An outer-pore gate modulates the pharmacology of the TMEM16A channel

Abstract

Significance The TMEM16A calcium-gated chloride channels participate in a range of vital physiological functions. TMEM16A channels are desirable new drug targets as their dysfunction can lead to pathology. In spite of this, their pharmacology is still in its infancy. Gaining insight into the mode of action and binding sites for test compounds forms the basis for the development of new TMEM16A-interacting drugs. Here, we demonstrate that intracellular calcium triggers a conformational change in the outer mouth of the channel, which enables entry of a small molecule into the pore. Characterization of this structural rearrangement defines a critical site for pharmacological intervention and reveals an aspect of calcium gating in the TMEM16A channel.

Inhibition mechanism of the chloride channel TMEM16A by the pore blocker 1PBC

“…The Ca 2+ -dependent remodeling of the outer pore is also reflected in the lower potency of the blocker in the absence of Ca 2+ in the constitutively active mutants observed here (Fig. 2c) and in a related study reporting the inhibition by the compound 9-AC, whose binding site was proposed to be located further towards the cytoplasm 54 . In contrast, the predicted location of inhibitors based on docking studies would be extracellular to the described site of 1PBC 47,48 .…”

“…22 ), the flexibility of equivalent residues on α3 and α4 facilitates the rearrangement of these helices during pore opening. These structural changes are reminiscent of an outer-pore gate that was proposed to open upon Ca 2+ binding during activation, providing access to the inhibitor 9-AC 54 .…”

The Ca2+-gated Cl- channel TMEM16A amplifies capillary pericyte contraction reducing cerebral blood flow after ischemia

“…Our work suggests that Ani9 could form the basis for the design of drugs suitable for use in humans and with adequate BBB permeability. The recently solved cryo-EM structure of the TMEM16A channel, and determination of small molecule binding sites may help this drug discovery effort (73)(74)(75)(76). Elucidating the mode of action of Ani9 may aid the design of specific modulators of TMEM16A channel activity for medical therapies to address a range of neurological conditions in which pericytes restrict cerebral blood flow (77,78).…”

The pharmacology of the TMEM16A channel: therapeutic opportunities

“…An additional modulatory Ca 2+ -binding site was also found in TMEM16x proteins [29,30]. Pore opening in response to Ca 2+ binding requires the rearrangement of a gate comprising a cytoplasm-facing portion of TM6 ('steric gate') [8,31] and opening of the outer pore [11,32]. The Ca 2+ -binding sites are formed by a series of negatively charged residues; the unoccupied sites produce an electrostatic repulsion to permeating anions ('electrostatic gate') [33].…”

“…Our detailed understanding of CaCC physiology and pharmacology has long been impeded by not knowing the channel's molecular structure. The identification of TMEM16A as a gene encoding CaCC [3][4][5] electrified the ion channel field and triggered a wealth of investigations leading to the determination of the TMEM16A 3D structure [6][7][8], the identification of binding sites for endogenous [9,10] and synthetic [11][12][13] ligands or natural products [14,15], and the elucidation of the role of the channel in a range of cell types [1,2,16]. While research efforts enabled the discovery of potent test compounds (inhibitors and activators), no therapeutic drugs acting on TMEM16A have yet reached use in clinical practice.…”