Figure 3.
MK LRRC8 channels permeate ATP. (A-C) Current-time relationship of inward IATP (ATP efflux) and outward VRAC in MKs isolated from Lrrc8afl/fl (WT) mice induced by HYPO (210 mOsm) swelling with an intracellular ATP concentration of 1 mM (A) or 50 mM (B) followed by application of 10 μM SN-401 (DCPIB), and in MKs isolated from Pf4-Cre;Lrrc8afl/fl (cKO) mice (C) with an intracellular ATP concentration of 50 mM. The inward component of the current represents IATP generated from ATP efflux. (D-F) Respective current-voltage relationships of inward IATP (ATP efflux) and outward VRAC elicited from voltage ramps from −140 mV to +80 mV. (G) Mean current densities of inward IATP (ATP efflux) at −140 mV in MKs isolated from WT mice after HYPO swelling, with an intracellular concentration of 1 mM ATP (n = 7) or 50 mM ATP (n = 6), and inhibition of ATP efflux by 10 μM SN-401 (n = 7 for 1 mM ATP + SN-401; n = 6 for 50 mM ATP + SN-401), and mean current densities of inward IATP in MKs isolated from cKO mice after HYPO swelling, with an intracellular concentration of 50 mM ATP (n = 7). (H-I) Current-voltage relationship of inward IATP (ATP efflux) and outward VRAC in MEG-01 cells elicited from voltage ramps from −140 mV to +80 mV before and after HYPO swelling with an intracellular ATP concentration of 1 mM (H) or 50 mM (I), followed by application of 10 μM SN-401 (DCPIB). The inward component of the current represents IATP generated from ATP efflux. (J) Mean current densities of inward IATP (ATP efflux) in MEG-01 cells at −140 mV after HYPO swelling, with an intracellular concentration of 1 mM ATP (n = 5) or 50 mM ATP (n = 4), and inhibition of ATP efflux by 10 μM SN-401 (n = 5 for 1 mM ATP + SN-401; n = 4 for 50 mM ATP + SN-401). (K-L) Current-time relationship of inward IATP and outward VRAC induced by HYPO (210 mOsm) swelling in MEG-01 cells transduced with adenoviral shSCR (K) or shLRRC8A (L). (M) Current-voltage relationship of inward IATP (ATP efflux) and outward VRAC during voltage ramps from −140 mV to +80 mV after HYPO swelling in MEG-01 cells treated with either shSCR or shLRRC8A. (N) Mean current densities of inward IATP (ATP efflux) in MEG-01 cells treated with shSCR (n = 7) or shLRRC8A (n = 7) at −140 mV after HYPO swelling. Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test for panels G,J,N. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. shLRRC8A, short hairpin RNA–targeting LRRC8A; shSCR, short hairpin control.

MK LRRC8 channels permeate ATP. (A-C) Current-time relationship of inward IATP (ATP efflux) and outward VRAC in MKs isolated from Lrrc8afl/fl (WT) mice induced by HYPO (210 mOsm) swelling with an intracellular ATP concentration of 1 mM (A) or 50 mM (B) followed by application of 10 μM SN-401 (DCPIB), and in MKs isolated from Pf4-Cre;Lrrc8afl/fl (cKO) mice (C) with an intracellular ATP concentration of 50 mM. The inward component of the current represents IATP generated from ATP efflux. (D-F) Respective current-voltage relationships of inward IATP (ATP efflux) and outward VRAC elicited from voltage ramps from −140 mV to +80 mV. (G) Mean current densities of inward IATP (ATP efflux) at −140 mV in MKs isolated from WT mice after HYPO swelling, with an intracellular concentration of 1 mM ATP (n = 7) or 50 mM ATP (n = 6), and inhibition of ATP efflux by 10 μM SN-401 (n = 7 for 1 mM ATP + SN-401; n = 6 for 50 mM ATP + SN-401), and mean current densities of inward IATP in MKs isolated from cKO mice after HYPO swelling, with an intracellular concentration of 50 mM ATP (n = 7). (H-I) Current-voltage relationship of inward IATP (ATP efflux) and outward VRAC in MEG-01 cells elicited from voltage ramps from −140 mV to +80 mV before and after HYPO swelling with an intracellular ATP concentration of 1 mM (H) or 50 mM (I), followed by application of 10 μM SN-401 (DCPIB). The inward component of the current represents IATP generated from ATP efflux. (J) Mean current densities of inward IATP (ATP efflux) in MEG-01 cells at −140 mV after HYPO swelling, with an intracellular concentration of 1 mM ATP (n = 5) or 50 mM ATP (n = 4), and inhibition of ATP efflux by 10 μM SN-401 (n = 5 for 1 mM ATP + SN-401; n = 4 for 50 mM ATP + SN-401). (K-L) Current-time relationship of inward IATP and outward VRAC induced by HYPO (210 mOsm) swelling in MEG-01 cells transduced with adenoviral shSCR (K) or shLRRC8A (L). (M) Current-voltage relationship of inward IATP (ATP efflux) and outward VRAC during voltage ramps from −140 mV to +80 mV after HYPO swelling in MEG-01 cells treated with either shSCR or shLRRC8A. (N) Mean current densities of inward IATP (ATP efflux) in MEG-01 cells treated with shSCR (n = 7) or shLRRC8A (n = 7) at −140 mV after HYPO swelling. Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test for panels G,J,N. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. shLRRC8A, short hairpin RNA–targeting LRRC8A; shSCR, short hairpin control.

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