MgATP activates the beta cell KATP channel by interaction with its SUR1 subunit

Abstract

ATP-sensitive potassium (KATP) channels in the pancreatic beta cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca2+ influx and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The beta cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg2+ caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg2+.

Figure 1

(A) Macroscopic currents recorded from inside-out patches excised from oocytes coinjected with Kir6.2 and SUR1 mRNAs (a) or injected with Kir6.2ΔC36 mRNA (b) in response to a series of voltage ramps from −110 mV to +100 mV. ATP (100 μM) was added to the internal solution as indicated. (B) Mean ATP dose-response relationships for Kir6.2/SUR1 currents (a) or Kir6.2ΔC36 currents (b) in the presence (•) or absence (○) of Mg²⁺. Test solutions were alternated with control solutions, and the slope conductance (G) is expressed as a percentage of the mean (G_c) of that obtained in control solution before and after exposure to ATP. Conductance was measured between −20 and −100 mV and is the mean of five voltage ramps. The number of patches was (a) • = 7; ○ = 15 and (b) ▪ = 11; ○ = 15. The lines are the best fit of the data to the Hill equation by using the mean values for K_i and h given in the text.

Figure 2

Mean ATP dose-response relationships for Kir6.2/SUR1 (▪, n = 15), Kir6.2/SUR1-D853N (○, n = 4) or Kir6.2/SUR1-D1505N (•, n = 5) currents, measured in the presence of Mg²⁺. Test solutions were alternated with control solutions, and the slope conductance (G) is expressed as a percentage of the mean (G_c) of that obtained in control solution before and after exposure to ATP. Conductance was measured between −20 and −100 mV and is the mean of five voltage ramps. The lines are the best fit of the data to the Hill equation by using the mean values for K_i and h given in the text.

Figure 3

(A) Macroscopic currents recorded from the same inside-out patch from an oocyte coinjected with Kir6.2-R50G and SUR1. Currents were elicited by a series of voltage ramps from −110 mV to +100 mV. ATP (100 μM) and ADP (100 μM) were added to the internal solution as indicated. Mg²⁺ (1.4 mM) was present throughout so ATP and ADP will exist as the Mg²⁺ salts. (B) Mean macroscopic slope conductance for Kir6.2-R50G/SUR1 currents, recorded in the presence of MgATP (solid bars) or MgADP (hatched bars), expressed as a percentage of the slope conductance in control solution (no additions). The dashed line indicates the conductance level in control solution. The number of oocytes is given above the bars.

Figure 4

(A) Macroscopic currents recorded from inside-out patches excised from oocytes coinjected with Kir6.2-R50G and either SUR1 or K719A/K1384M-SUR1 mRNAs. Currents were elicited by a series of voltage ramps from −110 mV to +100 mV. ATP (100 μM) was added to the internal solution as indicated. Mg²⁺ (1.4 mM) was present throughout so ATP will exist as the Mg²⁺ salt. (B) Mean ATP dose-response relationships for Kir6.2-R50G/SUR1 or Kir6.2-R50G/SUR1-K719A/K1384M currents, measured in the presence of Mg²⁺. The slope conductance (G) is expressed as a percentage of the mean (G_c) of that obtained in control (ATP-free) solution. The lines are drawn through the points by eye, and the number of patches is indicated next to each data point. The dotted line indicates the effect of ATP on Kir6.2/SUR1 currents and is the same data as in Fig. 1B. (C) Mean macroscopic slope conductance recorded in the presence of 100 μM or 1 mM MgATP, expressed as a percentage of the slope conductance in the absence of ATP, for channels comprising Kir6.2-R50G and SUR1 containing the mutations indicated. The dashed line indicates the conductance level in control solution. The number of oocytes is given above the bars.