No abstract

Insulin exocytosis is regulated by ion channels that control excitability and Ca2+ influx. Channels also play an increasingly appreciated role in microdomain structure. In this study, we examine the mechanism by which the voltage-dependent K+ (Kv) channel Kv2.1 (KCNB1) facilitates depolarization-induced exocytosis in INS 832/13 cells and β-cells from human donors with and without type 2 diabetes (T2D). We find that Kv2.1, but not Kv2.2 (KCNB2), forms clusters of 6–12 tetrameric channels at the plasma membrane and facilitates insulin exocytosis. Knockdown of Kv2.1 expression reduces secretory granule targeting to the plasma membrane. Expression of the full-length channel (Kv2.1-wild-type) supports the glucose-dependent recruitment of secretory granules. However, a truncated channel (Kv2.1-ΔC318) that retains electrical function and syntaxin 1A binding, but lacks the ability to form clusters, does not enhance granule recruitment or exocytosis. Expression of KCNB1 appears reduced in T2D islets, and further knockdown of KCNB1 does not inhibit Kv current in T2D β-cells. Upregulation of Kv2.1-wild-type, but not Kv2.1-ΔC318, rescues the exocytotic phenotype in T2D β-cells and increases insulin secretion from T2D islets. Thus, the ability of Kv2.1 to directly facilitate insulin exocytosis depends on channel clustering. Loss of this structural role for the channel might contribute to impaired insulin secretion in diabetes.

Aims/hypothesis It is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function. Methods Wild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1-syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca 2+ -responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured.

Insulin secretion from pancreatic ß cells is a multistep process that requires the coordination of exocytotic proteins that integrate diverse signals. These include signals derived from metabolic control of post-translational SUMOylation and depolarization-induced rises in intracellular Ca2+. Here we show that tomosyn, which suppresses insulin exocytosis by binding syntaxin1A, does so in a manner which requires its SUMOylation. Glucose-dependent de-SUMOylation of tomosyn1 at K298 releases syntaxin1A and controls the amplification of exocytosis in concert with a recently-identified tomosyn1-interacting partner; the Ca2+-binding protein secretagogin, which dissociates from tomosyn1 in response to Ca2+-raising stimuli and is required for insulin granule trafficking and exocytosis downstream of Ca2+ influx. Together our results suggest that tomosyn acts as a key signaling hub in insulin secretion by integrating signals mediated by metabolism-dependent de-SUMOylation and electrically-induced entry of Ca2+ to regulate the availability of exocytotic proteins required for the amplification of insulin secretion.

Aims/hypothesis Phosphatidylinositol 3-OH kinases (PI3Ks) regulate beta cell mass, gene transcription, and function, although the contribution of the specific isoforms is unknown. As reduced type 1A PI3K signalling is thought to contribute to impaired insulin secretion, we investigated the role of the type 1A PI3K catalytic subunits α and β (p110α and -β) in insulin granule recruitment and exocytosis in rodent and human islets. Methods The p110α and p110β subunits were inhibited pharmacologically or by small hairpin (sh)RNA-mediated knockdown, and were directly infused or overexpressed in mouse and human islets, beta cells and INS-1 832/13 cells. Glucose-stimulated insulin secretion (GSIS), single-cell exocytosis, Ca 2+ signalling, plasma membrane granule localisation, and actin density were monitored. Results Inhibition or knockdown of p110α increased GSIS. This was not due to altered Ca 2+ responses, depolymerisation of cortical actin or increased cortical granule density, but to enhanced Ca 2+ -dependent exocytosis. Intracellular infusion of recombinant PI3Kα (p110α/p85β) blocked exocytosis. Conversely, knockdown (but not pharmacological inhibition) of p110β blunted GSIS, reduced cortical granule density and impaired exocytosis. Exocytosis was rescued by direct intracellular infusion of recombinant PI3Kβ (p110β/p85β) even when p110β catalytic activity was inhibited. Conversely, both the wild-type p110β and a catalytically inactive mutant directly facilitated exocytosis. Conclusions/interpretation Type 1A PI3K isoforms have distinct and opposing roles in the acute regulation of insulin secretion. While p110α acts as a negative regulator of beta cell exocytosis and insulin secretion, p110β is a positive regulator of insulin secretion through a mechanism separate from its catalytic activity.