A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity
- University of California, San Francisco, United States
- Howard Hughes Medical Institute, University of California, San Fransisco, United States
Figures
Figure 1 with 1 supplement
Calmodulin (CaM) is not responsible for the calcium-dependent activation of TMEM16A calcium-activated chloride channels (CaCC).
(A) Two competing models to explain TMEM16A calcium sensitivity have been proposed. It is unclear whether calcium directly binds to TMEM16A-CaCCs (upper panel) or whether CaM is required to mediate …
Figure 1—figure supplement 1
Calmodulin (CaM) is not involved in the calcium-dependent activation of TMEM16A-CaCC.
(A) Acute application of 50 µM W7, a CaM antagonist, to the cytosolic face of the inside-out patches failed to inhibit endogenous Xenopus TMEM16A-CaCC currents. (B) Chronic incubation of Xenopus …
Figure 2
Screen for potential calcium-binding residues in TMEM16A-CaCC.
(A and B) Quantification of the apparent calcium sensitivity of E698Q and E701Q (Yu et al., 2012) mutant TMEM16A channels. (A) Representative current traces of E698Q and E701Q mutants in response to …
Figure 3
Systematic alanine scan of highly conserved intracellular acidic residues identified five mutations that dramatically reduced the apparent calcium sensitivity of TMEM16A-CaCC.
(A) Representative current traces of the E650A, E698A, E701A, E730A and D734A mutant channels in response to different intracellular calcium solutions recorded at +60 mV. Table indicates the …
Figure 4
The effects of different amino acid side chains on the calcium sensitivity of mutant TMEM16A-CaCC channels indicate that E698, E701, E730 and D734 might be directly involved in binding calcium.
(A–E) Calcium dose–response curves of (A) E650, (B) E698, (C) E701, (D), E730, and (E) D734 mutant mTMEM16A channels at +60 mV. Smooth curves represent fits to the Hill equation. Maximum activity …
Figure 5
TMEM16A channel sensitivity to strontium ions is disrupted by mutations of the identified calcium-binding sites.
(A) Representative current trace of wildtype mTMEM16A in response to different intracellular strontium solutions recorded at +60 mV. Table indicates the concentration of strontium used. (B) …
Figure 6
TMEM16A channel sensitivity to cadmium ions is disrupted by mutations at the identified calcium-binding sites.
(A) Representative current trace of wildtype mTMEM16A in response to different intracellular cadmium solutions recorded at +60 mV. Table indicates the concentration of cadmium used. (B) Cadmium …
Figure 7
Cysteine crosslinking suggests that the calcium-binding residues in TMEM16A-CaCC form a metal ion binding pocket that is exposed to the cytoplasm.
(A) Representative traces of E701C/E730C, E701C, and E730C mTMEM16A mutants recorded under reducing (DTT) and oxidizing (H2O2) conditions. (B) Comparison of currents recorded in oxidizing conditions …
Author response image 1
Barium dose-response curve of wildtype and mutant mTMEM16A channels at +60 mV. E730D and E730Q were the only mutants that produced appreciable activity out of the channels tested.
The pattern of bariumdependentactivation is not similar to that of cadmium-dependent activation.
