Abstract
TMEM16A and TMEM16B encode for Ca2+-activated Cl− channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca2+-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B.
Highlights
Ca2+-activated Cl− channels (CaCCs) are widely expressed in different cell types where they play a variety of important physiological roles
Toward anion permeability even though the sodium permeability significantly increased about 100 fold (Fig 2C). These results show that both R573 and K540 residues are important structural elements to control the permeation in the TMEM16B channel
With 1.5 μM instead of 0.5 μM [Ca2+]i. These results show that R573E and K540Q residues in TMEM16B channel modify anion permeability only at high [Ca2+]i
Summary
Ca2+-activated Cl− channels (CaCCs) are widely expressed in different cell types where they play a variety of important physiological roles. In olfactory and vomeronasal sensory neurons, CaCCs mediate a large component of transduction current [2,3,4,5] and in other neuronal cell types they can control excitability [6]. They regulate the fluid transport in different types of epithelia [7] and modulate the activity of smooth muscles of the blood vessels [8,9]. CaCCs are interesting because of their various hallmark features They are directly gated by sub-micromolar/micromolar concentrations of intracellular Ca2+ and the apparent Ca2+-sensitivity depends on membrane voltage [10]. The pore of CaCCs shows a relatively poor selectivity among anions following the “lyotropic” sequence SCN−>I−>Br−>Cl−>F−
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