Ion channels

Abstract:

1. We have examined the mechanism by which nucleotides modulate the tolbutamide block of the beta-cell ATP-sensitive K+ channel (KATP channel), using wild-type and mutant KATP channels heterologously expressed in Xenopus oocytes. This channel is composed of sulphonylurea receptor (SUR1) and pore-forming (Kir6.2) subunits. 2. The dose-response relation for tolbutamide block of wild-type KATP currents in the absence of nucleotide showed both a high-affinity (Ki = 2.0 microM) and a low-affinity (Ki = 1.8 mM) site. 3. The dose-response relation for tolbutamide block of Kir6.2 delta C36 (a truncated form of Kir6.2 which is expressed independently of SUR1) was best fitted with a single, low-affinity site (Ki = 1.7 mM). This indicates that the high-affinity site resides on SUR1, whereas the low-affinity site is located on Kir6.2. 4. ADP (100 microM) had a dual effect on wild-type KATP currents: the nucleotide enhanced the current in the presence of Mg2+, but was inhibitory in the absence of Mg2+. Kir6.2 delta C36 currents were blocked by 100 microM ADP in the presence of Mg2+. 5. For wild-type KATP currents, the blocking effect of 0.5 mM tolbutamide appeared greater in the presence of 100 microM MgADP (84 +/- 2%) than in its absence (59 +/- 4%). When SUR1 was mutated to abolish MgADP activation of KATP currents (K719A or K1384M), there was no difference in the extent of tolbutamide inhibition in the presence or absence of MgADP. 6. The Ki for tolbutamide interaction with either the high- or low-affinity site was unaffected by 100 microM MgADP, for both wild-type and K719A-K1384M currents. 7. MgGDP (100 microM) enhanced wild-type KATP currents and was without effect on K719A-K1384M currents. It did not affect the Ki for tolbutamide block at either the high- or low-affinity site. 8. Our results indicate that interaction of tolbutamide with the high-affinity site (on SUR1) abolishes the stimulatory action of MgADP. This unmasks the inhibitory effect of ADP and leads to an apparent increase in channel inhibition. Under physiological conditions, abolition of MgADP activation is likely to constitute the principal mechanism by which tolbutamide inhibits the KATP channel.

Abstract:

The ATP-sensitive potassium channel (K-ATP channel) plays a key role in insulin secretion from pancreatic beta-cells. It is closed by glucose metabolism, which stimulates secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and MgADP concentration, which inhibit and potentiate channel activity, respectively. The beta-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. The site at which ATP mediates channel inhibition lies on Kir6.2, while the potentiatory action of MgADP involves the nucleotide-binding domains of SUR1. K-ATP channels are also activated by MgGTP and MgGDP. Furthermore, both nucleotides support the stimulatory actions of diazoxide. It is not known, however, whether guanine nucleotides mediate their effects by direct interaction with one or more of the K-ATP channel subunits or indirectly via a GTP-binding protein. We used a truncated form of Kir6.2, which expresses independently of SUR1, to show that GTP blocks K-ATP currents by interaction with Kir6.2 and that the potentiatory effects of GTP are endowed by SUR1. We also showed that mutation of the lysine residue in the Walker A motif of either the first (K719A) or second (K1384M) nucleotide-binding domain of SUR1 abolished both the potentiatory effects of GTP and GDP on K-ATP currents and their ability to support stimulation by diazoxide. This argues that the stimulatory effects of guanine nucleotides require the presence of both Walker A lysines.

Abstract:

ATP-sensitive potassium channels (K-ATP channels) couple cell metabolism to electrical activity and are important in the physiology and pathophysiology of many tissues. In pancreatic beta-cells, K-ATP channels link changes in blood glucose concentration to insulin secretion. They are also the target for clinically important drugs such as sulphonylureas, which stimulate secretion, and the K+ channel opener diazoxide, which inhibits insulin release. Metabolic regulation of K-ATP channels is mediated by changes in intracellular ATP and Mg-ADP levels, which inhibit and activate the channel, respectively. The beta-cell K-ATP channel is a complex of two proteins: an inward-rectifier K+ channel subunit, Kir6.2, and the sulphonylurea receptor, SUR1. We show here that the primary site at which ATP acts to mediate K-ATP channel inhibition is located on Kir6.2, and that SUR1 is required for sensitivity to sulphonylureas and diazoxide and for activation by Mg-ADP.

Abstract:

The ATP-sensitive K-channel (K-ATP channel) plays a key role in insulin secretion from pancreatic beta-cells. It is closed by glucose metabolism, which stimulates insulin secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and Mg-ADP, which inhibit and potentiate channel activity, respectively. The beta-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. We have mutated (independently or together) two lysine residues in the Walker A (W(A)) motifs of the first (K719A) and second (K1384M) nucleotide-binding domains (NBDs) of SUR1. These mutations are expected to inhibit nucleotide hydrolysis. Our results indicate that the W(A) lysine of NBD1 (but not NBD2) is essential for activation of K-ATP currents by diazoxide. The potentiatory effects of Mg-ADP required the presence of the W(A) lysines in both NBDs. Mutant currents were slightly more sensitive to ATP than wild-type currents. Metabolic inhibition led to activation of wild-type and K1384M currents, but not K719A or K719A/K1384M currents, suggesting that there may be a factor in addition to ATP and ADP which regulates K-ATP channel activity.