ATP Channels Functional Coupling of Kir6.2 and SUR1 Subunits

The sensitivity of K ATP channels to high-affinity block by sulfonylureas and to stimulation by K 1 channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit. Phosphatidylinositol 4,5bisphosphate (PIP 2 ) profoundly antagonized ATP inhibition of K ATP channels expressed from cloned Kir6.2 1 SUR1 subunits, but also abolished high affinity tolbutamide sensitivity. By stabilizing the open state of the channel, PIP 2 drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition. Mutant K ATP channels (Kir6.2[ D N30] or Kir6.2[L164A], coexpressed with SUR1) also displayed an “uncoupled” phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability. Conversely, Kir6.2[R176A] 1 SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block. In addition to antagonizing high-affinity block by tolbutamide, PIP 2 also altered the stimulatory action of the PCOs, diazoxide and MgADP. With time after PIP 2 application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP 2 . The net effect of increasing open state stability, either by PIP 2 or mutagenesis, is an apparent “uncoupling” of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with polyl -lysine. key words: K 1 current • sulfonylurea • MgADP • diazoxide • PIP 2 i n t r o d u c t i o n The 10 yr that followed the discovery of ATP-sensitive (K ATP ) channels (Noma, 1983) led to the delineation of complex regulation by intracellular nucleotides and pharmacological agents (reviewed in Ashcroft, 1988; Nichols and Lederer, 1991; Terzic et al., 1994). The last 3 yr have seen a renewed interest in the regulation of ATP-sensitive potassium channels as a result of the cloning of the constituent subunits (Aguilar-Bryan et al., 1995; Inagaki et al., 1995, 1996). Uniquely, K ATP channels are normally formed as a complex of sulfonylurea receptor (SURx) 1 and inward rectifier (Kir6.x) subunits (Inagaki et al., 1995, 1997; Clement et al., 1997; Shyng and Nichols, 1997). Recent studies demonstrate that the Kir6.x subunits form the pore, and control the hallmark inhibition by ATP (Shyng et al., 1997a; Tucker et al., 1997, 1998; Drain et al., 1998), whereas the SURx subunit controls the sensitivity to the inhibitory sulfonylurea drugs, and to activating nucleotide diphosphates and potassium channel opening drugs (Aguilar-Bryan et al., 1995; Inagaki et al., 1996; Isomoto et al., 1996; Nichols et al., 1996; Shyng et al., 1997b; Gribble et al., 1997a,b; Schwanstecher et al., 1998). Deletion of up to z 36 amino acids from the COOH terminus of Kir6.2 results in the generation of ATP-sensitive channels in the absence of SURx subunits (Tucker et al., 1997; Zerangue et al., 1999), but these channels are not activated by MgADP or potassium channel openers (PCOs), nor are they inhibited at high affinity by sulfonylurea drugs, consistent with these agents acting through the SURx subunit (Gribble et al., 1997a). MgATP clearly binds to the nucleotide binding folds of SUR1 (Ueda et al., 1997), and ATP hydrolysis seems to be required for binding PCOs (Schwanstecher et al., 1998) and transduction (Nichols et al., 1996; Gribble et al., 1997b; Shyng et al., 1997a) of the stimulatory PCO signal to the channel. The physical nature of the coupling of SURx to Kir6.x subunits is essentially unknown at the present time, although intriguingly, Clement et al. (1997) demonstrated that Kir6.2 could be labeled with azido-glibenclamide only in the presence of SUR1 subunits, consistent with a tight physical association of the two subunits (Lorenz et al., 1998). In the present study, we have explored the functional coupling of SUR1 to Kir6.2 The results demAddress correspondence to C.G. Nichols, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. Fax: 314-362-7463; E-mail: [email protected] 1 Abbreviations used in this paper: Kir, inward rectifier; PCO, potassium channel opener; PIP 2 , phosphatidylinositol 4,5-bisphosphate; SUR, sulfonylurea receptor. on Jne 1, 2017 D ow nladed fom Published August 1, 1999