Kir6.2-dependent high-affinity repaglinide binding to beta-cell K(ATP) channels

Abstract

1. The beta-cell K(ATP) channel is composed of two types of subunit - the inward rectifier K(+) channel (Kir6.2) which forms the channel pore, and the sulphonylurea receptor (SUR1), which serves as a regulatory subunit. The N-terminus of Kir6.2 is involved in transduction of sulphonylurea binding into channel closure, and deletion of the N-terminus (Kir6.2DeltaN14) results in functional uncoupling of the two subunits. In this study, we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with SUR1 and the effect of Kir6.2 on this interaction. We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of SUR1 and Kir6.2 in Kir6.2DeltaN14/SUR1 channels. All binding experiments are performed on membranes in ATP-free buffer at 37 degrees C. 2. Repaglinide was found to bind with low affinity (K(D)=59+/-16 nM) to SUR1 alone, but with high affinity (increased approximately 150-fold) when SUR1 was co-expressed with Kir6.2 (K(D)=0.42+/-0.03 nM). Glibenclamide, tolbutamide and nateglinide all bound with marginally lower affinity to SUR1 than to Kir6.2/SUR1. 3. Repaglinide bound with low affinity (K(D)=51+/-23 nM) to SUR1 co-expressed with Kir6.2DeltaN14. In contrast, the affinity for glibenclamide, tolbutamide and nateglinide was only mildly changed as compared to wild-type channels. 4. In whole-cell patch-clamp experiments inhibition of Kir6.2DeltaN14/SUR1 currents by both repaglinide and nateglinde is abolished. 5. The results suggest that Kir6.2 causes a conformational change in SUR1 required for high-affinity repaglinide binding, or that the high-affinity repaglinide-binding site includes contributions from both SUR1 and Kir6.2. Glibenclamide, tolbutamide and nateglinide binding appear to involve only SUR1.

Figure 1

Saturation analysis of [³H]glibenclamide (a) and [³H]repaglinide (b) binding to SUR1 expressed alone (open circles) or co-expressed with Kir6.2 (filled circles). Inset: Scatchard analysis of data. Single representative experiments.

Figure 2

Inhibition of [³H]glibenclamide binding to SUR1 expressed alone (open circles) or co-expressed with Kir6.2 (filled circles) by repaglinide (a), glibenclamide (b), tolbutamide (c) and nateglinide (d).

Figure 3

Whole-cell currents through Kir6.2ΔN14/SUR1 channels (upper panel) or Kir6.2/SUR1 channels (lower panel, from Hansen et al., 2002) expressed in HEK293 cells. The dotted line is the zero current level. Application of repaglinide, nateglinide and BaCl₂ is indicated by solid horizontal lines.

Figure 4

Repaglinide displacement of [³H]glibenclamide from membranes expressing two different ratios of Kir6.2 to SUR1 (a). Data are from single representative experiments performed in triplicate. SUR1 data are taken from Figure 2. Log IC₅₀ values (b) and Hill coefficients (c) for repaglinide displacement of [³H]glibenclamide (n=4–6).