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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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The RNAi knockdown effect on the DMR of 40 μM pinacidil in C3A cells.(a,d) The DMR of pinacidil in untreated, mock transfection treated (mock) and scrambled RNAi (sc. RNAi) treated C3A cells, in comparison with the negative control. (b) The DMR of pinacidil in the mock transfection and three SUR1 RNAi treated cells. (c) The DMR of pinacidil in the mock transfection and three SUR2 RNAi treated cells. (e) The DMR of pinacidil in the mock transfection and three Kir6.1 RNAi treated cells. (f) The DMR of pinacidil in the mock transfection and three Kir6.2 RNAi treated cells. (g,h) statistical analysis of the effects of RNAi knockdown on the pinacidil DMR. *, p < 0.05; ***, p < 0.001. (i,j) The DMR of 32 μM pinacidil in C3A cells as a function of KATP blockers. The cells were first treated with each blocker at different doses for 1 hr, followed by stimulation with 32 μM pinacidil. (k) The impact of co-existence of 40 μM tolazamide on the potency of pinacidil. For (g–k), the pinacidil DMR amplitudes at 50 min post stimulation were plotted. Data represents mean ± s.d. (n = 6 for a–h; n = 3 for i–k).
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f5: The RNAi knockdown effect on the DMR of 40 μM pinacidil in C3A cells.(a,d) The DMR of pinacidil in untreated, mock transfection treated (mock) and scrambled RNAi (sc. RNAi) treated C3A cells, in comparison with the negative control. (b) The DMR of pinacidil in the mock transfection and three SUR1 RNAi treated cells. (c) The DMR of pinacidil in the mock transfection and three SUR2 RNAi treated cells. (e) The DMR of pinacidil in the mock transfection and three Kir6.1 RNAi treated cells. (f) The DMR of pinacidil in the mock transfection and three Kir6.2 RNAi treated cells. (g,h) statistical analysis of the effects of RNAi knockdown on the pinacidil DMR. *, p < 0.05; ***, p < 0.001. (i,j) The DMR of 32 μM pinacidil in C3A cells as a function of KATP blockers. The cells were first treated with each blocker at different doses for 1 hr, followed by stimulation with 32 μM pinacidil. (k) The impact of co-existence of 40 μM tolazamide on the potency of pinacidil. For (g–k), the pinacidil DMR amplitudes at 50 min post stimulation were plotted. Data represents mean ± s.d. (n = 6 for a–h; n = 3 for i–k).

Mentions: Third, we applied RNAi knockdown to determine the functional KATP channels in C3A cells. Results showed that the mock or scrambled RNAi transfection had little impact on the pinacidil DMR (Fig. 5a). The treatment with three RNAi against SUR1 had little impact on the pinacidil DMR (Fig. 5b), consistent with the absence of SUR1 mRNA in C3A cells. However, knockdown of SUR2 with three RNAi all markedly suppressed the pinacidil DMR (Fig. 5c). Similar to the mock or scrambled RNAi transfection (Fig. 5d), three RNAi knockdown of Kir6.1 had little impact on the pinacidil DMR (Fig. 5e). In contrast, the knockdown of Kir6.2 with three RNAi all markedly suppressed the pinacidil DMR (Fig. 5f). One-way ANOVA analysis suggests that RNAi for SUR2 and Kir6.2, but neither SUR1 or Kir6.1, significantly altered the pinacidil DMR (Fig. 5g and h). Given the low expression level of these proteins and the moderate efficiency of typical RNAi knockdown in our laboratory26 (also see below), we didnot attempt to use Western blot to quantify the knockdown efficiency of respective RNAi.


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)

The RNAi knockdown effect on the DMR of 40 μM pinacidil in C3A cells.(a,d) The DMR of pinacidil in untreated, mock transfection treated (mock) and scrambled RNAi (sc. RNAi) treated C3A cells, in comparison with the negative control. (b) The DMR of pinacidil in the mock transfection and three SUR1 RNAi treated cells. (c) The DMR of pinacidil in the mock transfection and three SUR2 RNAi treated cells. (e) The DMR of pinacidil in the mock transfection and three Kir6.1 RNAi treated cells. (f) The DMR of pinacidil in the mock transfection and three Kir6.2 RNAi treated cells. (g,h) statistical analysis of the effects of RNAi knockdown on the pinacidil DMR. *, p < 0.05; ***, p < 0.001. (i,j) The DMR of 32 μM pinacidil in C3A cells as a function of KATP blockers. The cells were first treated with each blocker at different doses for 1 hr, followed by stimulation with 32 μM pinacidil. (k) The impact of co-existence of 40 μM tolazamide on the potency of pinacidil. For (g–k), the pinacidil DMR amplitudes at 50 min post stimulation were plotted. Data represents mean ± s.d. (n = 6 for a–h; n = 3 for i–k).
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Related In: Results  -  Collection

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f5: The RNAi knockdown effect on the DMR of 40 μM pinacidil in C3A cells.(a,d) The DMR of pinacidil in untreated, mock transfection treated (mock) and scrambled RNAi (sc. RNAi) treated C3A cells, in comparison with the negative control. (b) The DMR of pinacidil in the mock transfection and three SUR1 RNAi treated cells. (c) The DMR of pinacidil in the mock transfection and three SUR2 RNAi treated cells. (e) The DMR of pinacidil in the mock transfection and three Kir6.1 RNAi treated cells. (f) The DMR of pinacidil in the mock transfection and three Kir6.2 RNAi treated cells. (g,h) statistical analysis of the effects of RNAi knockdown on the pinacidil DMR. *, p < 0.05; ***, p < 0.001. (i,j) The DMR of 32 μM pinacidil in C3A cells as a function of KATP blockers. The cells were first treated with each blocker at different doses for 1 hr, followed by stimulation with 32 μM pinacidil. (k) The impact of co-existence of 40 μM tolazamide on the potency of pinacidil. For (g–k), the pinacidil DMR amplitudes at 50 min post stimulation were plotted. Data represents mean ± s.d. (n = 6 for a–h; n = 3 for i–k).
Mentions: Third, we applied RNAi knockdown to determine the functional KATP channels in C3A cells. Results showed that the mock or scrambled RNAi transfection had little impact on the pinacidil DMR (Fig. 5a). The treatment with three RNAi against SUR1 had little impact on the pinacidil DMR (Fig. 5b), consistent with the absence of SUR1 mRNA in C3A cells. However, knockdown of SUR2 with three RNAi all markedly suppressed the pinacidil DMR (Fig. 5c). Similar to the mock or scrambled RNAi transfection (Fig. 5d), three RNAi knockdown of Kir6.1 had little impact on the pinacidil DMR (Fig. 5e). In contrast, the knockdown of Kir6.2 with three RNAi all markedly suppressed the pinacidil DMR (Fig. 5f). One-way ANOVA analysis suggests that RNAi for SUR2 and Kir6.2, but neither SUR1 or Kir6.1, significantly altered the pinacidil DMR (Fig. 5g and h). Given the low expression level of these proteins and the moderate efficiency of typical RNAi knockdown in our laboratory26 (also see below), we didnot attempt to use Western blot to quantify the knockdown efficiency of respective RNAi.

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