Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP2) - A study of channel rundown and reactivation

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6.2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K-ATP channels by adenine nucleotides, pho sphatidylinositol bisphosphate (PIP2), and phosphorylation. Upon excision o f inside-out patches into a Ca2+ and MgATP-Tree solution, the activity of K ir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minu te, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. T hus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while s low rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels a nd is due, at least in part, to Kir6.2 alone. Kir6.2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Ex cising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation c onferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca2+ accelerated thi s process, suggesting a role for PIP, hydrolysis mediated by a Ca2+-depende nt phospholipase C. PIP, could reactivate channel activity after a brief ex posure to Ca2+, but not after prolonged exposure. However, in both cases, P IPE reversed the increase in ATP sensitivity, indicating that PIP2 lowers t he ATP sensitivity by increasing P-o as well as by decreasing the channel a ffinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgA DP stimulation and sulfonylurea inhibition, suggesting functional uncouplin g of SUR1 from Kir6.2-GFP. Ca2+ facilitated the loss of sensitivity to MgAD P, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIPE and M gADP. Phosphorylation of Kir6.2 may thus be required for the channel to rem ain PIP2 responsive, while phosphorylation of Kir6.2 and/or SUR1 is require d for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP, and phos phorylation.