblanc N, Britton FC (2008) Expression, localization and functional properties of Bestrophin 3 channel isolated from mouse heart. Am J Physiol Cell Physiol. 295: C1610–C1624 © the American Physiological Society (APS). Whole-cell niflumic acid (NFA)-insensitive cGMP-dependent Cl_Ca current recorded in a mesenteric artery SMC [5]. Adapted with kind permission from Springer Science+Business Media: Pflügers Archiv European Journal of Physiology, Vasomotion has chloride-dependency in rat mesenteric small arteries, 457, 2008, 389–404, Boedtkjer DM, Matchkov VV, Boedkjer E, Nilsson H, Aalkjaer C, Figure 7.

Fig 4. Recombinant ClC-3 and SMC volume-regulated Cl⁻ currents

Osmotic regulation of whole-cell currents recorded from gpClC3-transfected NIH/3T3 cells under isotonic, hypotonic and hypertonic conditions [27]. Adapted by permission from Macmillan Publishers Ltd: [NATURE] (Duan D, Winter C, Cowley S, Hume JR, Horowitz B. Molecular identification of a volume-regulated chloride channel 390:417–421), copyright (1997). Volume regulation of whole-cell currents recorded from A10 vascular SMCs under similar conditions [149]. Reproduced with permission from Zhou JG, Ren JL, Qiu QY, He H, Guan YY (2005) Regulation of intracellular Cl− concentration through volume-regulated ClC-3 chloride channels in A10 vascular smooth muscle cells. J Biol Chem 280:7301–730. © 2008 The American Society for Biochemistry and Molecular Biology. All rights reserved.”

Fig 5. Cl⁻ channels present in vascular SMCs

Cl⁻ accumulates in SMCs due to the Na⁺-K⁺-Cl⁻ co-transporter (NKCC1) and the Cl⁻-HO₃⁻ exchanger-2 (AE2). cGMP-dependent and independent Cl_Ca channels, a volume-sensitive Cl⁻ channel and the cystic fibrosis transmembrane conductance regulator (CFTR) have been identified. The molecular identity of the first three channels has been proposed to be bestrophin, TMEM16A/Ano1 and ClC-3, respectively. Numerous mechanisms of Ca²⁺ activation of Cl_Ca channels in vascular SMCs have been suggested, including IP₃R- or RyR-mediated SR Ca²⁺ release, Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCC) and local Ca²⁺ influx through non-selective cation channels (NSCC). Activation of these channels leads to Cl⁻ efflux and subsequent depolarization of the cell membrane that activates voltage-dependent Ca²⁺ channels (VDCC). ClC-3 channels have been proposed to be activated by membrane swelling. ClC-3 is present in the plasma membrane and in intracellular compartments, including endosomes. Endosomal ClC-3 channels may regulate volume-regulated Cl⁻ channels via ROS production. CaMKII inhibits TMEM16A and activates ClC-3 channels. CFTR is a cAMP-activated ATP-gated anion channel that appears to be functional when the SMC membrane potential becomes more positive than the Cl⁻ equilibrium potential. Under this condition, CFTR channel activation would lead to Cl⁻ influx and oppose vasoconstriction.