Hyperinsulinism in infancy (HI) is the most common cause of recurrent or persistent hypoglycaemia in early childhood. The disease is principally caused by defects in the ATP-sensitive K channel (K_ATP) genes ABCC8 (SUR1) and KCNJ11 (Kir6.2), which can result in altered nucleotide regulation, channel gating, subunit assembly or subunit trafficking. Loss of K_ATP channels causes inappropriate Ca²⁺ channel activity and uncontrolled insulin release as a consequence. In this study we have investigated the cell surface expression of functional K_ATP channels in β-cells isolated from patients with HI by maintaining isolated cells under a variety of controlled conditions designed to modulate post-translational events associated with the trafficking of membrane proteins. Insulin-secreting cells were isolated from two patients with diffuse HI. Patients N79 and N94 were unrelated, and failed to respond adequately to diazoxide and/or Sandostatin or Octreotide in vivo. As a result both patients required a subtotal pancreatectomy to control hypoglycaemia.

Following surgery (with informed consent and Local Ethics Committee approval), a controlled collagenase digestion procedure was used to isolate intact islets of Langerhans, and single β-cells were liberated by mechanical agitation in a standard Ca-free extracellular solution. Single cells were subsequently maintained under standard tissue culture conditions at 37 °C or at either 37 or 25 °C in the presence or absence of: (1) 10 nM phorbol myristate acetate (PMA), 2 mM forskolin and 100 mM 3-isobutyl-1-methylxanthine (IBMX); (2) 2.5 mM 4-phenylbutryate; or (3) the K_ATP channel agonist 10 mM BPDZ 154, for up to 40 h. The surface expression of functional K_ATP channels was assessed by patch-clamp methods using isolated patches of cell membrane. RNA was extracted from isolated tissue using standard protocols and RT-PCR performed to document the expression of K_ATP channel mRNAs using specific oligonucleotide primers directed against SUR1 and Kir6.2. In N79 β-cells maintained at 37 °C, limited K_ATP channel activity was seen in only 38 % of cells (n = 3/8) as a consequence of defects in the C-terminal region of SUR1 (oligonucleotide primers to Kir6.2 and all three regions of SUR1 generated PCR products in control cells, whilst in N79 only Kir6.2 and the N-terminal region of SUR1 were amplified). By contrast when N79 β-cells were maintained at 25 °C (either with or without exposure to 2.5 mM 4-phenylbutyrate), 73 % of cells (n = 8/11) expressed functional channels that responded to ADP (0.5 mM) and diazoxide (0.5 mM). Maintenance of N79 β-cells at 37 °C in the presence of 100 mM IBMX, 10 nM PMA and 2 mM forskolin did not enhance expression of functional K_ATP channels (n = 3). Under standard cell culture conditions at 37 °C, there were no operational K_ATP channels in N94 β-cells, n = 6. However, when cells were maintained at 37 °C in tissue culture media supplemented with either IBMX, PMA and forskolin or 10 mM BPDZ 154, this led to a recovery of K_ATP channel currents that were inhibited by ATP, n = 4/10 cells.

These data document that modulation of post-translational events can potentially lead to the recovery of endogenous K_ATP channel function in HI β-cells.

All procedures accord with current local guidelines.