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

Abstract

The calcium-activated TMEM16 proteins and the mechanosensitive/osmolarity-activated OSCA/TMEM63 proteins belong to the Transmembrane Channel/Scramblase (TCS) superfamily. Within the superfamily, OSCA/TMEM63 proteins, as well as TMEM16A and TMEM16B, are thought to function solely as ion channels. However, most TMEM16 members, including TMEM16F, maintain an additional function as scramblases, rapidly exchanging phospholipids between leaflets of the membrane. Although recent studies have advanced our understanding of TCS structure-function relationships, the molecular determinants of TCS ion and lipid permeation remain unclear. Here we show that single mutations along the transmembrane helix (TM) 4/6 interface allow non-scrambling TCS members to permeate phospholipids. In particular, this study highlights the key role of TM 4 in controlling TCS ion and lipid permeation and offers novel insights into the evolution of the TCS superfamily, suggesting that, like TMEM16s, the OSCA/TMEM63 family maintains a conserved potential to permeate ions and phospholipids.

Figure 1:. 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 with side chains shown as yellow sticks (PDB 6QPB). (b) Representative images of TMEM16F knockout (KO) HEK293T cells expressing eGFP-tagged TMEM16F wildtype (WT), I521K, M522K, and T526K (center column). CF 594-conjugated Annexin V (AnV, right column) labelled PS exposing cells. BF denotes bright field images (left column). (c) Quantification of the percentage of cells with AnV labelling for TMEM16F WT (n=5), I521K (n=5), M522K (n=5), and T526K (n=5) transfected cells. Values were derived from images of biological replicates, with error bars representing standard error of the mean (SEM). Statistical comparisons to T526K were done using unpaired t-tests with Welch’s correction (ns: p>0.05, ****: 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), M522K (n=5), and T526K (n=7). 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 to T526K were done using unpaired t-tests with Welch’s correction (*: p<0.05, **: p<0.01). (g) Lysine mutations along TM 4 in TMEM16F enable spontaneous phospholipid permeation in the absence of calcium.

Figure 2:. I611K on TM 6 enables TMEM16F channel and scramblase activities in the absence of calcium stimulation.

(a) I611 highlighted on the TMEM16F CaPLSase structure with side chains shown as yellow sticks (PDB 6QPB). (b) Representative images of TMEM16F knockout (KO) HEK293T cells expressing eGFP-tagged TMEM16F wildtype (WT) and I611K (center column). CF 594-conjugated Annexin V (AnV, right column) labelled PS exposing cells. BF denotes bright field images (left column). (c) Quantification of the percentage of cells with AnV labelling for TMEM16F WT (n=5) and I611K (n=5) transfected cells. Values were derived from images of biological replicates, with error bars representing standard error of the mean (SEM). Statistical comparison was done using an unpaired t-test 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) and I611K (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 comparison was done using an unpaired t-test with Welch’s correction (***: p<0.001). (g) A lysine mutation on TM 6 in TMEM16F enables spontaneous phospholipid permeation in the absence of calcium.

Figure 3:. 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 with side chains shown as yellow sticks (PDB 5OYG). (b) Representative images of TMEM16F knockout (KO) HEK293T cells expressing eGFP-tagged TMEM16A wildtype (WT), I546K, I547K, and E551K (center column). CF 594-conjugated annexin V (AnV, right column) labelled PS exposing cells. BF denotes bright field images (left column). (c) Quantification of the percentage of cells with AnV labelling for TMEM16A WT (n=4), I546K (n=4), I547K (n=4), and E551K (n=5) 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), I547K (n=5), and E551K (n=6). Currents were elicited by the voltage protocol shown with the pipette 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, ****: p<0.0001). (g) Lysine mutations along TM 4 in TMEM16A enable spontaneous phospholipid permeation in the absence of calcium.

Figure 4:. Lysine mutations along TM 4 enable OSCA1.2 channel and scramblase activities.

(a) Top: OSCA1.2 is a cation non-selective ion channel gated by membrane tension. Bottom: TM 4 mutant locations mapped onto 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 OSCA1.2 WT, L438K, or A439K mutants (center column). CF 594-conjugated AnV (right column) labelled PS exposing cells. BF denotes bright field images (left column). 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 unpaired t-tests 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 lines denote 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 unpaired t-tests with Welch’s correction (***: p<0.001, ****: p<0.0001). (g) Quantification of activation ${\tau }_{\text{on}}$ 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 unpaired t-tests with Welch’s correction (***: p<0.001, ****: p<0.0001). (h) A lysine mutation along TM 4 converts the OSCA1.2 channel into a phospholipid scramblase with spontaneous phospholipid permeability.

Figure 5:. 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 columns). CF 594-conjugated AnV (right columns) labelled PS exposing cells. BF denotes bright field images (left columns). Each row 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-test with Welch’s correction (**: p<0.01). (d) The A439K mutation converts OSCA1.2 to an osmolarity-activated phospholipid scramblase.

Figure 6:. Lysine mutations along TM 4 enable TMEM63A channel and scramblase activities.

(a)Top: TMEM63A is an ion channel gated by high threshold 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 TMEM63A WT, W472K, S475K, and A476K (center column). CF 594-conjugated AnV (right column) labelled PS exposing cells. BF denotes bright field images (left column). (c) Quantification of the percentage of cells with AnV labelling for TMEM63A WT (n=4), W472K (n=4), S475K (n =4), and A476K-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 either eGFP mock-transfected (n=7) or eGFP-tagged TMEM63A WT (n=7), W472K (n=7), S475K (n=5), or A476K (n=7). Currents represent the subtraction of voltage alone from currents elicited by the voltage and pressure protocols shown. Dotted line denotes zero current. Note that the mock control was normalized to the mean maximal current elicited from WT-transfected cells. (f) Quantification of half-maximal voltage at −80 mmHg for WT (122 mV), W472K (96 mV), S475K (92 mV), and A476K (97 mV). Error bars represent standard error of the mean (SEM) calculated from independent patches. Statistical comparison was conducted with unpaired t-tests with Welch’s correction (*: p<0.05, **: p<0.01, ***: p<0.001). (g) Lysine mutations along TM 4 in TMEM63A enable spontaneous phospholipid permeability.