Dataset Information

Specific PIP₂ binding promotes calcium activation of TMEM16A chloride channels.

ABSTRACT: TMEM16A is a widely expressed Ca²⁺-activated Cl^- channel that regulates crucial physiological functions including fluid secretion, neuronal excitability, and smooth muscle contraction. There is a critical need to understand the molecular mechanisms of TMEM16A gating and regulation. However, high-resolution TMEM16A structures have failed to reveal an activated state with an unobstructed permeation pathway even with saturating Ca²⁺. This has been attributed to the requirement of PIP₂ for preventing TMEM16A desensitization. Here, atomistic simulations show that specific binding of PIP₂ to TMEM16A can lead to spontaneous opening of the permeation pathway in the Ca²⁺-bound state. The predicted activated state is highly consistent with a wide range of mutagenesis and functional data. It yields a maximal Cl^- conductance of ~1 pS, similar to experimental estimates, and recapitulates the selectivity of larger SCN^- over Cl^-. The resulting molecular mechanism of activation provides a basis for understanding the interplay of multiple signals in controlling TMEM16A channel function.

SUBMITTER: Jia Z

PROVIDER: S-EPMC7910439 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

Specific PIP2 binding promotes calcium activation of TMEM16A chloride channels.

Jia Zhiguang Z Chen Jianhan J

Communications biology 20210226 1

TMEM16A is a widely expressed Ca2+-activated Cl- channel that regulates crucial physiological functions including fluid secretion, neuronal excitability, and smooth muscle contraction. There is a critical need to understand the molecular mechanisms of TMEM16A gating and regulation. However, high-resolution TMEM16A structures have failed to reveal an activated state with an unobstructed permeation pathway even with saturating Ca2+. This has been attributed to the ...[more]

PMID: 33637964

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Project description:The inwardly rectifying potassium current of the cardiomyocyte (IK1) is the main determinant of the resting potential. Ion channels Kir2.1, Kir2.2, and Kir2.3 form tetramers and are the molecular correlate of macroscopic IK1 current. Verapamil is an antiarrhythmic drug used to suppress atrial and ventricular arrhythmias. Its primary mechanism of action is via blocking calcium channels. In addition, it has been demonstrated to block IK1 current and the Kir2.1 subunit. Its effect on other subunits that contribute to IK1 current has not been studied to date. We therefore analyzed the effect of verapamil on the Kir channels 2.1, 2.2, and 2.3 in the Xenopus oocyte expression system. Kir2.1, Kir2.2, and Kir2.3 channels were heterologously expressed in Xenopus oocytes. Respective currents were measured with the voltage clamp technique and the effect of verapamil on the current was measured. At a concentration of 300 µM, verapamil inhibited Kir2.1 channels by 41.36% ± 2.7 of the initial current, Kir2.2 channels by 16.51 ± 3.6%, and Kir2.3 by 69.98 ± 4.2%. As a verapamil effect on kir2.3 was a previously unknown finding, we analyzed this effect further. At wash in with 300 µM verapamil, the maximal effect was seen within 20 min of the infusion. After washing out with control solution, there was only a partial current recovery. The current reduction from verapamil was the same at - 120 mV (73.2 ± 3.7%), - 40 mV (85.5 ± 6.5%), and 0 mV (61.5 ± 10.6%) implying no voltage dependency of the block. Using site directed mutations in putative binding sites, we demonstrated a decrease of effect with pore mutant E291A and absence of verapamil effect for D251A. With mutant I214L, which shows a stronger affinity for PIP2 binding, we observed a normalized current reduction to 61.9 ± 0.06% of the control current, which was significantly less pronounced compared to wild type channels. Verapamil blocks Kir2.1, Kir2.2, and Kir2.3 subunits. In Kir2.3, blockade is dependent on sites E291 and D251 and interferes with activation of the channel via PIP2. Interference with these sites and with PIP2 binding has also been described for other Kir channels blocking drugs. As Kir2.3 is preferentially expressed in atrium, a selective Kir2.3 blocking agent would constitute an interesting antiarrhythmic concept.

Dataset Information

Specific PIP₂ binding promotes calcium activation of TMEM16A chloride channels.

Publications

Specific PIP<sub>2</sub> binding promotes calcium activation of TMEM16A chloride channels.

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