Barth D and Fronius M (2019), Shear force modulates the activity of acid-sens...

XB-ART-55930

Sci Rep 2019 May 01;91:6781. doi: 10.1038/s41598-019-43097-7.

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Shear force modulates the activity of acid-sensing ion channels at low pH or in the presence of non-proton ligands.

Barth D , Fronius M .

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Acid-sensing ion channels (ASICs) belong to the degenerin/epithelial sodium channel protein family that form mechanosensitive ion channels. Evidence as to whether or not ASICs activity is directly modulated by mechanical force is lacking. Human ASICs (hASIC1V3, hASIC2a and hASIC3a) were heterologously expressed as homomeric channels in Xenopus oocytes and two-electrode voltage-clamp recordings were performed. hASIC3a was expressed in HEK-293 cells and currents measured by whole-cell patch-clamp recordings. ASIC currents in response to shear force (SF) were measured at pH 7.4, acidic pH, or in the presence of non-proton ligands at pH 7.4. SF was applied via a fluid stream generated through a pressurized perfusion system. No effect was observed at pH 7.4. Increased transient currents for each homomeric channel were observed when elevated SF was applied in conjunction with acidic pH (6.0-4.0). The sustained current was not (hASIC2a) or only slightly increased (hASIC1V3 and hASIC3a). SF-induced effects were not seen in water injected oocytes and were blocked by amiloride. Non-proton ligands activated a persistent current in hASIC1V3 and cASIC1 (MitTx) and hASIC3a (GMQ) at pH 7.4. Here SF caused a further current increase. Results suggest that ASICs do have an intrinsic ability to respond to mechanical force, supporting their role as mechanosensors in certain local environments.

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Species referenced: Xenopus laevis
Genes referenced: asic1 asic2

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	Figure 1. Illustration of SF application via two different systems. (A) Oocytes were placed in a custom made flow chamber where SF and acidic pH was applied at the same time via a pressurized perfusion system. (B) Oocytes were placed in 96 well plates where SF and acidic pH was applied via a tube directed at the oocyte.
	Figure 2. SF applied at pH 7.4 does not activate ASICs. Voltage-clamp experiments of ASIC expressing oocytes. Low (0.1 dyn/cm2) and high (0.35 dyn/cm2) SF rates were applied at normal pH conditions (pH 7.4) followed by pH 5 to confirm ASIC expression. Current trace and statistical analysis of hASIC1V3 at pH 7.4 when exposed from 0 to 0.1 dyn/cm2 SF (light blue) (A) and 0 to 0.35 dyn/cm2 (dark blue) (B). No change in transmembrane current was observed. The same results were observed for hASIC2a (C,D) and hASIC3a (E,F). Paired t test; ns: p > 0.05; n = 6–13.
	Figure 3. SF modulates the pH-induced activation of hASIC1V3. Oocytes were perfused with ORi (oocyte ringer’s solution) at pH 7.4 unless otherwise stated. (A) Representative current trace of hASIC1V3 being activated at acidic pH (application indicated by black bars) during low (0.1 dyn/cm2, light blue bar) and elevated (0.35 dyn/cm2, blue bar) SF conditions. (B) Summarized data of the pH-induced transient currents. (C) Analyses of the AUC and (D) the sustained currents. Transient (E) and sustained (E’) currents exhibited by pH 5 and SF are decreased by amiloride (0.1 and 1 mM). Two-way ANOVA; p < 0.05; p < 0.01; **p < 0.001; n = 8–13.
	Figure 4. SF affects activation/inactivation kinetics and is ASIC specific. (A) Activation kinetics of acidic pH-induced currents in ASIC1V3 are faster with elevated SF. (B) Inactivation of pH-induced currents are faster with elevated SF. (C) Comparison of pH 5 evoked currents of hASIC1V3 injected oocytes were elevated at high SF (blue) compared to low SF (light blue) when incubated in Ca2+ free ORi or clamped at the calculated Cl− reversal potential (−20 mV). (D) It may be noted that the small pH-induced currents observed with pH4.5 and 4 in water-injected oocytes are in agreement with previous reports53. However, these pH-induced responses were not influenced by SF. (A,B,D): two-way ANOVA ns: p > 0.05, ***p < 0.0001; (C) paired t test: p < 0.05; p < 0.01; *p < 0.001; n ≥ 6.
	Figure 5. The activity of hASIC2a is modulated by SF. (A) Current trace of hASIC2a activity in response to acidic pH, during low (0.1 dyn/cm2) and elevated (0.35 dyn/cm2) SF. Comparison of the transient current (B), AUC (C) and the sustained current (D). Transient (E) and sustained (E’) ASIC2a currents evoked by pH 5 and SF is blocked by amiloride (0.1 and 1 mM). Two-way ANOVA; ns: p > 0.05; p < 0.01; **p < 0.0001; n = 8).
	Figure 6. SF modulates the pH-induced activation of hASIC3a. (A) Current trace of hASIC3a being activated by acidic pH (black bars) during low (0.1 dyn/cm2) and elevated (0.35 dyn/cm2) SF conditions. Statistical quantification of the transient current (B) and AUC (C) during hASIC3a activation via different acidic pH and SF conditions. 0.35 dyn/cm2 SF leads to a stronger activation of hASIC3a compared to 0.1 dyn/cm2 under acidic pH conditions. (D) Statistical quantification of the sustained current of hASIC3a during different pH and SF conditions showed a significant increased sustained current when exposed to elevated SF (0.35 dyn/cm2) compared to low SF (0.1 dyn/cm2). (E,E’) hASIC3a currents evoked by pH 5 at low and high SF were blocked by amiloride. Two-way ANOVA analysis; p < 0.05; *p < 0.01; n = 6–9.
	Figure 7. SF modulates the pH-induced activation of hASIC3a in HEK-293 cells. HEK-293 transfected with hASIC3a were perfused with pH 7.4 unless stated otherwise. A low (light blue) and high (blue) SF rate combined with pH 5 was applied via a tube directed at the cell. pH 5-induced currents (A,B) and AUC (C) were significantly increased with high SF compared with low SF. Additional application of 1 mM amiloride (orange bar) in the presence of pH 5 did not induce currents. Paired t test; *p < 0.05; n = 6.
	Figure 8. SF modulation of ASIC activity is reproducible in a different SF application system. Oocytes were perfused with ORi (pH 7.4) unless otherwise stated. A low and high SF rate combined with pH 5 was applied via a tube directed at the oocyte. The transient current and the AUC of hASIC1V3 (A) and hASIC2a (B) activation by pH 5 is significantly increased at the high SF rate. The sustained current was unaffected by different SF rates. (C) pH 5 applied at high SF significantly increased the transient and sustained current of hASIC3a activation as well as the AUC compared with low SF. Paired t test; ns: p > 0.05; p < 0.05; p < 0.01; **p < 0.001; n = 7–11.
	Figure 9. SF increases ASIC currents after pre-incubation with MitTx or GMQ at pH 7.4. Oocytes were perfused with ORi at pH 7.4 in absence or presence of MitTx (20 nM) or GMQ (1 mM). SF (0.35 dyn/cm2, blue bar) was applied via the perfusion system and initially used for 1 min to wash in MitTx or GMQ followed by a stop of the perfusion (no SF). After reaching a plateau, SF was applied again and the values before and after SF application were used for statistical analysis. 1 mM Amiloride (ami, black bar) was used to block ASIC mediated currents. Oocytes expressing hASIC1V3 (A,B) and cASIC1 (C,D) showed an increase in membrane current after the application of MitTx, which was even further increased by the additional application of SF. (E,F) Similar to MitTx, application of GMQ caused an activation of hASIC3a which was further increased by SF. Paired t test; *p < 0.001; **p < 0.0001; n = 10–20.

References [+] :

Alijevic, Subtype-specific modulation of acid-sensing ion channel (ASIC) function by 2-guanidine-4-methylquinazoline. 2012, Pubmed, Xenbase

	Figure 1. Illustration of SF application via two different systems. (A) Oocytes were placed in a custom made flow chamber where SF and acidic pH was applied at the same time via a pressurized perfusion system. (B) Oocytes were placed in 96 well plates where SF and acidic pH was applied via a tube directed at the oocyte.
	Figure 2. SF applied at pH 7.4 does not activate ASICs. Voltage-clamp experiments of ASIC expressing oocytes. Low (0.1 dyn/cm2) and high (0.35 dyn/cm2) SF rates were applied at normal pH conditions (pH 7.4) followed by pH 5 to confirm ASIC expression. Current trace and statistical analysis of hASIC1V3 at pH 7.4 when exposed from 0 to 0.1 dyn/cm2 SF (light blue) (A) and 0 to 0.35 dyn/cm2 (dark blue) (B). No change in transmembrane current was observed. The same results were observed for hASIC2a (C,D) and hASIC3a (E,F). Paired t test; ns: p > 0.05; n = 6–13.
	Figure 3. SF modulates the pH-induced activation of hASIC1V3. Oocytes were perfused with ORi (oocyte ringer’s solution) at pH 7.4 unless otherwise stated. (A) Representative current trace of hASIC1V3 being activated at acidic pH (application indicated by black bars) during low (0.1 dyn/cm2, light blue bar) and elevated (0.35 dyn/cm2, blue bar) SF conditions. (B) Summarized data of the pH-induced transient currents. (C) Analyses of the AUC and (D) the sustained currents. Transient (E) and sustained (E’) currents exhibited by pH 5 and SF are decreased by amiloride (0.1 and 1 mM). Two-way ANOVA; p < 0.05; p < 0.01; **p < 0.001; n = 8–13.
	Figure 4. SF affects activation/inactivation kinetics and is ASIC specific. (A) Activation kinetics of acidic pH-induced currents in ASIC1V3 are faster with elevated SF. (B) Inactivation of pH-induced currents are faster with elevated SF. (C) Comparison of pH 5 evoked currents of hASIC1V3 injected oocytes were elevated at high SF (blue) compared to low SF (light blue) when incubated in Ca2+ free ORi or clamped at the calculated Cl− reversal potential (−20 mV). (D) It may be noted that the small pH-induced currents observed with pH4.5 and 4 in water-injected oocytes are in agreement with previous reports53. However, these pH-induced responses were not influenced by SF. (A,B,D): two-way ANOVA ns: p > 0.05, ***p < 0.0001; (C) paired t test: p < 0.05; p < 0.01; *p < 0.001; n ≥ 6.
	Figure 5. The activity of hASIC2a is modulated by SF. (A) Current trace of hASIC2a activity in response to acidic pH, during low (0.1 dyn/cm2) and elevated (0.35 dyn/cm2) SF. Comparison of the transient current (B), AUC (C) and the sustained current (D). Transient (E) and sustained (E’) ASIC2a currents evoked by pH 5 and SF is blocked by amiloride (0.1 and 1 mM). Two-way ANOVA; ns: p > 0.05; p < 0.01; **p < 0.0001; n = 8).
	Figure 6. SF modulates the pH-induced activation of hASIC3a. (A) Current trace of hASIC3a being activated by acidic pH (black bars) during low (0.1 dyn/cm2) and elevated (0.35 dyn/cm2) SF conditions. Statistical quantification of the transient current (B) and AUC (C) during hASIC3a activation via different acidic pH and SF conditions. 0.35 dyn/cm2 SF leads to a stronger activation of hASIC3a compared to 0.1 dyn/cm2 under acidic pH conditions. (D) Statistical quantification of the sustained current of hASIC3a during different pH and SF conditions showed a significant increased sustained current when exposed to elevated SF (0.35 dyn/cm2) compared to low SF (0.1 dyn/cm2). (E,E’) hASIC3a currents evoked by pH 5 at low and high SF were blocked by amiloride. Two-way ANOVA analysis; p < 0.05; *p < 0.01; n = 6–9.
	Figure 7. SF modulates the pH-induced activation of hASIC3a in HEK-293 cells. HEK-293 transfected with hASIC3a were perfused with pH 7.4 unless stated otherwise. A low (light blue) and high (blue) SF rate combined with pH 5 was applied via a tube directed at the cell. pH 5-induced currents (A,B) and AUC (C) were significantly increased with high SF compared with low SF. Additional application of 1 mM amiloride (orange bar) in the presence of pH 5 did not induce currents. Paired t test; *p < 0.05; n = 6.
	Figure 8. SF modulation of ASIC activity is reproducible in a different SF application system. Oocytes were perfused with ORi (pH 7.4) unless otherwise stated. A low and high SF rate combined with pH 5 was applied via a tube directed at the oocyte. The transient current and the AUC of hASIC1V3 (A) and hASIC2a (B) activation by pH 5 is significantly increased at the high SF rate. The sustained current was unaffected by different SF rates. (C) pH 5 applied at high SF significantly increased the transient and sustained current of hASIC3a activation as well as the AUC compared with low SF. Paired t test; ns: p > 0.05; p < 0.05; p < 0.01; **p < 0.001; n = 7–11.
	Figure 9. SF increases ASIC currents after pre-incubation with MitTx or GMQ at pH 7.4. Oocytes were perfused with ORi at pH 7.4 in absence or presence of MitTx (20 nM) or GMQ (1 mM). SF (0.35 dyn/cm2, blue bar) was applied via the perfusion system and initially used for 1 min to wash in MitTx or GMQ followed by a stop of the perfusion (no SF). After reaching a plateau, SF was applied again and the values before and after SF application were used for statistical analysis. 1 mM Amiloride (ami, black bar) was used to block ASIC mediated currents. Oocytes expressing hASIC1V3 (A,B) and cASIC1 (C,D) showed an increase in membrane current after the application of MitTx, which was even further increased by the additional application of SF. (E,F) Similar to MitTx, application of GMQ caused an activation of hASIC3a which was further increased by SF. Paired t test; *p < 0.001; **p < 0.0001; n = 10–20.