Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic beta-cells and HIT cells

Area of Science:

• Cellular physiology
• Ion channel function
• Metabolic regulation

Background:

• ATP-inhibited potassium channels (K(ATP)) are crucial in regulating pancreatic beta-cell function.
• The precise mechanisms governing K(ATP) channel activity, particularly the role of nucleotides, remain incompletely understood.
• Existing models often rely on the ATP/ADP ratio, potentially oversimplifying complex nucleotide interactions.

Purpose of the Study:

• To investigate the direct effects of ADP on K(ATP) channels in pancreatic beta-cells and HIT cells.
• To elucidate the biphasic dose-response relationship of ADP on K(ATP) channel activity.
• To develop a refined model of nucleotide-channel interactions accounting for magnesium dependency and competition.

Main Methods:

• Utilized excised, inside-out membrane patches from cultured mouse pancreatic beta-cells and HIT cells.
• Applied varying concentrations of ADP and ATP in controlled solutions, including creatine/creatine phosphate systems to fix nucleotide levels.
• Analyzed channel activity using electrophysiological recordings and developed a new computational model for nucleotide-channel interactions.

Main Results:

• ADP demonstrated a biphasic effect: activation at 10-500 microM and inhibition at >500 microM in mouse beta-cells.
• Magnesium was required for ADP-induced channel opening but not for inhibition.
• A novel model with distinct ADP binding sites (MgADP for activation, ATP/ADP site for inhibition) accurately predicted experimental data.
• The model predicted that physiological ADP levels (10-100 microM) could significantly influence beta-cell membrane potential by competing with ATP.

Conclusions:

• K(ATP) channel activity is not solely determined by the ATP/ADP ratio; absolute nucleotide concentrations and specific binding sites are critical.
• ADP acts as a significant regulator of K(ATP) channels, even at physiologically relevant low micromolar concentrations.
• The findings necessitate a revised understanding of nucleotide regulation of K(ATP) channels in pancreatic beta-cells, impacting insights into insulin secretion and metabolic control.