Figure 6.
LRRC8 channel small-molecule inhibitors suppress agonist-induced aggregation, ATP release, and calcium influx. (A) Molecular structure of the small-molecule LRRC8 channel complex modulator SN-401 (DCPIB) and SN-406, a derivative of SN-401. (B) Current-time relationship of inward IATP (ATP efflux) and outward VRAC induced by HYPO swelling in MEG-01 cells and subsequent inhibition by application of 10 μM SN-406. (C-D) Current-voltage relationship of inward IATP (ATP efflux) and outward VRAC during voltage ramps from −140 mV to +80 mV in MEG-01 cells after HYPO swelling in the absence or presence of 10 μM SN-406 (C), with mean current densities of inward IATP at −140 mV (n = 7-15) (D). (E-H) Aggregometry of platelets isolated from WT C57BL/6J mice stimulated with Thr (0.02 U/mL) in the presence of vehicle (0.02% dimethyl sulfoxide [DMSO]; n = 37), SN-401 (10 μM; n = 7), or SN-406 (10 μM; n = 5) (E), with concurrent ATP release (n = 5-30) (F-H). (I) Cytosolic calcium measured using the ratiometric dye Fura-2 in platelets isolated from WT C57BL/6J mice stimulated with PAR4-AP (400 μM; n = 8) in the presence of vehicle only (0.02% DMSO) or SN-406 (10 μM). (J-K) Aggregometry of human platelets stimulated with PAR1-AP and PAR4-AP (10 μM and 100 μM, respectively) in the presence of vehicle (0.02% DMSO; n = 10 from 3 healthy volunteers) or SN-406 (10 μM; n = 11 from 3 healthy volunteers) (J), with measurements of cytosolic calcium (K). (L) Aggregometry of PAR4-AP (400 μM) stimulated mouse platelets in the presence of vehicle (0.02% DMSO; n = 35), and SN-406 at 0.1 μM (n = 5), 1 μM (n = 5), or 10 μM (n = 6). (M) SN-406 dose-dependent inhibition of PAR4-AP (400 μM) stimulated mouse platelet aggregation (SN-406: 0-10 μM; n = 5-6). (N) Aggregometry of platelets isolated from Pf4-Cre;Lrrc8afl/fl (cKO) and Lrrc8afl/fl (WT) littermate controls stimulated by PAR4-AP (400 μM) in the presence of vehicle (0.02% DMSO; n = 5 for WT; n = 7 for cKO) or 1.1 μM SN-406 (n = 6 for WT; n = 8 for cKO). (O) Current-time relationship of inward IATP and outward VRAC induced by HYPO (210 mOsm) swelling in MEG-01 cells and subsequent inhibition by application of 10 μM dicoumarol. (P-Q) Current-voltage relationship of inward IATP and outward VRAC during voltage ramps from −140 mV to +80 mV in MEG-01 cells after HYPO swelling in the absence or presence of 10 μM dicoumarol (P), with mean current densities of inward IATP –140 mV (Q) (n = 4 for both groups). (R) Aggregometry of platelets isolated from WT C57BL/6J mice stimulated with PAR4-AP (400 μM) in the presence of vehicle (0.1% DMSO; n = 4) or dicoumarol (20 μM; n = 3), with subsequent application of ATP (40 μM). Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test for panels D,G,H,Q; and by 2-way ANOVA for E, F, I-K,N,R. Thr used in this experiment was sourced from the Chrono-Log Corporation. Representative traces for panels E-F,J,N,R are provided in supplemental Figure 10. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. a.u., arbitrary unit.

LRRC8 channel small-molecule inhibitors suppress agonist-induced aggregation, ATP release, and calcium influx. (A) Molecular structure of the small-molecule LRRC8 channel complex modulator SN-401 (DCPIB) and SN-406, a derivative of SN-401. (B) Current-time relationship of inward IATP (ATP efflux) and outward VRAC induced by HYPO swelling in MEG-01 cells and subsequent inhibition by application of 10 μM SN-406. (C-D) Current-voltage relationship of inward IATP (ATP efflux) and outward VRAC during voltage ramps from −140 mV to +80 mV in MEG-01 cells after HYPO swelling in the absence or presence of 10 μM SN-406 (C), with mean current densities of inward IATP at −140 mV (n = 7-15) (D). (E-H) Aggregometry of platelets isolated from WT C57BL/6J mice stimulated with Thr (0.02 U/mL) in the presence of vehicle (0.02% dimethyl sulfoxide [DMSO]; n = 37), SN-401 (10 μM; n = 7), or SN-406 (10 μM; n = 5) (E), with concurrent ATP release (n = 5-30) (F-H). (I) Cytosolic calcium measured using the ratiometric dye Fura-2 in platelets isolated from WT C57BL/6J mice stimulated with PAR4-AP (400 μM; n = 8) in the presence of vehicle only (0.02% DMSO) or SN-406 (10 μM). (J-K) Aggregometry of human platelets stimulated with PAR1-AP and PAR4-AP (10 μM and 100 μM, respectively) in the presence of vehicle (0.02% DMSO; n = 10 from 3 healthy volunteers) or SN-406 (10 μM; n = 11 from 3 healthy volunteers) (J), with measurements of cytosolic calcium (K). (L) Aggregometry of PAR4-AP (400 μM) stimulated mouse platelets in the presence of vehicle (0.02% DMSO; n = 35), and SN-406 at 0.1 μM (n = 5), 1 μM (n = 5), or 10 μM (n = 6). (M) SN-406 dose-dependent inhibition of PAR4-AP (400 μM) stimulated mouse platelet aggregation (SN-406: 0-10 μM; n = 5-6). (N) Aggregometry of platelets isolated from Pf4-Cre;Lrrc8afl/fl (cKO) and Lrrc8afl/fl (WT) littermate controls stimulated by PAR4-AP (400 μM) in the presence of vehicle (0.02% DMSO; n = 5 for WT; n = 7 for cKO) or 1.1 μM SN-406 (n = 6 for WT; n = 8 for cKO). (O) Current-time relationship of inward IATP and outward VRAC induced by HYPO (210 mOsm) swelling in MEG-01 cells and subsequent inhibition by application of 10 μM dicoumarol. (P-Q) Current-voltage relationship of inward IATP and outward VRAC during voltage ramps from −140 mV to +80 mV in MEG-01 cells after HYPO swelling in the absence or presence of 10 μM dicoumarol (P), with mean current densities of inward IATP –140 mV (Q) (n = 4 for both groups). (R) Aggregometry of platelets isolated from WT C57BL/6J mice stimulated with PAR4-AP (400 μM) in the presence of vehicle (0.1% DMSO; n = 4) or dicoumarol (20 μM; n = 3), with subsequent application of ATP (40 μM). Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test for panels D,G,H,Q; and by 2-way ANOVA for E, F, I-K,N,R. Thr used in this experiment was sourced from the Chrono-Log Corporation. Representative traces for panels E-F,J,N,R are provided in supplemental Figure 10. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. a.u., arbitrary unit.

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