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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 sensitivity of the pinacidil DMR in C3A cells to kinase inhibition.(a) The 32 μM pinacidil DMR amplitudes at 50 min post stimulation were plotted as a function of compound. The pinacidil DMR was obtained after pretreatment with inhibitors, all at 10 μM, for 1 hr. (b) The dose-dependent inhibition of the 32 μM pinacidil DMR amplitudes at 50 min post stimulation by H-89 and H-7. Data represents mean ± s.d. (n = 4 for all).
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f6: The sensitivity of the pinacidil DMR in C3A cells to kinase inhibition.(a) The 32 μM pinacidil DMR amplitudes at 50 min post stimulation were plotted as a function of compound. The pinacidil DMR was obtained after pretreatment with inhibitors, all at 10 μM, for 1 hr. (b) The dose-dependent inhibition of the 32 μM pinacidil DMR amplitudes at 50 min post stimulation by H-89 and H-7. Data represents mean ± s.d. (n = 4 for all).

Mentions: The regulation of KATP channels has been extensively investigated; in particular, its upstream regulation mediated by protein kinase A (PKA) has been well documented49505152. The phosphorylation of ion channels by protein kinases is an important mechanism by which membrane excitability is regulated by signaling pathways. However, a clear understanding about the signaling of KATP is still lacking. Thus, we investigated the impact of a library of known kinase inhibitors on the pinacidil DMR in C3A cells. Results showed that the majority of kinase inhibitors had little impact on the pinacidil DMR (Fig. 6a). However, a subset of inhibitors markedly suppressed the DMR of pinacidil. Among them, H-89, HA-1077, H-7, and H-8 are known PKA inhibitors, while AG490 is a putative Janus activated kinase (JAK) inhibitor, Y27632 is a ROCK inhibitor, and the alkaloid staurosporine is a broad spectrum, high affinity kinase inhibitor with highest affinity for protein kinase C (0.7 nM). DMR inhibition assay showed that H-89 and H-7 dose-dependently and completely blocked the DMR of 32 μM pinacidil, leading to a logIC50 of −5.32 ± 0.09 and −5.17 ± 0.10, respectively (Fig. 6b). The sensitivity of the pinacidil DMR to these PKA inhibitors were consistent with the previous findings, which suggest that PKA is anchored in proximity to KATP channels in the caveolae49, and more than one site in Kir6.2 (e.g., S372 or T224 in Kir6.2)51 and SUR (e.g., T633 and S1465 in SUR2B)49 have been implicated in PKA phosphorylation. The positive regulation of KATP by PKA was evidenced by the observed attenuation of KATP channel currents in the presence of PKA inhibitors49505152. Of note, compounds containing 5-isoquinolinsulfonyl moieties such as H-89 also bind directly to the SUR subunit but with relatively low potency53. At the concentration range tested it is not possible to distinguish between direct blocking of the KATP channel and indirect inhibition through abrogation of PKA activity. The inhibition of the pinacidil DMR by staurosporine may also be linked to PKA (Ki ~ 7 nM), but the prevalence of other mechanisms cannot be ruled out5455. Nonetheless, these results suggest that the pinacidil DMR can be modulated by kinase activity.


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 sensitivity of the pinacidil DMR in C3A cells to kinase inhibition.(a) The 32 μM pinacidil DMR amplitudes at 50 min post stimulation were plotted as a function of compound. The pinacidil DMR was obtained after pretreatment with inhibitors, all at 10 μM, for 1 hr. (b) The dose-dependent inhibition of the 32 μM pinacidil DMR amplitudes at 50 min post stimulation by H-89 and H-7. Data represents mean ± s.d. (n = 4 for all).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: The sensitivity of the pinacidil DMR in C3A cells to kinase inhibition.(a) The 32 μM pinacidil DMR amplitudes at 50 min post stimulation were plotted as a function of compound. The pinacidil DMR was obtained after pretreatment with inhibitors, all at 10 μM, for 1 hr. (b) The dose-dependent inhibition of the 32 μM pinacidil DMR amplitudes at 50 min post stimulation by H-89 and H-7. Data represents mean ± s.d. (n = 4 for all).
Mentions: The regulation of KATP channels has been extensively investigated; in particular, its upstream regulation mediated by protein kinase A (PKA) has been well documented49505152. The phosphorylation of ion channels by protein kinases is an important mechanism by which membrane excitability is regulated by signaling pathways. However, a clear understanding about the signaling of KATP is still lacking. Thus, we investigated the impact of a library of known kinase inhibitors on the pinacidil DMR in C3A cells. Results showed that the majority of kinase inhibitors had little impact on the pinacidil DMR (Fig. 6a). However, a subset of inhibitors markedly suppressed the DMR of pinacidil. Among them, H-89, HA-1077, H-7, and H-8 are known PKA inhibitors, while AG490 is a putative Janus activated kinase (JAK) inhibitor, Y27632 is a ROCK inhibitor, and the alkaloid staurosporine is a broad spectrum, high affinity kinase inhibitor with highest affinity for protein kinase C (0.7 nM). DMR inhibition assay showed that H-89 and H-7 dose-dependently and completely blocked the DMR of 32 μM pinacidil, leading to a logIC50 of −5.32 ± 0.09 and −5.17 ± 0.10, respectively (Fig. 6b). The sensitivity of the pinacidil DMR to these PKA inhibitors were consistent with the previous findings, which suggest that PKA is anchored in proximity to KATP channels in the caveolae49, and more than one site in Kir6.2 (e.g., S372 or T224 in Kir6.2)51 and SUR (e.g., T633 and S1465 in SUR2B)49 have been implicated in PKA phosphorylation. The positive regulation of KATP by PKA was evidenced by the observed attenuation of KATP channel currents in the presence of PKA inhibitors49505152. Of note, compounds containing 5-isoquinolinsulfonyl moieties such as H-89 also bind directly to the SUR subunit but with relatively low potency53. At the concentration range tested it is not possible to distinguish between direct blocking of the KATP channel and indirect inhibition through abrogation of PKA activity. The inhibition of the pinacidil DMR by staurosporine may also be linked to PKA (Ki ~ 7 nM), but the prevalence of other mechanisms cannot be ruled out5455. Nonetheless, these results suggest that the pinacidil DMR can be modulated by kinase activity.

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