Conformational changes upon pore blocker removal reveal conductive states of TMEM16A - PubMed (original) (raw)
Conformational changes upon pore blocker removal reveal conductive states of TMEM16A
Christina A Stephens et al. bioRxiv. 2026.
Abstract
TMEM16A is a Ca2+-activated anion channel that provides direct electrical feedback to the plasma membrane in response to intracellular Ca2+. Its conductive state remains unresolved, leaving questions about gating, Cl- permeation, and modulation by Ca2+, depolarization, and lipids. To investigate the open state, we performed molecular dynamics simulations of TMEM16A bound to the putative open-state blocker 1PBC. After inhibitor removal, the putative, pore-lining helix TM4 developed kinks at two sites: an upper site that opens the pore for Cl- permeation, and a deeper site causing constriction. A conserved hydrophobic network between TM3 and TM4 persisted in most open structures but separated during extreme dilation, allowing lipids to transiently block the pore. Patch-clamp recordings indicated that the intact network promotes activation. Further simulations yielded >60 Cl- permeation events and a single-channel conductance matching experiments. Additional electrostatic and kinetic modeling indicated that TMEM16A's transition from outward-rectification to Ohmic conductance with increasing Ca2+ results from a weak voltage dependence of Ca2+ binding, which act cooperatively to open the pore.
Conflict of interest statement
DECLARATION OF INTERESTS M.G. and F.V.M are employees of the software company Berkeley Madonna.
Figures
Figure 1.. TMEM16A pore constricts or dilates the ion pathway when 1PBC is removed.
(A) Cryo-EM structure of 1PBC (green)/Ca2+-bound (blue) TMEM16A (PDB ID 7ZK3). (B) Overlay of cryo-EM structure (gray) with snapshot from an unrestrained simulation (pink). (C) Enlarged view of Cl− (green sphere) pathway constriction point (dashed box in panel B) for the structures in panel B. (D) Representative snapshots of 4 different degrees of gate separation. I Histogram of the minimum distances between gating residues L547 and I641 aggregated from all simulations. Vertical dotted line is the distance from cryo-EM structure, and the gray line and shaded region are the mean and standard deviation of distances from restrained simulation, respectively. Labeled peaks correspond to labels over snapshots in D (F, G) Average Cl− and K+ densities along the pore separated into 4 categories based on the L547-I641 distance: 0–3 Å (constricted), 3–6 Å (1), 6–9 Å (2), and 9–20 Å (3). Densities from the restrained simulation are gray. These panels share the same x axis. Vertical dashed line is the position of L547 in the pore.
Figure 2.. Spontaneous chloride permeation through dilated states of TMEM16A.
(A) Z-positions of chloride ions in the pore, each color represents a unique ion. The y-axis is zeroed at the cryo-EM z-position of the L547 Cα. Only simulations containing complete permeation events (numbered) are shown. Event 2 is the only inward permeation. The gray bars indicate when the L547-I641 heavy atom distance is > 9 Å. (B) Snapshots of the open TMEM16A groove with zoomed-in images of two Cl− interaction sites: site A (top) and site C (bottom). (C) Zoomed-in snapshot of a third Cl− interaction site (site B). (D) Plot of the percentage of simulation time basic residues interact with Cl− ions (left y-axis, black bars) and dwell times of each interaction instance (right y-axis, green dots).
Figure 3.. TMEM16A pore opening involved rearrangement of hydrophobic contacts.
(A) All simulation data projected onto the first two tIC eigenvectors colored by log density. tIC 1 describes hydrophobic gate opening and tIC 2 distance between TM3 and TM4. Circles indicate cluster centroids sized by relative population; star denotes starting conformation. See Methods for details. (B) Histograms of water flux (number of waters/ns) through the center of the pore in select clusters. (C) Representative structures from selected clusters as labeled below each column. Top: hydrophobic network at TM3/4 extracellular termini. Bottom: hydrophobic contacts between TM4 and 6. Cryo-EM structures of 1PBC/Ca2+ -bound, Ca2+ -bound and apo TMEM16A shown in gray, brown, and cyan respectively. Structures for clusters 2, 7, and 14 are medoids. The non-pink colored portions of helix indicate locations on TM4 that bend to support opening (yellow) or further constricting (cyan) the channel.
Figure 4.. Mutations in the TM3-TM4 hydrophobic network shift TMEM16A activation gating.
(A) Whole cell voltage clamp recordings from wild-type TMEM16A and L522A and I534A mutants expressed in HEK293 cells. Cells were held at −80 mV, then pulsed for 200 ms to potentials from −120 to +140 mV. As I534A exhibited negligible current amplitude at our initial base [Ca2+], a second, higher concentration was tested. (B) Current-voltage (I-V) plots for steady-state current amplitudes as in panel A. (C) Conductance-voltage (G-V) plots generated from data in A and normalized to the current amplitude at +140 mV. Two-state Boltzmann equations were fit to each trace. Conductance from the I534A mutant at the lower [Ca2+] was too low to properly fit a Boltzmann curve, so these data are not shown in panel C. n=4–8 for all conditions.
Figure 5.. Permeation properties of several TMEM16A conformations.
(A) Structures from TMEM16A clusters (MSM states): 2, 7, 8, 11 and 12. Only TM 3–8 are shown and TM4 is colored green without its solvent accessible surface shown (transparent pink elsewhere) and L547 (TM4, red) and R515 (TM3, blue) sidechains shown as sticks. Each structure shown with a 3D spline (gray) fit to the averaged pore center coordinates and radii from multiple simulation frames using HOLE2. (B) Cartoon representation of the cluster 12 structure during simulations under an applied −350 mV (left) and 350 mV voltage with overlay of all Cl− positions (in green) every 0.5 ns. (C) Currents calculated from simulations of cluster 12 plotted as a function of voltage. A linear fit was made to the current data and error bars indicate the 95% confidence interval taken from a Poisson distribution of event rates. Positional restraints were applied to the pore-lining helices of each simulation.
Figure 6.. Cl− permeation energies are strongly influenced by Ca2+ binding and can switch from Ohmic to rectifying conduction states.
(A) The permeation pathway (pink tube) through the cluster 12 representative structure (gray) predicted with HOLE2 fit to a 2D spline and extended manually into solution. Phosphorous (red) and nitrogen (blue) atoms of the POPC headgroups are shown. (B) Free energy profiles for Cl− permeation calculated from ion density along the pathway in A with both Ca2+ ions bound (black), the electrostatic component of the lower Ca2+ removed (blue), or the electrostatic components of both Ca2+ removed (red). (C) A 3-site kinetic model for Cl− permeation based on observations from our simulations. Currents are estimates of single-channel values in 140 mM symmetric Cl−. Only 2 Cl− (green circles) occupy the pore at any given time and the rate constants obey detailed balance based on the energy profiles in panel B. The Cl− permeation model was coupled to an equilibrium Ca2+ (yellow circles) binding model (dashed oval) to determine the probability of the channel to be in the 0, 1, or 2 Ca2+ bound state. Supplemental Kinetic Model for details and all parameters. (D) Single channel currents from model with 2 Ca2+ bound (black) or 1 Ca2+ bound (blue). (E) Predicted ensemble averaged, single channel currents from model in panel C along with the equilibrium Ca2+ binding model. The model was computed at moderate 400 nM (green) and high 10 μM (yellow) intracellular Ca2+ levels. (F) The probability that the channel adopts the 0 Ca2+ (P0) and 2 Ca2+ (P2) bound states in intermediate (green curves) and high (yellow curves) calcium. The singly bound Ca2+ state is negligible in both conditions.
Figure 7.. Lipids block ion permeation in most dilated TMEM16A states.
(A) Traces of chloride z-positions in the TMEM16A pore during simulations of the cluster 12 structure with 350 mV applied voltage and (B) snapshots from the same simulation at timepoints indicated by the black arrows. (C) Traces of chloride z-positions in the TMEM16A pore during simulations of the cluster 12 structure with −350 mV applied voltage and (D) snapshots from the same simulation at timepoints indicated by the black arrows. (E) Traces of chloride z-positions in the TMEM16A pore during simulations of the cluster 12 structure with 350 mV applied voltage and (F) snapshots from the same simulation in panel E at timepoints indicated by the black arrow. The gray bars in panel A and C indicate when a lipid phosphorous atom is within 5 Å of the R515 sidechain. POPC lipids are shown in yellow, and chloride is shown as a green sphere.
References
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