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, phosphatidylinositol bisphosphate (PIP₂), and phosphorylation. Upon excision of inside-out patches into a Ca²⁺- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, 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. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and 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. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred 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 this process, suggesting a role for PIP₂ hydrolysis mediated by a Ca²⁺-dependent phospholipase C. PIP₂ could reactivate channel activity after a brief exposure to Ca²⁺, but not after prolonged exposure. However, in both cases, PIP₂ reversed the increase in ATP sensitivity, indicating that PIP₂ lowers the ATP sensitivity by increasing P_o as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca²⁺ facilitated the loss of sensitivity to MgADP, 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 PIP₂ and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP₂ responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP₂ and phosphorylation.