Regulation of the Ca2+-activated chloride channel Anoctamin-1 (TMEM16A) by Ca2+-induced interaction with FKBP12 and calcineurin
Graphical abstract
Introduction
The calcium-activated chloride (Cl−) channel Anoctamin-1 or TMEM16A controls blood pressure, gastro-intestinal motility, sperm capacitation and motility, pain sensation and secretion of saliva, mucin, insulin and melatonin [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]]. TMEM16A is a homodimer harbouring one permeation pathway per monomer [[12], [13], [14], [15]]. It is gated by direct binding of intracellular calcium (Ca2+) to a pocket composed of acidic residues that locates near the end of the pore towards the cytoplasmic side [[14], [15], [16], [17]].
TMEM16A is subjected to various mechanisms of regulation. For instance, extracellular protons tune the activity of TMEM16A by titrating residue E623 [18]. On the contrary, intracellular protons inhibit Ca2+ gating by competing with Ca2+ for binding sites within the Ca2+ pocket [19,20]. Also, the elevation of temperature results in TMEM16A activation even in the absence of intracellular Ca2+ [21]. We have shown recently that depletion of PI(4,5)P2 by a voltage-sensitive phosphatase partially abolish TMEM16A activity [22,23]. To maintain channel activity, PI(4,5)P2 binds to the channel via a network of PI(4,5)P2 binding sites [[24], [25], [26]]. Interestingly, long-chain poly-unsaturated fatty acids that help control blood pressure also down regulate TMEM16A activity [22].
In addition, TMEM16A activity appears to be contingent on its phosphorylation state. Phosphorylation reduces channel activity whereas dephosphorylation increases channel activity [27,28]. The phosphorylation state of the channel is controlled by the concerted action of the Ca2+/calmodulin-dependent kinase II (CaMKII) and by calcium/calmodulin-dependent calcineurin (CaN) and Ca2+-independent PP1 and PP2A phosphatases [[29], [30], [31], [32]]. CaMKII phosphorylates TMEM16A in Ser525 and/or Ser727; these residues are presumably also the targets for CaN, PP1 or PP2A [31,[30], [31], [32]]. Smooth muscle cells express the A-α and A-β CaN isoforms, but only the A-α isoform seems to be required to increase channel activity in rabbit pulmonary artery smooth muscle cells [29]. In addition, the Cl- currents activated by Ca2+ in rabbit pulmonary arterial myocytes are diminished after CaN inhibition with cyclosporine A (CsA) [29]. However, this regulatory mechanism is not fully explained yet. Some observations do not support this mechanism, for example in CaN A-α null mouse, the volume of pilocarpine-induced saliva secretion, which depends on TMEM16A activation, is not diminished [33]. In rabbit portal vein, the Ca2+-activated Cl- current is not enhanced after inhibition of CaMKII [27]. Moreover, there is no consensus on whether TMEM16A activity is regulated by calmodulin, a cytosolic protein required by both CaN and CaMKII [[34], [35], [36]].
Upon an intracellular Ca2+ increase, TMEM16A would need to circumvent the phosphorylation-dependent down-regulation due to CaMKII activation in order to fulfil its physiological role. To do so, TMEM16A could interact directly or indirectly with CaN, PP1 or PP2A. Of these CaN stands out because requires Ca2+ and calmodulin to be active and regulates several ion channels [36,37]. Furthermore, CaN binds to the ubiquitous FK506-binding cis-trans peptidyl-prolyl isomerase FKBP12 in a Ca2+-dependent manner [[38], [39], [40]] and regulates the function of ryanodine receptors, for example [[40], [41], [42], [43]]. Thus, we hypothesize that CaN could regulate TMEM16A activity through FKBP12 when intracellular Ca2+ increases.
Here we show that intracellular Ca2+ drives the formation of a ternary complex consisting of TMEM16A-FKBP12-CaN. CsA and FK506 are inhibitors of CaN-Aα and CaN-Aβ [44] and partially diminished TMEM16A activity. FK506 abolished heterotrimer formation by binding to FKBP12 without hindering channel activation but effectively preventing the effect of CsA on TMEM16A. We propose that under physiological conditions an elevation of intracellular Ca2+ induces association of FKBP12 and CaN with TMEM16A in order to enhance TMEM16A activity, however, the formation of this ternary complex is not required for the channel’s activation by Ca2+.
Section snippets
Materials and methods
The biological and chemical reagents, DNA plasmids, equipment, and software used in this work are listed in Table 1.
Regulation of TMEM16A activity by hetero-multimerization
To test the idea that TMEM16A could form a ternary complex with FKBP12 and CaN following an increase in intracellular Ca2+ we performed co-immunoprecipitation assays [48]. HEK-293 cells expressing mouse TMEM16A-3XFlag (ac variant) were bathed in a solution containing 0 or 0.5 mM Ca2+ and then exposed to 1 μM ionomycin during 30 s; immediately after the cells were lysed for protein isolation. Fig. 1A shows that a CaN antibody precipitated both TMEM16A (revealed by a monoclonal anti-Flag M2
Discussion
In this work, we demonstrate a Ca2+-dependent heteromultimerization of TMEM16A with the cytosolic proteins CaN and FKBP12. In addition, we found that TMEM16A activity decreased after removal of FKBP12-CaN with FK506 or by inhibiting CaN with CsA. In the ternary complex FKBP12 seems to bind both TMEM16A and CaN. This idea is supported by experiments in cells treated with FK506. Those experiments showed a lack of effect of CsA on TMEM16A, no co-immunoprecipitation between TMEM16A and FKBP12, and
Credit statement
Conceptualization: A Sánchez-Solano, P Pérez-Cornejo, J Arreola
Methodology: A Sánchez-Solano, S Cruz-Rangel, N Corral, I Aréchiga-Figureroa, G Segura-Covarrubias, ML Guzmán-Hernández, P Pérez-Cornejo, J Arreola
Validation: A Sánchez-Solano, S Cruz-Rangel, N Corral, I Aréchiga-Figureroa, G Segura-Covarrubias, ML Guzmán-Hernández, P Pérez-Cornejo, J Arreola
Formal analysis:A Sánchez-Solano, S Cruz-Rangel, N Corral, I Aréchiga-Figureroa, G Segura-Covarrubias, ML Guzmán-Hernández, P Pérez-Cornejo, J
Funding
Funding was provided by grants CB-219949-2014 and FC 2016-01-1955 from the National Council for Research and Technology, Mexico, to JA, IAF, and PPC.
Declaration of Competing Interest
The authors declare that they have no conflicts of interest with the contents of this article and have approved its submission to Cell Calcium.
Acknowledgements
We would like to thank Carmen Y. Hernandez-Carballo for technical assistance and Dr. Sergio Sanchez-Armass for generous assistance with calcium measurements and the use of his facility.
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Present address: Department of Medicine, University of Pittsburgh, Pittsburgh PA, United States.