Temperature-dependent increase in the calcium sensitivity and acceleration of activation of ANO6 chloride channel variants

Figures

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  Figure 1. Membrane expression and currents of hANO6 transcript variant V1, V2, V3, and V5. (a) Surface biotinylation assay in HEK293T cells transfected with pcDNA3.1 expressing hANO6 V1, V2, V3, and V5. (b) Summary of membrane expression of each variant normalised to V1 (n = 4). *P < 0.05, ***P < 0.001, compared to V1. (c) Representative trace recordings of ANO6 currents (IANO6) with whole-cell patch clamping with 100 μM free calcium in pipette solution at 27 °C for the mock (upper left panel), ANO6 variants V1 (upper middle panel), V2 (upper right panel), V3 (lower left panel), and V5 (lower right panel) expressed in HEK293T cells. The relative step pulse trace for each variant is presented in the right upper panel of the ramp pulse chart trace. Under the whole-cell patch clamp, a ramp-like pulse from −100 mV to 100 mV (duration time 3 s) was applied every 20 s (holding voltage, −60 mV) and the step pulse protocol was started from a holding potential of −60 mV and then depolarised for 0.5 s from −100 mV to +100 mV with 20 mV increments. (d) The time-based plots of activated outward currents at 100 mV recorded in V1, V2, and V5-transfected HEK293T cells. Time 0 represents the beginning of…

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  Figure 2. Increased calcium sensitivity of ANO6 variants at 37 °C. (a,b) Representative traces of ANO6 currents (IANO6) with whole-cell patch clamping with 1 μM free calcium in pipette solution at 27 °C (a) and 37 °C (b) for the ANO6 variants V1 (left panel), V2 (middle panel), and V5 (right panel) expressed in HEK293T cells. The voltage protocol is already described in Fig. 1. (c) I-V relation curve obtained from the peak current of each variant (V1-left panel, V2-middle panel, and V5 -right panel) at 27 °C (open-square) and 37 °C (solid-square). (d) The time-based plots of activated outward currents at 100 mV recorded in V1, V2, and V5 transfected HEK293T cells at 37 °C with 1 μM [Ca2+]i. (e) Summary bar graph of the t1/2,peak of IANO6 detected at 37 °C. Data are presented as the means ± SEM (n = 13 for V1 and V2, n = 15 for V5). ***P < 0.001 compared to the control.

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  Figure 3. Reversible effect of temperature changes on IANO6. (a) Representative current traces of IANO6 of each variant (ANO6 V1 - left panel, V2 - middle panel, and V5 - right panel) expressed in HEK293T cells from 37 °C to 27 °C and returned to 37 °C. (b) The peak current (I)–voltage (V) relationship curve of ANO6 variants V1 (left panel), V2 (middle panel), and V5 (right panel) detected at first 37 °C (solid-square), 27 °C (x, centre-square), and second 37 °C (open-square). The same whole-cell patch protocol was used as described in Fig. 2 (n = 8 for V1, n = 6 for V2, and n = 7 for V5).

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  Figure 4. Temperature-dependent activation of ANO6 variants by submicromolar [Ca2+]i. (a,b) Representative trace of the IANO6 of V1 (left), V2 (middle), and V5 (right) with 300 nM free calcium in pipette solution at 37 °C (a) and 42 °C (b) expressed in HEK293T cells. The pulse protocol is the same as that described in Fig. 2. (c) I-V relationship curves for the peak amplitude of each variant (V1 - left, V2 – middle, and V5 - right) detected at 37 °C (open-square) and 42 °C (solid-square) with 300 nM [Ca2+]i. (d) The plots of activated outward currents at 100 mV recorded in V1, V2, and V5-transfected HEK293T cells with 300 nM [Ca2+]i. Right panel shows different cases of V1-induced currents at 42 °C, middle and left panels show V2 and V5-induced currents, respectively, at 37 °C (open-square) and 42 °C (solid-square). (e) Summary bar graph of t1/2,peak values of V1 detected at 42 °C; V2 and V5 detected at 37 °C and 42 °C. Data are presented as the means ± SEM (n = 4 for V1, n = 7 for V2, and V5 at 37 °C; n = 5 for V1, n = 8 for V2, and n = 7 for V5 at 42 °C).

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  Figure 5. Partial activation of ANO6 variants 1, 2, and 5 by resting [Ca2+]i at 42 °C (a,b) Representative trace of the IANO6 of mock, V1, V2, and V5 at 37 °C (a) and 42 °C (b) with 100 nM [Ca2+]i expressed in HEK293T cells. The pulse protocol is the same as that described in Fig. 2. (c) The peak I-V relationship curves obtained from mock and each variant detected at 37 °C (open-square) and 42 °C (solid-square) (n = 5 for all variants at 37 °C; n = 4 for V1, n = 7 for V2 and V5 at 42 °C).

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  Figure 6. Accelerated activation of IANO6 at 37 °C. (a,b) Representative current traces of IANO6 generated from three variants (V1, left panel; V2, middle panel; and V5, right panel) with 3 μM free calcium in pipette solution at 27 °C (a) and 37 °C (b) in ANO6 variant-overexpressing HEK293T cells. The whole-cell patch recording pulse protocol was the same as that described in Fig. 2. (c) Time-based activated outward currents at 100 mV obtained from each variant detected at 27 °C (open-square) and 37 °C (solid-square). (d) Summary bar graph of t1/2,peak of IANO6 of each variant estimated from the trace chart. (e) Peak currents elicited by 3 µM [Ca2+]i at +100 mV holding potential from each variant at 27 °C (white bar) and 37 °C (grey bar). Data represent the means ± SEM (n = 10 for 27 °C and n = 15 for 37 °C, for all variants). NS indicates not significant, **P < 0.01, ***P < 0.001 compared to the control.

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  Figure 7. Calcium sensitivity and activation kinetics of ANO6 in excised patches. (a) Representative macroscopic currents of the IANO6 of ANO6 variants V1 (left panel), V2 (middle panel), and V5 (right panel) in an inside-out patch clamp from ANO6 variant-transfected HEK293T cells. The patches were exposed for 40–60 s to Ca2+ concentrations of 300 nM, 1 μM, and 3 μM at a holding potential of +60 mV. (b) Statistics of the peak IANO6 with different calcium concentrations at 27 °C (open-square) and 37 °C (solid-square); graphs displayed at the bottom represent the expansion of the boxed parts of the upper graphs. Data are presented as the means ± SEM (n = 5 for V1, left; n = 6 for V2, middle; and n = 7 for V5, right). NS indicates not significant *P < 0.05, ***P < 0.001 comparing current values at 37 °C and 27 °C.

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  Figure 8. PLA2-independent activation of ANO6 variants 1, 2, and 5. (a,b) Representative current traces of IANO6 generated from the three variants (V1, left panel; V2, middle panel; and V5, right panel) with 1 μM free calcium and 30 mM Cl− in pipette solution at 37 °C, (a) control conditions and (b) ACA (20 μM)-treated conditions, in ANO6 variant-expressed HEK293T cells. (c) The peak current (I) - voltage (V) relation curve obtained from each variant detected under control conditions (open-square) and ACA-treated conditions (solid-square). (d) Summary bar graph of t1/2,peak of IANO6 of each variant estimated from the trace chart. Data represent the means ± SEM (n = 7 for V1 and V2, n = 6 for V5). NS indicates not significant, compared to the control. (e) Representative trace of the IANO6 of V1 (left-panel), V2 (middle-panel), and V5 (right-panel) with 100 nM free calcium and 30 mM Cl− in pipette solution at 37 °C. (f) Corresponding current (I)–voltage (V) relationship curve obtained from the peak currents of V1- square, V2 - circle, V5 - up-triangle. Data represent the means ± SEM (n = 4 for V1, V2, and V5).

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  Figure 9. Phosphatidylserine scramblase activity of ANO6 variants V1, V2, and V5 with different [Ca2+]i. (a) Annexin V-binding (X-axis) and PI-binding (Y-axis) cell counts in ANO6 variant-transfected HEK293T cells from flow cytometry. Cells were treated with 10 μM of ionomycin for 15 min at 27 °C or 37 °C. The incubation solution was NT solution containing free calcium concentrations of 0 μM, 100 μM, or 200 μM for the 27 °C condition and 0 μM, 30 μM, or 100 μM for the 37 °C condition. (b) Summary bar graph of the response of Annexin V-positive cells [%, percentage of total cell counts (Q4-region)] to ionomycin treatment for each variant (V1- left panel, V2- middle panel, and V5- right panel). Data are presented as the means ± SEM (n = 5–10). NS means not significant; *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control.

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  Figure 10. Summary model for temperature-dependent channel activity and scramblase activity of ANO6 variants. (a) Calcium-dependent channel activity and (b), PS exposure response to raised temperature. Black arrow - 27 °C, Red arrow - 37 °C, Arrow with cross bar - no movement of ion or PS, Open arrow - ion movement or PS movement.