Does calmodulin regulate the bicarbonate permeability of ANO1/TMEM16A or not?
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چکیده
The role of calmodulin in the activation of Ca 2+-activated Cl channels (CaCCs) has been an important topic over the past three decades of CaCC research. Recently, the discovery of ANO1/TMEM16 as a component of CaCC raised the question of whether calmodulin modulates ANO1 activity (Tian et al. permeability of human ANO1 (hANO1) can be dynamically modulated by Ca 2+ /calmodulin (Jung et al., 2013). We read with interest the article titled " Calcium-calmodulin does not alter the anion permea-bility of the mouse TMEM16A calcium-activated chloride channel " (Yu et al., 2014b) that challenges the conclusions of our study (Jung et al., 2013). The two major concerns raised by Yu et al. are: (1) they were unable to reproduce the calmodulin-dependent ion permeabil-ity changes in inside-out patch recordings of mouse ANO1/TMEM16A (mANO1) channel, and (2) whole-cell recordings used in our study to measure the bi-ionic potentials are not suitable to obtain the reversal potential (E rev) because of the series resistance and/or ion accumulation problems. However, we believe that the conclusions of Yu et al. were based on several inappropriate assumptions and technical issues. First, our conclusion of Ca 2+ /calmodulin-induced regulation of ANO1 HCO 3 permeability is based on an integrated approach of biochemical and electrophysio-logical methods. For example, in whole-cell recordings we used (a) the calmodulin-binding inhibitor J-8, (b) calmodulin knockdown by siRNAs, and (c) mutation of calmodulin-binding domains (CBDs) in hANO1 to examine the involvement of calmodulin in the high Ca 2+-induced regulation of hANO1 HCO 3 permeability. In addition, inclusion of calmodulin to the cytoplasmic side of outside-out and inside-out patches reproduced the results of the whole-cell recordings. On the other hand, Yu et al. (2014b) used exclusively inside-out patches expressing mANO1 and recombinant tagged-bovine calmod-ulin, and concluded that calmodulin does not alter the anion permeability of ANO1. Yu et al. did not examine the effects of calmodulin using other approaches. Suspecting Correspondence to Min Goo Lee: m l e e @ y u h s. a c that the differences in calmodulin and approach used by Yu et al. may have led to the disparate findings, we attempted to reproduce their findings using similar inside-out patch recordings and, importantly, the same His 6-tagged recombinant bovine calmodulin used by Yu et al. (C4874; Sigma-Aldrich) and the calmodulin purified from human brain (208698; EMD Millipore). Although the human calmodulin reproduced our results of …
منابع مشابه
Purified human brain calmodulin does not alter the bicarbonate permeability of the ANO1/TMEM16A channel
Purified human brain calmodulin does not alter the bicarbonate permeability of the ANO1/TMEM16A channel Yawei Yu and Tsung-Yu Chen Volume 145, No. 1, January 5, 2015. Pages 79–81. In a revised version of Jung and Lee’s Letter to the Editor, two additional panels were added to their Fig. 1. Consequently, Yu and Chen’s references to Jung and Lee’s Fig. 1, B and C, should refer instead to Fig. 1, ...
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