The multiple expression of Ca²⁺-activated Cl⁻ channels via homo- and hetero-dimer formation of TMEM16A splicing variants in murine portal vein

Highlights

• TMEM16A splice variants were identified and cloned from murine portal vein.

• Two major splice variants were functionally identified as Ca²⁺ activated Cl⁻ channel.

• Single molecular images were obtained by total internal reflection fluorescence microscopy.

• Homo- and hetero-dimer formations of the variants were demonstrated in living cells.

• The homo/hetero-dimers are essential for the channel activity in smooth muscle cells.

Abstract

Ca²⁺-activated Cl⁻ channel (CaCC) often plays substantial roles in the regulation of membrane excitability in smooth muscle cells (SMCs). TMEM16A, a member of the TMEM16 family, has been suggested as the molecular entity responsible for CaCC in several types of SMCs. In this study, the expression of TMEM16A splicing variants and their contribution to CaCC activity were examined in murine portal vein SMCs (mPVSMCs). Four transcripts of TMEM16A splicing variants, which include four alternatively spliced segments (“a” and “b” in N-terminus and “c” and “d” in the first intracellular loop), were identified; the expression ratio of four transcripts of “abc”, “acd”, “abcd” and “ac” was 64.5, 25.8, 4.8 and 4.8%, respectively. The immunostaining of isolated mPVSMCs with anti-TMEM16A antibody indicates the abundant expression of TMEM16A on the cell membrane. CaCC currents recorded in mPVSMCs were markedly reduced by T16A_inh-A01, a specific TMEM16A inhibitor. When the two major TMEM16A splicing variants, abc and acd isoforms, were expressed separately in HEK293 cells, the CaCC currents, which possess similar electrophysiological characteristics to those in mPVSMCs were observed. The single-molecule photobleaching analyses using total internal reflection fluorescence (TIRF) microscope indicated that the distribution of stepwise photobleaching events was fit well with a binomial distribution for homodimer. Additionally, the heterodimer formation was suggested by fluorescence resonance energy transfer (FRET) analyses in HEK293 cells co-expressing CFP- or YFP-tagged variants. In conclusion, alternatively spliced variants of TMEM16A abc and acd in mPVSMCs are two major molecular entities of CaCC and may form hetero-/homo-dimers to be functional as CaCC in the regulation of membrane excitability and contractility in mPVSMCs.

Introduction

Ca²⁺-activated Cl⁻ channels (CaCCs) are expressed ubiquitously in various types of cells and play wide variety of physiological roles including fluid secretion, neuronal excitation, and smooth muscle (SM) contraction. Particularly in vascular SM cells (SMCs), the activation of CaCC contributes to the shift of resting membrane potential to positive direction and regulates Ca²⁺ influx through voltage-dependent Ca²⁺ channels, resulting in the increase in muscle tone in several arteries and veins [1]. Portal vein shows spontaneous contractions, which substantially contribute to the blood flow from mesenteric vascular beds to liver and the application of CaCC blockers markedly reduces the portal vein contractions [2]. Therefore, the identification of molecular entity of CaCC in portal vein, is one of the emergent issues for obtaining the comprehensive understanding of mechanisms underlying the physiological and pathophysiological regulations of gastro-liver circulation.

Recently, TMEM16A and TMEM16B, which belong to TMEM16 family, have been identified as the counterpart of classic CaCC [3], [4], [5]. TMEM16A is expressed in various tissues including vascular SMs and expected to regulate their functions [6], [7], [8]. The pharmacological blockade of TMEM16A leads to muscle relaxation in thoracic aorta and mesenteric artery [9]. TMEM16A has eight putative transmembrane domains and is presumed to form dimers as a functional CaCC [10], [11]. In addition, TMEM16A has at least four alternatively spliced segments (named segments: a, b, c, and d). The inclusion or skipping of these segments alters electrophysiological properties of TMEM16A CaCC activity, while the details have not been elucidated yet. The segment b includes putative calmodulin binding domain and segment c affects voltage dependence [12], [13]. Moreover, skipping of segment d changes activation kinetics [14]. Thus, TMEM16A can produce multiple protein isoforms and may differentially contribute to CaCC activity. However, the proportional expression of each TMEM16A splicing variant and the interaction between different variants have been totally unknown regardless of cell types.

The present study was undertaken to elucidate the molecular entities of CaCC in murine portal vein smooth muscle cells (mPVSMCs). We found that TMEM16A splicing variants were expressed and substantially responsible for CaCC activity in mPVSMCs. Results suggest that the mixed expression of homodimers and heterodimers may determine the features of CaCC activity in mPVSMCs.