Cobalt ion interaction with TMEM16A calcium-activated chloride channel: Inhibition and potentiation

Figures

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  Fig 1. Structures of TMEM16A and TMEM16F with bound Ca2+. (A) TMEM16F structure (PDB: 6QPC). (B) TMEM16A structure (PDB: 5OYB). On the left part of both A & B, the top view is from within the phospholipids for seeing both subunits. The view below is obtained by rotating the structure 90° clockwise around the two-fold axis. The boxed area in the lower view is expanded on the right using a stereo pair, and the subunit in the back is hidden for clarity. Purple spheres represent calcium ions, and the space-filled residue in red represents Y589 of TMEM16A or Y564 of TMEM16F. Helices in color are labeled with numbers representing the six transmembrane helices (TM3-8) encompassing the transport pathway.

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  Fig 2. Effects of Co2+ on the WT TMEM16A channel. (A) Effects of intracellular Co2+ (left) and Ca2+ (right) when applied alone. The membrane voltage began at 0 mV, and was then stepped from -60 mV to +60 mV in +20 mV steps. [Co2+]i (pink line) and [Ca2+]i (green line) were applied as indicated on the same membrane patch. (B) Effects of various [Co2+]i on the TMEM16A current activated by 30 μM [Ca2+]i. The membrane voltage was 0 mV at the start of the recording and then stepped to -20 mV (black traces) and +20 mV (orange traces), respectively, at the time indicated by the capacitive spikes. [Ca2+]i (30 μM) was then applied, followed by applying Co2+ in the presence of the same [Ca2+]i as indicated by horizontal lines. Ipeak, I0 and ICo are defined as in the Data Analysis section in METHODS.

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  Fig 3. Competitive inhibition by Co2+ on [Ca2+]i-induced TMEM16A current. (A) Inhibition by 2 mM Co2+ of the TMEM16A current activated by various [Ca2+]i at -20 mV (black) and +20 mV (orange). (B) Concentration-dependent Co2+ inhibition of the TMEM16A current activated by various [Ca2+]i at +20 mV (upper panel) and -20 mV (lower panel). Numbers next to each individual dose-response curves represent the [Ca2+]i (in μM) used in activating the current. Data points were fitted to the Langmuir function defined in METHODS (Eq 1). The fitted K1/2 values of Co2+ inhibition at -20 mV and +20 mV in various [Ca2+]i are presented in Table 1.

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  Fig 4. Effects of Co2+ on the kinetics of TMEM16A activation/deactivation. The indicated [Co2+]i was present throughout the entire recording period in all recordings. TMEM16A current was induced by (A) 1 μM [Ca2+]i and (B) 1 mM [Ca2+]i. All recording traces in A were from the same patch, while the recording traces in B were from another patch. Notice intracellular Co2+ slows down the current activation upon applying [Ca2+]i and the current reduction upon [Ca2+]i washout in a dose-dependent way. At the same time Ca2+ antagonizes the effects of Co2+ on the activation/deactivation kinetics. Similar results were observed in another 4 patches (two patches each with 1 μM [Ca2+]i and 1 mM [Ca2+]i).

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  Fig 5. Comparing Co2+ effects between WT TMEM16A and the Y589A mutant. (A) Effects of various [Co2+]i on the Y589A mutant. [Ca2+]i = 30 μM for all patches. (B) Comparison of the dose-response curves of Co2+ inhibition between WT TMEM16A and the Y589A mutant. (C) Representative traces comparing the current-reduction process upon washout of [Ca2+]i at +20 mV between WT TMEM16A and the Y589A mutant. Recorded currents were normalized to the current right before the washout of [Ca2+]i. The current reduction processes were fitted to Eq 2 with the averaged τoff of 144 ± 5 ms (n = 6) and 601 ± 29 ms (n = 4) for the WT channel and the Y589A mutant, respectively. (D) Comparison of the dose-response curves of Co2+ potentiation between WT TMEM16A and the Y589A mutant at -20 mV (left) and +20 mV (right).

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  Fig 6. Dependence of Co2+ inhibition on the current reduction time (τoff) upon Ca2+ washout. Recording traces illustrate the effects of 2 mM Co2+ on various Y589 mutants activated by 30 μM [Ca2+]i at +20 mV. Dissociation rates of Ca2+ were evaluated from the current reduction process upon Ca2+ washout at the end of each recording by fitting the current reduction process with a single-exponential decay function (Eq 2). Notice that the degree of Co2+ inhibition was reduced with the increase of the time constant (τoff) of the current reduction upon removing [Ca2+]i (see correlation plot in the left panel of Fig 7A).

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  Fig 7. Correlation of Co2+ inhibition with τoff, the hydrophobicity index, or the molecular volume of the amino acid placed at position 589. Here, experiments were similar to those shown in Fig 6. the remaining current fraction after 6-sec Co2+ application (ICo/Ipeak) was calculated and was plotted against the three different parameters. (A) Results obtained at +20 mV. (B) Results obtained at -20 mV. All data points were obtained from the effects of 2 mM [Co2+]i on the WT TMEM16A current induced by 30 μM [Ca2+]i.

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  Fig 8. Correlation of Co2+ potentiation with τoff, the hydrophobicity index, or the molecular volume of the amino acid at position 589. (A) Results obtained at +20 mV. (B) Results obtained at -20 mV. All data points were obtained from the effects of 2 mM [Co2+]i on the 30 μM [Ca2+]i-induced TMEM16A current. The degree of Co2+ potentiation was defined as Ipeak/I0. Notice a rough correlation between the potentiation and the volume of the amino acid placed at position 589—the larger the side-chain volume, the smaller the Co2+ potentiation.

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  Fig 9. Dose-dependent Co2+ potentiation of the WT TMEM16A current. (A) Recording traces depicting Co2+ potentiation of the TMEM16A current activated by 100 μM, 300 μM, and 1 mM [Ca2+]i, respectively. To focus on the Co2+ potentiation effect, only the traces from 450 ms before to 550 ms after the application of Co2+ are shown. (B) Averaged Co2+ potentiation of the WT TMEM16A current as a function of [Co2+]i. Notice the difference in the degree of potentiation between the experiments at -20 mV and +20 mV.

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  Fig 10. Potentiation of TMEM16A current by mM concentrations of [Ca2+]i. Recording traces of (A) WT TMEM16A and (B) Y589A. Both panels show a larger current induced by 2 mM (left panel) or 20 mM [Ca2+]i (middle panel) than that induced by 20 μM [Ca2+]i. Numbers below each pulse represent [Ca2+]i in mM. Orange and black traces are from +20 mV and -20 mV, respectively. Notice the fast and the slow current reduction upon washout of 2 or 20 mM [Ca2+]i. Right panels show dose-dependent Ca2+ potentiation of WT TMEM16A and Y589A at ±20 mV compared to the current induced by 20 μM [Ca2+]i. Current activated by various test [Ca2+]i (middle pulse) was normalized to the average of the control currents at 20 μM [Ca2+]i before and after the test [Ca2+]i.

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  Fig 11. Current potentiation in various Y589 mutants by high [Ca2+]i. Dose-response curves at +20 mV (orange triangle) and -20 mV (black circle) were constructed from data obtained by the three-pulse protocol as that shown in Fig 10 (A & B). The dose-response curves of WT TMEM16A are plotted as dash curves in other panels for comparison.

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  Fig 12. Correlation of Ca2+ potentiation with Co2+ potentiation for various Y589 mutants. Y-axis represents the potentiation by 20 mM [Ca2+]i (namely, I20 mM/I20 μM) while X-axis is the potentiation of the [Ca2+]i (300 μM)-induced current by 20 mM [Co2+]i. Aromatic mutants are highlighted in yellow.