Structural Biology and Molecular Biophysics

TMEM16 and TMEM63/OSCA proteins share a conserved potential to permeate ions and phospholipids

Augustus J. Lowry
Pengfei Liang
Y.C. Serena Wan
Zhen-Ming Pei
Huanghe Yang author has email address
Yang Zhang author has email address

Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA
Department of Biology, Duke University, Durham, NC 27710, USA
Department of Neurobiology, Duke University School of Medicine, Durham, NC 27710, USA

https://doi.org/10.7554/eLife.96957.1

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Figures and data

Lysine mutations along TM 4 enable TMEM16F channel and scramblase activities in the absence of calcium stimulation.
(a) Top: TMEM16F is a calcium-activated phospholipid scramblase. Bottom: TM 4 mutant locations mapped on TMEM16F CaPLSase structure (PDB 6QPB). (b) Representative images of TMEM16F knockout (KO) HEK293T cells expressing eGFP-tagged TMEM16F wildtype (WT), I521K, and M522K (center row). CF 594-conjugated Annexin V (AnV, bottom row) labelled PS exposing cells. BF denotes bright field images (top row). (c) Quantification of the percentage of cells with AnV labelling for TMEM16F WT (n=5), I521K (n=5), and M522K (n=5) transfected cells. Values were derived from images of biological replicates, with error bars representing standard error of the mean (SEM). Statistical comparisons were done using unpaired t-tests with Welch’s correction (****: p<0.0001). (d) Representative current recordings and (e) current-voltage (I-V) relationships of whole cell patches from TMEM16F KO HEK293T cells expressing eGFP-tagged TMEM16F WT (n=6), I521K (n=5), and M522K (n=5). Currents were elicited by the voltage protocol shown with the pipette solution containing 5 mM EGTA. Dotted line denotes zero current. (f) Quantification of current at +160 mV. Currents in (e) and (f) were normalized to cell capacitance with the mean ± SEM calculated from independent patches. Statistical comparisons were done using unpaired t-tests with Welch’s correction (*: p<0.05). (g) Lysine mutations along TM 4 in TMEM16F enable spontaneous phospholipid permeation.

Lysine mutations along TM 4 enable TMEM16A channel and scramblase activities in the absence of calcium stimulation.
(a) Top: TMEM16A is a calcium-activated chloride channel Bottom: TM 4 mutant locations mapped on TMEM16A CaCC structure (PDB 5OYG). (b) Representative images of TMEM16F knockout (KO) HEK293T cells expressing eGFP-tagged TMEM16A wildtype (WT), I546K, and I547K (center rows). CF 594-conjugated annexin V (AnV, bottom row) labelled PS exposing cells. BF denotes bright field images (top row). (c) Quantification of the percentage of cells with AnV labelling for TMEM16A WT (n=4), I546K (n=4), and I547K (n=4) transfected cells. Values were derived from images of biological replicates, with error bars representing the standard error of the mean (SEM). Statistical comparisons were done using unpaired t-tests with Welch’s correction (*: p<0.05, **: p<0.01). (d) Representative whole-cell current recordings and (e) current-voltage (I-V) relationships of whole cell patches from TMEM16F KO HEK293T cells expressing eGFP-tagged TMEM16A WT (n=14), I546K (n=5), and I547K (n=5). Currents were elicited by the voltage protocol shown with the pipette containing an EGTA solution. Dotted line denotes zero current. (f) Quantification of current at +160 mV. Currents in (e) and (f) were normalized to cell capacitance with the mean ± SEM calculated from independent patches. Statistical comparisons were done using unpaired t-tests with Welch’s correction (*: p<0.05, ****: p<0.0001). (g) Lysine mutations along TM 4 in TMEM16A enable spontaneous phospholipid permeation.

Lysine mutations along TM 4 enable OSCA1.2 channel and scramblase activities.
Top: OSCA1.2 is a cation non-selective ion channel gated by membrane tension. Bottom: the TM 4/6 interface of OSCA1.2 (PDB 6MGV) with key residues shown as yellow sticks. (b) Representative images of TMEM16F KO HEK293T cells expressing eGFP-tagged (middle row) OSCA1.2 WT (left column), L438K (middle column), or A439K mutants (right column). CF 594-conjugated AnV (bottom row) labelled PS exposing cells. BF denotes bright field images (top row). Asterisk highlights a PS positive cell for the A439K mutant. (c) Quantification of the percentage of cells with AnV labelling for OSCA1.2 WT (n = 4), L438K (n = 7), and A439K-transfected cells (n = 6). Statistical comparisons were conducted with an unpaired t-test with Welch’s correction (*: p<0.05, ****: p<0.0001). (d) Representative current recordings and (e) normalized conductance-voltage (G-V) relationships of inside-out patches from TMEM16F KO HEK293T cells expressing eGFP-tagged OSCA1.2 WT (n=8), L438K (n=8), and A439K (n = 6). Currents were elicited by the voltage protocol shown next to the listed pressures. Dotted line denotes zero current. (f) Quantification of half-maximal voltage at −50 mmHg for WT (109 mV), L438K (67 mV), and A439K (63 mV). Error bars represent standard error of the mean (SEM) calculated from independent patches. Statistical comparison was conducted with an unpaired t-tests with Welch’s correction (***: p<0.001, ****: p<0.0001). (g) Quantification of activation τ_on at −50 mmHg and 160 mV for WT (41 ms), L438K (13 ms), and A439K (16 ms). Error bars represent standard error of the mean (SEM) calculated from independent patches. Statistical comparison was conducted with an unpaired t-tests with Welch’s correction (***: p<0.001, ****: p<0.0001). (h) A lysine mutation along TM 4 converts OSCA1.2 channel into a phospholipid scramblase with spontaneous phospholipid permeability.

OSCA1.2 A439K is an osmolarity-activated scramblase.
(a-b) Representative images of hypotonic osmolarity stimulation of TMEM16F KO HEK293T cells expressing eGFP-tagged OSCA1.2 (a) WT or (b) the A439K mutant (center rows). CF 594-conjugated AnV (bottom rows) labelled PS exposing cells. BF denotes bright field images (top rows). Each column of representative images corresponds to the indicated time after hypo-osmotic stimulation. (c) Quantification of AnV intensity for OSCA1.2 WT (n=5) and A439K (n=5) after hypo-osmotic stimulation. Statistical comparison was conducted with an unpaired t-tests with Welch’s correction (**: p<0.01). (d) The A439K mutation converts OSCA1.2 to an osmolarity-activated phospholipid scramblase.

Lysine mutations along TM 4 enable TMEM63A channel and scramblase activities.
Top: TMEM63A is a cation non-selective ion channel gated by membrane tension. Bottom: the TM 4/6 interface of HsTMEM63A (PDB 8GRS) with key residues shown as yellow sticks using amino acid numbering corresponding to the mouse ortholog. (b) Representative images of TMEM16F KO HEK293T cells expressing eGFP-tagged (middle row) TMEM63A WT (left column), W472K (middle column), or S475K mutants (right column). CF 594-conjugated AnV (bottom row) labelled PS exposing cells. BF denotes bright field images (top row). (c) Quantification of the percentage of cells with AnV labelling for TMEM63A WT (n = 4), W472K (n = 4), and S475K-transfected cells (n = 4). Statistical comparisons were conducted with unpaired t-tests with Welch’s correction (**: p<0.01, ****: p<0.0001). (d) Representative current recordings and (e) normalized conductance-voltage (I-V) relationships of cell attached patches from TMEM16F KO HEK293T cells expressing eGFP-tagged TMEM63A WT (n = 7), W472K (n = 7), and S475K (n = 5). Currents represent the subtraction of voltage alone from currents elicited by the voltage and pressure protocols shown. Dotted line denotes zero current. (f) Quantification of half-maximal voltage at −80 mmHg for WT (122 mV), W472K (96 mV), and S475K (92 mV). Error bars represent standard error of the mean (SEM) calculated from independent patches. Statistical comparison was conducted with an unpaired t-tests with Welch’s correction (*: p<0.05, **: p<0.01). (g) Lysine mutations along TM 4 in TMEM63A enable spontaneous phospholipid permeability.

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