کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1944968 | 1053247 | 2009 | 11 صفحه PDF | دانلود رایگان |
Investigation of the mechanistic bases and physiological importance of cAMP regulation of HCN channels has exploited an arginine to glutamate mutation in the nucleotide-binding fold, an approach critically dependent on the mutation selectively lowering the channel's nucleotide affinity. In apparent conflict with this, in intact Xenopus oocytes, HCN and HCN-RE channels exhibit qualitatively and quantitatively distinct responses to the tyrosine kinase inhibitor, genistein — the estrogenic isoflavonoid strongly depolarizes the activation mid-point of HCN1-R538E, but not HCN1 channels (+ 9.8 mV ± 0.9 versus + 2.2 mV ± 0.6) and hyperpolarizes gating of HCN2 (− 4.8 mV ± 1.0) but depolarizes gating of HCN2-R591E (+ 13.2 mV ± 2.1). However, excised patch recording, X-ray crystallography and modeling reveal that this is not due to either a fundamental effect of the mutation on channel gating per se or of genistein acting as a mutation-sensitive partial agonist at the cAMP site. Rather, we find that genistein equivalently moves both HCN and HCN-RE channels closer to the open state (rendering the channels inherently easier to open but at a cost of decreasing the coupling energy of cAMP) and that the anomaly reflects a balance of these energetic effects with the isoform-specific inhibition of activation by the nucleotide gating ring and relief of this by endogenous cAMP. These findings have specific implications with regard to findings based on HCN-RE channels and kinase antagonists and general implications with respect to interpretation of drug effects in mutant channel backgrounds.
Journal: Biochimica et Biophysica Acta (BBA) - Biomembranes - Volume 1788, Issue 9, September 2009, Pages 1939–1949