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The structure of insulin contains determinants of foldability, trafficking, self-assembly, and receptor binding. Insulin is the biosynthetic product of a single-chain precursor, preproinsulin, whose proteolytic processing is coupled to trafficking between cellular compartments. The connecting (C) domain of proinsulin is removed by a specialized set of endoproteases and a carboxypeptidase activity, acting mainly within maturing secretory granules. Insulin is stored as microcrystalline arrays of zinc insulin hexamers within specialized glucose-regulated secretory vescicles. Regulation of insulin secretion is coupled to metabolism and electrophysiologic events involving plasma membrane depolarization and calcium-ion homeostasis. The insulin receptor is a transmembrane protein containing an extracellular hormone-binding domain and intracellular tyrosine kinase domain. Binding of insulin to the insulin receptor (an (αβ) 2 dimer) is mediated by side chains in both the A- and B chains of insulin. The primary hormone-binding site (Site 1) in the extracellular domain of the insulin receptor contains the L1 β -helix of one α -subunit and the C-terminal α CT α -helix of the other α -subunit. Dominant mutations in the insulin gene cause monogenic syndromes of diabetes mellitus, prominently including permanent neonatal-onset diabetes, due to toxic misfolding of proinsulin variants.

Insulin plays a central role in the regulation of human metabolism. The hormone is a 51-residue anabolic protein that is secreted by the β-cells in the Islets of Langerhans. Containing two chains (A and B) connected by disulfide bonds, the mature hormone is the post-translational product of a single-chain precursor, designated proinsulin . Extensive studies of the three-dimensional structure of insulin, pioneered by D. C. Hodgkin , have enabled the development of therapeutic analogs for the treatment of the metabolic disorder diabetes mellitus (DM) . The insulin gene is the site of dominant mutations associated with DM . Although such mutations are uncommon, their molecular analysis has provided important insights into the biochemical bases of the hormone’s pathway of biosynthesis and mechanism of receptor binding . The largest class of mutations is associated with the impaired folding of proinsulin, which leads in turn to progressive endoplasmic-reticular (ER) stress, β-cell death and DM, usually with onset in the neonatal period .

Key complementary functions of insulin are (a) stimulation of glucose uptake from the systemic circulation and (b) suppression of hepatic gluconeogenesis, together regulating glucose homeostasis . DM is characterized by decreased glucose tolerance resulting from a relative deficiency of insulin or a lack of sensitivity to the endogenous hormone. Insufficient insulin, or decreased insulin sensitivity, results in hyperglycemia. Long-term exposure of tissues to elevated ambient glucose concentrations is associated with the development of complications, including macro- and microvascular disease. Of particular concern are coronary heart disease, cerebrovascular disease, and the characteristic retinopathy, nephropathy, and neuropathy of this disorder .

The history of the discovery of insulin and its therapeutic utility defined a paradigm for the integration of physiologic and biochemical approaches in experimental medicine . At the end of the 19 th century Von Mering and Minkowski noted that removal of the pancreas led to the development of DM in dogs . In 1916 Schafer first speculated that an antidiabetic hormone, which he named “insuline,” was secreted from pancreatic islets . Barron noted in 1920 that ligation of the pancreatic duct, with destruction of the exocrine pancreas, only resulted in DM if the islets, so named by Langerhans in 1869 , were also destroyed . Subsequently, the work of Banting, Best, Collip and MacCleod in the early 1920′s resulted in the identification of a substance in extracts of pancreas that had the remarkable ability to reduce blood glucose levels in diabetic animals . By 1923 these pancreatic extracts were employed to successfully treat patients with DM. The dramatic clinical utility of insulin encouraged broad public support for medical research .

The modern view of insulin is as a ligand that activates a specific cellular receptor, designated the insulin receptor (IR) . The IR belongs to a superfamily of receptor tyrosine kinase whose activation modulates multiple post-receptor signaling pathways . Insulin thus regulates a host of other cellular processes, such as protein and fat synthesis, RNA and DNA synthesis, as well as cell growth and differentiation . It is, however, the regulation of glucose uptake that is of primary concern in the clinical manifestations of diabetes; therefore, this chapter will begin with a brief description of how plasma glucose homeostasis is achieved.

Specific membrane transporters facilitate the movement of glucose into cells to reduce plasma glucose concentrations in response to insulin stimulation. The transported glucose is subsequently used as metabolic fuel or stored as a complex polymeric structure, designated glycogen. Two major types of glucose transporters are known: Na + -dependent and Na + -independent. Only the Na + -independent transporters possess an insulin-responsive isoform. We describe each in turn. The Na + -dependent glucose transporter family has been identified in several tissues, particularly in small intestinal epithelium (SGLT1) and the renal proximal tubule (SGLT2) as well as in other kidney tubule cells . These transporters are located on the lumenal side of intestinal and kidney cells and act to absorb glucose against its concentration gradient by coupling the movement of glucose into these cells with the concomitant movement of Na + into the cell. Since Na + is moving down its electrochemical gradient this energy can be used to cotransport glucose into the cells. Thus, this transporter is dependent on the concentrations of extracellular and intracellular sodium ions, which are maintained by a Na + /K + -ATPase ion pump.

The Na + -independent glucose transporter family, consisting of several isoforms, facilitates the movement of glucose down its concentration gradient across a plasma membrane. Although seven isoforms have been identified (designated Glut1-7) , only one will be discussed in detail here, Glut4, because it is the transporter that is in highest concentration in insulin-sensitive tissues, including skeletal muscle, cardiac muscle, and adipose tissue (fat) . Glut4, and to a lesser extent Glut1 enable these cells to increase their uptake of glucose, thereby lowering circulating concentrations. Because the intracellular concentration of glucose is low due to the rapid phosphorylation of glucose to glucose-6-phosphate (not a substrate of the Glut transporters) and its conversion to other metabolic products, the presence of active transporters in the plasma membrane favors the movement of glucose into cells.

Insulin enhances glucose uptake by increasing the number of transporters in the plasma membrane of target cells. This was first demonstrated in adipocytes and subsequently in skeletal and cardiac muscle . Insulin stimulation of such cells mobilizes transporters from intracellular compartments to the plasma membrane to facilitate glucose transport. Translocation of receptors to the plasma membrane has been demonstrated to occur within 30 seconds of insulin stimulation ; as the stimulus dissipates the decrease in the number of plasma membrane receptors declines coincident with a decline in glucose transport . Whereas glucose transport via Glut4 is a passive process (limited only by the chemical potential of the glucose gradient and the V max of the transporters), translocation and reverse reinternalization of receptors are energy-dependent processes . The impaired ability of insulin, on binding and activation of the IR, to signal Glut4 translocation from intracellular stores contributes to postprandial hyperglycemia in Type 2 DM . Animal studies have also demonstrated that insulin resistance is associated with a decreased translocation of glucose transporters to the plasma membrane in muscle cells . In fact, decreased insulin levels in animal models of DM have been shown not only to decrease transporter translocation, but also to attenuate expression of Glut4 in muscle cells. Thus, it appears that insulin provides both a short-term signal to increase glucose-transporter translocation and a long-term signal to maintain a basal level of expression of such transporters in target cells. The combination of acute and basal actions provides a common mechanism in Type 1 DM (characterized by low or vanishing endogenous insulin levels) or Type 2 DM (characterized by insulin resistance) could cause pathologically high plasma glucose levels: loss of regulation and expression of transmembrane glucose transporters. Glut2, expressed on surface of β-cells, contributes to the regulation of insulin secretion . Accordingly, a β-cell specific IR knock-out (KO) model indicated that insulin likely positively regulates its own secretion from the β-cell .

Since its purification and clinical application in 1923 , the central importance of insulin in regulating glucose metabolism and the prevention of DM has stimulated research in attempts to understand the mechanism of action of this peptide hormone. This work has lead to the determination of the three-dimensional structure of insulin , identification of its precursor and the processing and secretion mechanisms that underlie its production . Complementary advances have seen the identification of the IR and of its mechanisms of signal transduction . Recent studies have defined three-dimensional structures of proinsulin and elements of the IR involved in hormone binding . In addition to their fundamental importance, these discoveries have deepened our understanding of the molecular basis of DM and its treatment as discussed below.

Insulin was the first peptide hormone discovered. Before Abel crystallized insulin in 1926 and Jensen and Evans in 1935 identified the N-terminal phenylalanine of the B-chain , proving that insulin was indeed a protein, all hormones were believed to be small molecules. With the elucidation of the amino-acid sequence of insulin by Sanger in the mid 1950′s (see Figure 1), it became known that insulin was a two-chain heterodimer consisting of a 21-residue A-chain linked to a 30-residue B chain by two disulfide bonds derived from cysteine residues (A7-B7 and A20-B19). An intrachain disulfide bond also exists within the A-chain (A6-A11). Figure 1 . Primary structures of porcine insulin and porcine proinsulin. The primary sequence of porcine insulin (a) as determined by Sanger and co-workers ; and proinsulin . The sequence of human insulin is identical to that of porcine insulin except for the change of Ala B30 to Thr B30 in human insulin.

Although this primary structure provided valuable information regarding the amino-acid composition and size (ca. 6,000 Daltons) of the insulin molecule , questions concerning the processes of insulin biosynthesis and secretion were not resolved until the late 1960′s with the discovery of proinsulin . This precursor protein (ca. 9,000 D) contains both the A- and B-chain of insulin in a continuous single chain joined through an intervening segment, designated the C domain . The C domain varies in length among vertebrate species (typically 30-35 residues) and is flanked at each end by dibasic residues (Arg-Arg and Lys-Arg) . Proinsulin is cleaved at those dibasic links by a trypsin-like enzyme to release the mature hormone and a free C-peptide (which lacks the dibasic residues). Figure 2. (a) Diagrammatic illustration of the processing of insulin. The transcription and translation of the human insulin gene, as well as processing of preproinsulin to insulin is illustrated. (b) Conversion of proinsulin to insulin demonstrating the secondary and tertiary structures of insulin; the flexible C domain in proinsulin and split proinsulins is shown in dotted line. Thick blue arrows indicated predominant path.

Chan et al. subsequently discovered that there was an additional and larger precursor of insulin, preproinsulin. This single-chain polypeptide (ca. 12,000 Daltons) consists of proinsulin extended at the amino-terminus by a 24-residue signal peptide of hydrophobic residues . Such a signal sequence is characteristic of proteins that enter the secretory pathway . The signal peptide of preproinsulin is cleaved coincident with its translocation into the ER , and thus proinsulin itself (together with the oxidative folding machinery of the ER) mediates proper disulfide pairing and three-dimensional protein folding .

The steps involved in the conversion of the information encoded within the 1500 bases of the human insulin gene (as sequenced by Bell and colleagues in 1980 ) into preproinsulin and its subsequent proteolytic conversion to insulin are illustrated in Figure 2A and 2B. The first set of steps occur at the nucleic-acid level: the initial mRNA transcript is modified via excision of the two intervening sequences, the 5′ terminus is capped by 7-methyl guanosine, and the 3’-terminus undergoes polyadenylation to produce a mature mRNA product. This mRNA product encodes preproinsulin, which is translated on the rough endoplasmic reticulum (rER) and subsequently translocated into the RER lumen via a series of interactions of the signal peptide with the signal recognition particle (SRP) and SRP-receptor in the rER membrane . The next set of steps occurs at the protein level. The signal peptide is cleaved in the lumen of the rER by a signal peptidase (located on the lumenal side of the rER membrane). Within the cisternae of the rER, proinsulin undergoes rapid folding and disulfide bond formation to generate the native tertiary structure, the direct precursor of insulin. The signal peptide is rapidly degraded in the rER and is therefore not a normal secretory product of the β-cells .

In the final series of steps proinsulin is transported to the Golgi apparatus where it is packaged into secretory granules and converted to native insulin and C-peptide. The conversion process may begin in the trans Golgi network but continues in the condensing vacuoles (early secretory granules), and the products are stored in mature secretory vesicles, and secreted in equimolar amounts along with small amounts (ca. 2-3%) of proinsulin and intermediate cleavage products . Glucose, in addition to stimulating insulin secretion by β-cells, also activates insulin gene transcription, enhances insulin mRNA stability, and stimulates its translation .

Proinsulin, despite its larger size, shares many of the physical properties of insulin. Proinsulin has been shown, for example, to form dimers and Zn 2+ -coordinated hexamers in a manner similar to insulin , has a comparable isoelectric point and solubility , and cross-reacts with insulin antisera . These findings motivated the hypothesis that the structure of the insulin moiety in proinsulin is similar, if not identical, to that of native insulin . Although the crystal structure of proinsulin has not been determined , presumably due to the flexibility of the C domain, its solution structure has recently been determined by multidimensional NMR methods . The insulin moiety indeed retains the conformation of insulin whereas the C domain is flexible (but not completely disordered). The solution structure rationalizes why proinsulin is a full agonist of insulin and displays 3-5% biological activity ; the binding regions of the insulin moiety are accessible even in the presence of the C domain .

In 1969 it was demonstrated via pulse-chase studies that proteolytic processing of proinsulin occurs in the Golgi apparatus (GA) and/or early secretory vesicles of the β-cells ; and subsequent studies have identified the trans Golgi as the initial compartment wherein proinsulin and its converting enzymes are brought together to form secretory granules . Monoclonal antibodies specific for intact proinsulin also demonstrated that proinsulin is transferred from the rER to the cis- and trans Golgi, where the precursor is concentrated to form prosecretory vesicles . Several studies have demonstrated that shuttling of proinsulin vesicles from the rER to the cis Golgi and through the GA is an energy-requiring process requiring ATP . Subsequent conversion of proinsulin to insulin, initiated in the trans Golgi, accelerates within prosecretory granules as they acidify and mature in the cytosol over a period of 1-3 hours in preparation for secretion. Residual proinsulin and intermediate cleavage products then comprise only 2-3 percent of total stored insulin-related protein. Insulin secretory granules turn over at a much slower rate of many hours or several days normally. In the intracellular pathway taken by proinsulin on budding from the rER, distinct times are required for individual stages of transfer. In rat islets the conversion of proinsulin to insulin begins about 30 min after ribosomal synthesis of preproinsulin and resembles first-order reaction kinetics with half-times of approximately 30-60 minutes .

The conversion of proinsulin to insulin occurs through the joint action of two types of proteases: one with trypsin-like endoprotease activity which cleave after the dibasic residues pairs at each end of the C domain, and another with exopeptidase activity resembling that of carboxypeptidase B to remove the basic residues left after tryptic-like cleavage . Previous studies have also demonstrated that mixtures of pancreatic trypsin and carboxypeptidase B could convert proinsulin to insulin in vitro . Two endoproteases were found within insulinoma secretory granules . Initially called Type I and Type II converting enzymes, each of these acidic endoproteases was found to be dependent on Ca 2+ ions. Type I was active in 1 mM Ca 2+ and cleaved at Arg31-Arg32 in proinsulin (the first two positions of the C domain) whereas Type II required 0.1 mM Ca 2+ and cleaved predominantly at Lys64-Arg65 (the last two positions of the C domain) . Each was also found to have an acidic pH optimum near 6.0.

The discovery in yeast Saccharomyces cerevisciae of an endoprotease, designated Kex2, that cleaves at dibasic residue sites in the yeast α -mating factor and in microbial toxin precursor polypeptides facilitated the search for homologous endoproteases in mammalian β-cells . Kex2 is a homologue of subtilisin, a bacterial serine protease. In yeast this integral membrane protein is localized in the trans Golgi network. Analysis of human insulinoma cDNA via PCR techniques lead to the discovery of PC2, an enzyme having an homologous catalytic domain . PC2 shares 49% amino-acid identity with Kex2, but importantly lacks its transmembrane (TM) segment, indicating that it was likely to be a soluble protease and thus was a candidate for one of the processing endoproteases in β-cell secretory vesicles. Similar screening methods led to the discovery of PC1/3, the second secretory-granule convertase . Subsequent work demonstrated that both PC2 and PC1/3 display optimal activity at pH 5.5, consistent with the internal pH of β-cell secretory granules and are also expressed in the brain and many other neuroendocrine cells. These two proprotein convertases are members of a larger 7-member family of kexin/subtilisin-like endoproteases, which normally function within the secretory pathway in most eukaryotic cells .

Immunocytochemical studies of pancreatic islets β-cells provided evidence for the presence of both PC2 and PC1/3 in β-cells pancreatic islets , and their ability to convert proinsulin to insulin was demonstrated by co-infection with vaccinia viruses expressing PC2 and PC1/3 in Cos7 cells expressing proinsulin . Whereas PC2 mediated cleavage only at the C-peptide/A-chain junction, PC1/3 cleaved at both dibasic sites with a preference for the B-chain/C-peptide site. Subsequent studies also established the identity of PC2 and PC1/3 with the calcium-dependent Type II and Type I insulinoma endoproteases, respectively, as discovered by Davidson and coworkers . The carboxypeptidase B-like exopeptidase, which removes COOH-terminal basic residues after cleavage by PC2 and PC3/PC1, was also found and is known as carboxypeptidase E . Although structurally homologous to pancreatic carboxypeptidases A and B , carboxypeptidase E has several unique features that differentiate it from other carboxypeptidases . In the maturing secretory granule of the B cell, PC2, PC1/3, and carboxypeptidase E work together to convert proinsulin to mature insulin and C-peptide. Studies have demonstrated that PC1/3 first cleaves proinsulin at the B chain-C peptide junction, generating an intermediate product that is preferentially cleaved by PC2 to yield insulin . Thus knockout of PC1/3 results in very high circulating proinsulin levels while PC2 nulls exhibit much lower levels .

The PC family of convertases are all synthesized as inactive precursors with a lengthy N-terminal propeptide that is autocatalytically cleaved at a tetrabasic cleavage site by the proenzyme as it passes from the slightly alkaline ER to the neutral and mildly acidic conditions of the Golgi apparatus and is then removed by a second cleavage within the propeptide which assists in its release and disposal, yielding the active full-length enzyme . In the case of PC1/3 the full length form is active , but undergoes C-terminal autocatalytic truncation to a smaller, more active form within the secretory granules . In the case of PC2 the removal of the propeptide is more complex and requires the presence of neuroendocrine protein 7B2 . Activation of PC2 thus does not occur in the Golgi but rather within the secretory granules where it also functions. The full length enzyme is the active form in the granules within the beta cells and other neuroendocrine tissues. Knockout of 7B2 thus provides a phenocopy of the PC2 null when on the same genetic background, or inbred mouse strain (for review, see Ref ).

Secretory granules in the β-cell undergo maturation in the cytosol. Electron-microscopic (EM) studies have demonstrated that mature granules have a dense crystalline-appearing core with a spacing similar to that of 2-Zn insulin crystals . Newly synthesized insulin is likely to forms crystals with zinc ions that are transported into the maturing secretory granules as demonstrated by a knockout of the zinc transporter (ZnT8) . The crystals reside in the dense core of the β-granules whereas the soluble C-peptide resides in the less dense or clear periphery of the granule . Proinsulin is also known to crystallize with insulin in small amounts, probably as mixed hexamers . Both proinsulin and insulin have the ability to bind zinc and form zinc-coordinated hexamers (two Zn 2+ axial atoms per hexameric unit); the side chain of histidine B10 coordinates axial Zn 2+ ions . The self-assembly and micro-crystallization of zinc-insulin hexamers may be regulated by compartmental pH. The secretory granules possess an intrinsic proton pump, which serves to lower the pH within the granule to pH 5.0-5.5: this is optimal for both prohormone processing and crystallization in vitro . The pH within the rER is less acidic, which promotes thiol-disulfide exchange and hence proinsulin folding with native disulfide pairing.

High ambient glucose concentration in the islets promotes insulin biosynthesis and is the primary regulator of secretion. Elevated glucose concentrations cause an increase in cAMP levels by a mechanism that does not appear to involve activation of adenylate cyclase . cAMP then exerts its effects via a mechanism involving protein kinase A (PKA), leading to the phosphorylation and activation of certain key proteins . Through this complex chain of events, glucose and cAMP (and possibly contributions from the rise in intracellular free calcium and IP3) rapidly increase translation and transcription of insulin mRNA . Insulin mRNA normally turns over slowly, with a half life of approximately 30 hours at normal or below normal levels. However, elevated ambient glucose concentrations increase the half life of insulin mRNA as much as threefold . Calcium dependent exocytosis of secretory granules is the main mechanism of secretion in both glucose-stimulated and basal states . Little or no direct secretion of proinsulin occurs from the rER to the plasma membrane by way of unregulated pathways . Other hormones and chemical substances also play an important role in the regulation of insulin secretion , including glucagon, which is secreted by α -cells in pancreatic islets as discussed in detail in another chapter; glucagon-like peptide (GLP-1: Ref. ); cholecystokinin ; and gastric inhibitory peptide all acting via specific receptors on the β-cell. Inhibitors of insulin secretion include catecholamines (adrenaline and noradrenaline) which interact with adrenergic receptors on the β-cell membrane , and somatostatin which is secreted by -cells of the pancreatic islets . Amylin is also secreted by the β -cell although the regulatory mechanisms for amylin co-secretion are not well understood.

The integrated regulation of insulin secretion in pancreatic β -cells provides an opportunity to develop quantitative data-based computer models relating metabolic sensing to electrophysiological events and to intracellular Ca 2+ dynamics, ultimately leading to exocytosis of the secretory granules . Such analyses provides fundamental insight into the biochemical and biophysical determinants of cytoplasmic transport of organelles .

Secretion of insulin from β -cells is not only an important step in the regulation of glucose homeostasis in healthy individuals, but has also been demonstrated to be impaired (for different reasons) in both Type 1 and Type 2 DM . In fact, in the prediabetic state of Type 1 DM as well as in various forms of Type 2 DM, abnormalities in insulin secretion are an integral component of the pathophysiology . In light of its extensive characterization the β-cell also serves as a model of the secretory process for other cell types.

Insulin is stored in large dense core vesicles (LDCV) and released by exocytosis as described above. Such release is a multistep process that consists of the transport of the secretory vesicles to the plasma membrane, then docking, priming, and finally fusion of the vesicle with the plasma membrane. This process is regulated cooperatively by nutrients, other hormones, and neurotransmitters in association with electrical depolarization of the β-cell and release of insulin. Only a small portion of the insulin stored in vesicles in the β-cell is released, however, even under maximum stimulation. This suggests that systemic insulin levels are regulated by secretion rather than by biosynthesis and is not ordinarily limited by the size of storage pools. However the mechanisms that regulate the directed transport of the insulin granules to the plasma membrane are also not well understood.

The best characterized mechanism of coupling glucose metabolism to insulin secretion resides in the electrical excitability of the β-cell. A large number of ion channels, pumps, and transporters contribute to intracellular calcium concentration, as well as other ions, to set the membrane potential (V m ) of the β-cell; this set point is near -70 mV when extracellular glucose is ~3 mM (see Figure 3). In 1968 Dean and Mathews demonstrated that β-cells are electrically excitable and that glucose controlled this excitability . They also showed that the action potentials of β-cells were increased by sulfonylureas. The use of patch-clamp techniques enabled the electrical activity of the β-cell to be studied in detail, and such studies by Ashcroft, Rorsman and others elucidated the key role of ATP-sensitive potassium (KATP) channels in the resting membrane potential of the β-cell as well as the importance of these channels in the mechanism of insulin secretion (for a recent perspective, see ).

Figure 3 . β -cell ion channels. KATP channel conductance predominates in the resting β-cell, maintaining the resting Vm near the electrochemical potential (EK) for K+ (~ -80 mV). These potassium channels belong to the inward rectifier (Kir) subfamily. They obtain their name since these channels conduct K + current into the cell more readily than to the outside of the cell (inward rectification, as a diode). KATP channels are, however, weak inward rectifiers because they pass a significant amount of current in the outward direction. At -70 mV open KATP channels carry a small amount of outward current, which maintains the hyperpolarized resting potential in the β-cells. A myriad of biochemical and biophysical structure-function studies of recombinant Kir channels has led to a more complete understanding of these channels. The crystal structure of a bacterial Kir analog, the Streptomyces lividens KcSA channel, has been determined ; and the inner pore of a mammalian Kir has likewise been crystallized, and its structure determined . The structures revealed that Kir channels consist of four subunits: each folds into the membrane to define two transmembrane domains (M1 and M2) surrounding a pore loop (P). The four P-loops line the central ion-conducting pore with the M1 and M2 subunits providing outer supports (Figure 4).

Elevation of glucose concentration to >8-10 mM results in depolarization of the β-cell. Glucose is taken up into the β-cell by the GLUT2 transporter and metabolized via glucokinase, glycolysis and in mitochondria to generate ATP. This alters the ATP/ADP ratio, which causes closure of KATP channels and depolarization of the cell via the decreased K + permeability. ATP inhibits KATP channels and ADP opens them . Other nucleotides generated by glucose metabolism (Ap3A: diadenosine triphosphate, and Ap4A: diadenosine tetraphosphate) have been implicated as second messengers mediating the closure of KATP channels, but their significance remains uncertain. Mutations in either the Kir, or SUR1, can result in persistent activation, leading to neonatal hyperinsulinemia and hypoglycemia . It is also known that some Kir channels in β-cells are activated via G-protein coupled receptors as reviewed below.

Figure 4 . Kir channels. KATP channels are unique in the inward rectifier family because they require an auxiliary subunit, the sulfonylurea receptor (SUR1), to function. The SUR1 is a member of the ATP binding cassette (ABC) family of membrane proteins, which includes the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel among others . It was named due to its binding to iodinated glyburide but clearly it is not actually an sufonylurea receptor. KATP channels in the β-cell consists of Kir6.2 subunits surrounded by their accompanying SUR1 subunit (not shown). Pioneering studies by Katz, Miledi, and Douglas first established intracellular Ca 2+ concentrations as a general coupling factor between membrane depolarization and vesicular exocytosis ; this mechanism operates in β-cells (for review, see ). Voltage dependent Ca 2+ channels (Cav) open on membrane depolarization as caused by Kir channel closure; and it is this Ca 2+ influx that leads in turn to exocytosis of the glucose-regulated secretory granules and insulin secretion.

Cav channels are classified one the basis of a low-voltage threshold (LV: activated at more negative potentials) or high-voltage threshold (HV: activated at relatively depolarized potentials). HV channels can be further divided into subclasses: L, N, P, Q, and R . Insulin secretion is inhibited by dihydropyridine-based calcium-channel blocking agents, which inhibit L-type Cav. Although activators of L-type Cav can stimulate insulin secretion, Cav1.3 KO mice exhibited perturbed islet function resulting in glucose intolerance; these mice were smaller than controls and yet maintained glucose-dependent insulin secretion . This phenotype is likely to be secondary either to upregulation of other Cav1.2 channels or to the existence of other mechanisms of insulin secretion unreliant on voltage gated channels as discussed next. It has been suggested that the neuronal type of Ca channels play a direct role in exocytosis .