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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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mRNA expression patterns of KATP channels in different cell lines.(a) C3A; (b) A549; (c) A431; (d) HT29 cells.
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f4: mRNA expression patterns of KATP channels in different cell lines.(a) C3A; (b) A549; (c) A431; (d) HT29 cells.

Mentions: Second, we performed reverse transcriptase PCR to determine the mRNA expression of KATP channels. Results showed that C3A endogenously expresses mRNAs for SUR2B, Kir6.1, and Kir6.2, to less extent SUR2A, but not SUR1 (Fig. 4a). A549 expresses mRNAs for SUR1, Kir6.1 and Kir6.2, but not SUR2A and SUR2B (Fig. 4b). A431 expresses mRNAs for SUR2A, Kir6.1 and Kir6.2, but not SUR1 and SUR2B (Fig. 4c). HT29 expresses mRNAs for primarily SUR2B, Kir6.1 and Kir6.2, and to less extent SUR1 and SUR2A (Fig. 4d). These results suggest that different cell lines have different expression patterns of KATP channels.


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)

mRNA expression patterns of KATP channels in different cell lines.(a) C3A; (b) A549; (c) A431; (d) HT29 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4017216&req=5

f4: mRNA expression patterns of KATP channels in different cell lines.(a) C3A; (b) A549; (c) A431; (d) HT29 cells.
Mentions: Second, we performed reverse transcriptase PCR to determine the mRNA expression of KATP channels. Results showed that C3A endogenously expresses mRNAs for SUR2B, Kir6.1, and Kir6.2, to less extent SUR2A, but not SUR1 (Fig. 4a). A549 expresses mRNAs for SUR1, Kir6.1 and Kir6.2, but not SUR2A and SUR2B (Fig. 4b). A431 expresses mRNAs for SUR2A, Kir6.1 and Kir6.2, but not SUR1 and SUR2B (Fig. 4c). HT29 expresses mRNAs for primarily SUR2B, Kir6.1 and Kir6.2, and to less extent SUR1 and SUR2A (Fig. 4d). These results suggest that different cell lines have different expression patterns of KATP channels.

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