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Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel.

Sun H, Wei Y, Deng H, Xiong Q, Li M, Lahiri J, Fang Y - Sci Rep (2014)

Bottom Line: Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells.Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling.The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

View Article: PubMed Central - PubMed

Affiliation: 1] Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, NY 14831, United States of America [2].

ABSTRACT
Current technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

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JAK2 and JAK3 were involved in the KATP signaling in C3A cells.(a) The DMR amplitudes of 40 μM pinacidil at 50 min post stimulation as a function of AG490 dose. The cells were treated with AG490 for 1 hr, followed by pinacidil stimulation. (b) The real-time DMR of 40 μM pinacidil in C3A cells with mock transfection (mock) or JAK siRNA. The buffer DMR was included as a negative control. (c) The pinacidil DMR amplitudes at 50 min post stimulation as a function of treatment. (d) Western blot using anti-JAK2 for cell lysate without (−) and with (+) 100 μM pinacidil treatment. (e) Western blot using anti-JAK3 for the lysate of mock transfected cells without (−) and with (+) 100 μM pinacidil treatment. (f) Western blot using anti-JAK3 for the lysate of the Kir6.2 siRNA treated cells without (−) and with (+) 100 μM pinacidil treatment. (g) The relative intensity of JAK3 protein in the mock transfected or the Kir6.2 siRNA treated cells without (Control) and with 100 μM pinacidil treatment (+Pinacidil). Data represents mean ± s.d. (n = 6 for a–c; n = 3 for g).
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f7: JAK2 and JAK3 were involved in the KATP signaling in C3A cells.(a) The DMR amplitudes of 40 μM pinacidil at 50 min post stimulation as a function of AG490 dose. The cells were treated with AG490 for 1 hr, followed by pinacidil stimulation. (b) The real-time DMR of 40 μM pinacidil in C3A cells with mock transfection (mock) or JAK siRNA. The buffer DMR was included as a negative control. (c) The pinacidil DMR amplitudes at 50 min post stimulation as a function of treatment. (d) Western blot using anti-JAK2 for cell lysate without (−) and with (+) 100 μM pinacidil treatment. (e) Western blot using anti-JAK3 for the lysate of mock transfected cells without (−) and with (+) 100 μM pinacidil treatment. (f) Western blot using anti-JAK3 for the lysate of the Kir6.2 siRNA treated cells without (−) and with (+) 100 μM pinacidil treatment. (g) The relative intensity of JAK3 protein in the mock transfected or the Kir6.2 siRNA treated cells without (Control) and with 100 μM pinacidil treatment (+Pinacidil). Data represents mean ± s.d. (n = 6 for a–c; n = 3 for g).

Mentions: Janus kinases (JAK1, 2, 3) are protein tyrosine kinases involved in cytokine mediated cellular signaling and are crucial for a variety of cellular functions including cellular survival, proliferation, differentiation and apoptosis5657. Given the attenuation of the pinacidil DMR by AG490, we examined the role of JAKs in KATP channel signaling using multiple assays. First, DMR inhibition assay showed that AG490 dose-dependently suppressed the pinacidil DMR, yielding a logIC50 of −4.77 ± 0.10 (n = 3) (Fig. 7a). Second, siRNA knockdown studies showed that the treatment with two siRNAs for JAK1 had little impact on the pinacidil DMR, but two siRNAs for JAK2 and two for JAK3 all markedly suppressed the pinacidil DMR (Fig. 7b and c). Third, western blotting showed that C3A cells primarily express JAK3, to less extend JAK2, but not JAK1. Moreover, the treatment of cells with 100 μM pinacidil markedly increased the level of JAK2 (Fig. 7d) and JAK3 (Fig. 7e). Importantly, the Kir6.2 siRNA treatment impaired the pinacidil induced increase of JAK3 protein level (Fig. 7f and g), suggesting that the pinacidil induced increase in JAK3 protein is due to the activation of Kir6.2 KATP. We also attempted, but failed, to detect p-JAK levels in the absence and presence of pinacidil. Several possible mechanisms are accounted for this; one may be due to the poor activity of anti-p-JAK antibodies used; another is that the pinacidil activated KATP did not cause phosphorylation of JAKs, but only increased the expression level of JAKs. Of note, we used moderately high dose (40 μM) of pinacidil for DMR profiling of the effect of kinase inhibitor and RNAi knockdown in order to improve sensitivity, but a higher dose (100 μM) for JAK expression analysis to achieve maximal effect. Nonetheless, these results suggest the important role of JAK3, to less extent JAK2, in the KATP channel signaling in C3A cells.


Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel.

Sun H, Wei Y, Deng H, Xiong Q, Li M, Lahiri J, Fang Y - Sci Rep (2014)

JAK2 and JAK3 were involved in the KATP signaling in C3A cells.(a) The DMR amplitudes of 40 μM pinacidil at 50 min post stimulation as a function of AG490 dose. The cells were treated with AG490 for 1 hr, followed by pinacidil stimulation. (b) The real-time DMR of 40 μM pinacidil in C3A cells with mock transfection (mock) or JAK siRNA. The buffer DMR was included as a negative control. (c) The pinacidil DMR amplitudes at 50 min post stimulation as a function of treatment. (d) Western blot using anti-JAK2 for cell lysate without (−) and with (+) 100 μM pinacidil treatment. (e) Western blot using anti-JAK3 for the lysate of mock transfected cells without (−) and with (+) 100 μM pinacidil treatment. (f) Western blot using anti-JAK3 for the lysate of the Kir6.2 siRNA treated cells without (−) and with (+) 100 μM pinacidil treatment. (g) The relative intensity of JAK3 protein in the mock transfected or the Kir6.2 siRNA treated cells without (Control) and with 100 μM pinacidil treatment (+Pinacidil). Data represents mean ± s.d. (n = 6 for a–c; n = 3 for g).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4017216&req=5

f7: JAK2 and JAK3 were involved in the KATP signaling in C3A cells.(a) The DMR amplitudes of 40 μM pinacidil at 50 min post stimulation as a function of AG490 dose. The cells were treated with AG490 for 1 hr, followed by pinacidil stimulation. (b) The real-time DMR of 40 μM pinacidil in C3A cells with mock transfection (mock) or JAK siRNA. The buffer DMR was included as a negative control. (c) The pinacidil DMR amplitudes at 50 min post stimulation as a function of treatment. (d) Western blot using anti-JAK2 for cell lysate without (−) and with (+) 100 μM pinacidil treatment. (e) Western blot using anti-JAK3 for the lysate of mock transfected cells without (−) and with (+) 100 μM pinacidil treatment. (f) Western blot using anti-JAK3 for the lysate of the Kir6.2 siRNA treated cells without (−) and with (+) 100 μM pinacidil treatment. (g) The relative intensity of JAK3 protein in the mock transfected or the Kir6.2 siRNA treated cells without (Control) and with 100 μM pinacidil treatment (+Pinacidil). Data represents mean ± s.d. (n = 6 for a–c; n = 3 for g).
Mentions: Janus kinases (JAK1, 2, 3) are protein tyrosine kinases involved in cytokine mediated cellular signaling and are crucial for a variety of cellular functions including cellular survival, proliferation, differentiation and apoptosis5657. Given the attenuation of the pinacidil DMR by AG490, we examined the role of JAKs in KATP channel signaling using multiple assays. First, DMR inhibition assay showed that AG490 dose-dependently suppressed the pinacidil DMR, yielding a logIC50 of −4.77 ± 0.10 (n = 3) (Fig. 7a). Second, siRNA knockdown studies showed that the treatment with two siRNAs for JAK1 had little impact on the pinacidil DMR, but two siRNAs for JAK2 and two for JAK3 all markedly suppressed the pinacidil DMR (Fig. 7b and c). Third, western blotting showed that C3A cells primarily express JAK3, to less extend JAK2, but not JAK1. Moreover, the treatment of cells with 100 μM pinacidil markedly increased the level of JAK2 (Fig. 7d) and JAK3 (Fig. 7e). Importantly, the Kir6.2 siRNA treatment impaired the pinacidil induced increase of JAK3 protein level (Fig. 7f and g), suggesting that the pinacidil induced increase in JAK3 protein is due to the activation of Kir6.2 KATP. We also attempted, but failed, to detect p-JAK levels in the absence and presence of pinacidil. Several possible mechanisms are accounted for this; one may be due to the poor activity of anti-p-JAK antibodies used; another is that the pinacidil activated KATP did not cause phosphorylation of JAKs, but only increased the expression level of JAKs. Of note, we used moderately high dose (40 μM) of pinacidil for DMR profiling of the effect of kinase inhibitor and RNAi knockdown in order to improve sensitivity, but a higher dose (100 μM) for JAK expression analysis to achieve maximal effect. Nonetheless, these results suggest the important role of JAK3, to less extent JAK2, in the KATP channel signaling in C3A cells.

Bottom Line: Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells.Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling.The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

View Article: PubMed Central - PubMed

Affiliation: 1] Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, NY 14831, United States of America [2].

ABSTRACT
Current technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

Show MeSH
Related in: MedlinePlus