Voltage-gated K+ channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

Bone homeostasis is maintained by a dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Since excessive osteoclast activity is implicated in pathological bone resorption, understanding the mechanism underlying osteoclast differentiation, function and survival is of both scientific and clinical importance. Osteoclasts are monocyte/macrophage lineage cells with a short life span that undergo rapid apoptosis, the rate of which critically determines the level of bone resorption in vivo. However, the molecular basis of rapid osteoclast apoptosis remains obscure. Here we report the role of a BH3-only protein, Noxa (encoded by the Pmaip1 gene), in bone homeostasis using Noxa-deficient mice. Among the Bcl-2 family members, Noxa was selectively induced during osteoclastogenesis. Mice lacking Noxa exhibit a severe osteoporotic phenotype due to an increased number of osteoclasts. Noxa deficiency did not have any effect on the number of osteoclast precursor cells or the expression of osteoclast-specific genes, but led to a prolonged survival of osteoclasts. Furthermore, adenovirus-mediated Noxa overexpression remarkably reduced bone loss in a model of inflammation-induced bone destruction. This study reveals Noxa to be a crucial regulator of osteoclast apoptosis, and may provide a molecular basis for a new therapeutic approach to bone diseases.     

Voltage-gated K channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

KCNQ1, located on 11p15.5, encodes a voltage-gated K⁺ channel with six transmembrane regions, and loss-of-function mutations in the KCNQ1 gene cause hereditary long QT syndrome. Recent genetic studies have identified that single nucleotide polymorphisms located in intron 15 of the KCNQ1 gene are strongly associated with type 2 diabetes and impaired insulin secretion. In order to understand the role of KCNQ1 in insulin secretion, we introduced KCNQ1 into the MIN6 mouse β-cell line using a retrovirus-mediated gene transfer system. In KCNQ1 transferred MIN6 cells, both the density of the KCNQ1 current and the density of the total K⁺ current were significantly increased. In addition, insulin secretion by glucose, pyruvate, or tolbutamide was significantly impaired by KCNQ1-overexpressing MIN6 cells. These results suggest that increased KCNQ1 protein expression limits insulin secretion from pancreatic β-cells by regulating the potassium channel current.     

Is the K permeability of the resting membrane controlled by the excitable K channel?

To test whether or not the potassium permeability of the resting membrane is controlled by the excitable K channels (delayed rectifier), we examined changes in the Na and K permeability ratio, PNa/PK, of the squid axon before and after the excitable K channels were blocked. The blockage of the K channels was accomplished by three independent methods: internal application of tetraethylammonium, internal application of 4-aminopyridine plus Cs, and prolong internal perfusion of NaF solution. The permeability ratio was determined using two different methods: the conventional electrophysiological method and a new method based on the measurements of the hyperpolarizing effect of Na removal. We found that blocking the K channels did not cause a proportional decrease in the K permeability of the resting membrane, suggesting that the semipermeable property of the resting membrane is not determined by the excitable K channels.     

Does decreasing extracellular Na inhibit in vitro renin secretion by affecting NaCa exchange?

It has been shown previously that in vitro renin secretion is inhibited by partial replacement of extracellular NaCl with either mannitol or choline chloride; the inhibitory effect is attributed to an increase in intracellular Ca, resulting from a decreased rate of Ca efflux via Na-Ca exchange. In the present experiments, we confirmed that partially replacing NaCl with choline chloride inhibited renin secretion from rat renal cortical slices, but we found that atropine completely blocked the effect, suggesting cholinergic mediation. Partially replacing NaCl with mannitol also inhibited renin secretion, but the effect could not be attributed specifically to a reduction in extracellular Na. Moreover, the stimulatory effect of Ca chelation on renin secretion was antagonized by either mannitol- or choline chloride -containing incubation media. These results do not support the hypothesis that lowering extracellular Na inhibits renin secretion by a mechanism involving decreased Ca efflux via Na-Ca exchange.     

Regulation of the Na+/K+-ATPase by insulin: Why and how?

The sodium-potassium ATPase (Na⁺/K⁺-ATPase or Na⁺/K⁺-pump) is an enzyme present at the surface of all eukaryotic cells, which actively extrudes Na⁺ from cells in exchange for K⁺ at a ratio of 3:2, respectively. Its activity also provides the driving force for secondary active transport of solutes such as amino acids, phosphate, vitamins and, in epithelial cells, glucose. The enzyme consists of two subunits ( and ) each expressed in several isoforms. Many hormones regulate Na⁺/K⁺ -ATPase activity and in this review we will focus on the effects of insulin. The possible mechanisms whereby insulin controls Na⁺/K⁺-ATPase activity are discussed. These are tissue- and isoform-specific, and include reversible covalent modification of catalytic subunits, activation by a rise in intracellular Na⁺ concentration, altered Na⁺ sensitivity and changes in subunit gene or protein expression. Given the recent escalation in knowledge of insulin-stimulated signal transduction systems, it is pertinent to ask which intracellular signalling pathways are utilized by insulin in controlling Na⁺/K⁺-ATPase activity. Evidence for and against a role for the phosphatidylinositol-3-kinase and mitogen activated protein kinase arms of the insulin-stimulated intracellular signalling networks is suggested. Finally, the clinical relevance of Na⁺/K⁺-ATPase control by insulin in diabetes and related disorders is addressed.     

The effect of ATP-sensitive K+ channels on the electrical burst activity and insulin secretion in pancreatic β-cells

In recent years, the electrical burst activity of the insulin releasing pancreatic β-cells has attracted many experimentalists and theoreticians, largely because of its functional importance, but also because of the nonlinear nature of the burst activity. The ATP-sensitive K⁺ channels are believed to play an important role in electrical activity and insulin release. In this paper, we show by computer simulation how ATP and antidiabetic drugs can lengthen the plateau fraction of bursting and how these chemicals can increase the intracellular Ca²⁺ level in the pancreatic β-cell.     

Activation of activin type IB receptor signals in pancreatic β cells leads to defective insulin secretion through the attenuation of ATP-sensitive K channel activity

In studies of gene-ablated mice, activin signaling through activin type IIB receptors (ActRIIB) and Smad2 has been shown to regulate not only pancreatic β cell mass but also insulin secretion. However, it still remains unclear whether gain of function of activin signaling is involved in the modulation of pancreatic β cell mass and insulin secretion. To identify distinct roles of activin signaling in pancreatic β cells, the Cre-loxP system was used to activate signaling through activin type IB receptor (ActRIB) in pancreatic β cells. The resultant mice (pancreatic β cell-specific ActRIB transgenic (Tg) mice; ActRIBCAβTg) exhibited a defect in glucose-stimulated insulin secretion (GSIS) and a progressive impairment of glucose tolerance. Patch-clamp techniques revealed that the activity of ATP-sensitive K⁺ channels (K_ATP channels) was decreased in mutant β cells. These results indicate that an appropriate level of activin signaling may be required for GSIS in pancreatic β cells, and that activin signaling involves modulation of K_ATP channel activity.     

Does the induction of macrophage lysosomal enzyme secretion by zymosan involve the mannose receptor?

Secretion of the lysosomal enzyme hexosaminidase induced by zymosan is inhibited by mannose and by high concentrations of mannose-6-phosphate and 2-deoxy-glucose but not by mannan. Secretion of hexosaminidase from cells storing previously endocytosed zymosan is unaffected by mannose. Exposure of the cells to mannose does not affect secretion caused by subsequent exposure to zymosan. These findings suggest a role for the mannose-glycoprotein receptor in initiation of lysosomal enzyme secretion by zymosan.     

The interaction of “K+-like” cations with the apical K+ channel in frog skin

The apparent permeability of the apical K+ channel in the abdominal skin of the frog (Rana temporaria) for different monovalent cations was tested by comparing the short-circuit current (SCC) obtained after imposition of serosally directed ionic concentration gradients. Furthermore, the SCC was subjected to noise analysis. Of various cations tested, only the "K+-like" ions NH+4, Rb+ and Tl+, besides K+, were found to permeate the apical K+ channel, as reflected by SCC- and fluctuation analysis: (i) The SCC could be depressed by addition of the K+-channel blocker Ba2+ to the mucosal solution. (ii) With the K+-like ions (Ringer's concentration), a spontaneous Lorentzian noise was observed. Plateau values were similar for K+ and Tl+, and smaller for NH+4 and Rb+. The corner frequencies clearly increased in the order K+ less than NH+4 less than Tl+ much less than Rb+. The SCC dose-response relationships revealed a Michaelis-Menten-type current saturation only for pure K+- or Tl+-Ringer's solutions as mucosal medium, whereas a more complicated SCC behavior was seen with Rb+ and especially, NH+4. For K+-Tl+ mixtures an anomalous mole-fraction relationship was observed: At low [Tl+]/[K+] ratios, Tl+ ions appeared to inhibit competitively the K+ current while, at high [Tl+]/[K+] ratios, Tl+ seemed to be a permeant cation. This feature was also detected in the noise analysis of K+-Tl+ mixtures. Long-term exposure to mucosal Tl+ resulted in an irreversible deterioration of the tissue. The SCC depression by Ba2+ was of a simple saturation-type characteristic with, however, different half-maximal doses (NH+4 less than K+ less than Rb+). Ba2+ induced a "blocker noise" in presence of all permeant cations with corner frequencies that depended on the Ba2+ concentration. A linear increase of the corner frequencies of the Ba2+-induced noise with increasing Ba2+ concentration was seen for NH+4, Rb+ and K+. With the assumption of a pseudo two-state model for the Ba2+ blockade the on- and off-rate constants for the Ba2+ interaction with the NH+4/Rb+/K+ channel were calculated and showed marked differences, dependent on the nature of the permeant ion. The specific problems with Tl+ prevented such an analysis but SCC- and noise data indicated a comparably poor efficiency of Ba2+ as Tl+-current inhibitor. We attempted a qualitative analysis of our results in terms of a "two-sites, three-barriers" model of the apical K+ channel in frog skin.     

L-histidine sensing by calcium sensing receptor inhibits voltage-dependent calcium channel activity and insulin secretion in β-cells

AimsOur goal was to test the hypothesis that the histidine-induced activation of calcium sensing receptor (CaR) can regulate calcium channel activity of L-type voltage dependent calcium channel (VDCC) due to increased spatial interaction between CaR and VDCC in β-cells and thus modulate glucose-induced insulin secretion.Main methodsRat insulinoma (RINr1046-38) insulin-producing β-cells were cultured in RPMI-1640 medium on 25 mm diameter glass coverslips in six-well culture plates in a 5% CO₂ incubator at 37 °C. The intracellular calcium concentration, [Ca²⁺]_i, was determined by ratio fluorescence microscopy using Fura-2AM. The spatial interactions between CaR and L-type VDCC in β-cells were measured by immunofluorescence confocal microscopy using a Nikon C1 laser scanning confocal microscope. The insulin release was determined by enzyme-linked immunosorbent assay (ELISA).Key findingsThe addition of increasing concentrations of L-histidine along with 10 mM glucose resulted in 57% decrease in [Ca²⁺]_i. The confocal fluorescence imaging data showed 5.59 to 8.62-fold increase in colocalization correlation coefficient between CaR and VDCC in β-cells exposed to L-histidine thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. The insulin ELISA data showed 54% decrease in the 1st phase of glucose-induced insulin secretion in β-cells exposed to increasing concentrations of L-histidine.SignificanceL-histidine-induced increased spatial interaction of CaR with VDCC can inhibit calcium channel activity of VDCC and consequently regulate glucose-induced insulin secretion by β-cells. The L-type VDCC could therefore be a potential therapeutic target in diabetes.     

Modulation of the inwardly rectifying K+ channel in isolated human atrial myocytes by α1-adrenergic stimulation

We have examined the ₁-adrenergic modulation of the inwardly-rectifying K⁺ channel (I _K1) in isolated human atrial myocytes using the patch clamp technique. ₁-Adrenergic agonist methoxamine produced action potential prolongation and a depolarization of the resting membrane potential. Under whole-cell voltage clamp conditions, bath application of methoxamine can inhibit macroscopic I _K1. The methoxamine-induced inhibition was reversible and concentration dependent, with the concentration for half-maximal inhibition being 18 m. The methoxamine-induced inhibition of I _K1 was prevented by bath application of ₁-adrenergic blocker prazosin. The current was similarly inhibited by phorbol ester (PMA), an activator of protein kinase C (PKC). In contrast, methoxamine failed to inhibit the current in the presence of a specific PKC inhibitor H-9, suggesting that PKC is involved in the methoxamine-induced inhibition of I _K1. In single channel recording from cell attached patches, bath-applied methoxamine could suppress I _K1 channels by decreasing the frequency and duration of bursting without affecting unitary amplitude. Direct application of purified PKC to excised inside-out patches inhibited channel activity similar to methoxamine in cell-attached patches. The PKC selective inhibitor, PKC19-36, prevented the PKC-induced inhibition of the channel. We conclude that human atrial I _K1 can be inhibited by ₁-adrenergic stimulation via PKC-dependent pathways.     

Does motilin control interdigestive pepsin secretion in the dog?

Pepsin output in the Heidenhain pouch, plasma motilin concentration, and contractile activity in the pouch and the main stomach were investigated in five dogs. During the interdigestive state, the pepsin output was significantly increased with a cyclic increase in contractile activity in both the pouch and main stomach at approximately 100-min intervals. The plasma immunoreactive motilin (IRM) concentration fluctuated during the interdigestive state, and, peaks of IRM concentration coincided with the maximum pepsin secretory activity. Exogenous administration of motilin (0.5 micrograms/kg-hr) increased contractile activity in the main stomach and pouch quite similar to the natural interdigestive migrating contractions (IMC), and increased pepsin output significantly. Atropine pre-treatment suppressed the naturally-occurring and motilin-induced pepsin output and contractions in the pouch. It is concluded that pepsin output and contractions in the Heidenhain pouch increase in close association with the IMC in the main stomach during the interdigestive state and these cyclic motor and secretory events in the vagally denervated fundic pouch are most likely regulated by motilin through the intramural cholinergic pathway.     

The Gárdos channel: a review of the Ca2+-activated K+ channel in human erythrocytes

Ca²⁺-dependent K⁺ efflux from human erythrocytes was first described in the 1950s. Subsequent studies revealed that a K⁺-specific membrane protein (the Gárdos channel) was responsible for this phenomenon (the Gárdos effect). In recent years several types of Ca-activated K⁺ channel have been identified and studied in a wide range of cells, with the erythrocyte Gárdos channel serving as both a model for a broader physiological perspective, and an intriguing component of erythrocyte function.The existence of this channel has raised a number of questions. For example, what is its role in the establishment and maintenance of ionic distribution across the red cell membrane? What role might it play in erythrocyte development? To what extent is it active in circulating erythrocytes? What are the cell-physiological implications of its dysfunction?This review summarises current knowledge of this membrane protein with respect to its function and structure, its physiological roles (some putative) and its contribution to various disease states, and it provides an introduction to adaptable NMR methods, which is our own area of technical expertise, for such ion transport analysis.     

Does magnesium dysbalance participate in the development of insulin resistance in early stages of renal disease?

We investigated the potential role of magnesium (Mg) dysbalance in the pathogenesis of insulin resistance (IR) in patients with mildly-to-moderately decreased renal function (creatinine: 142.8+/-11.0 mmol/l). The data were compared to those of 8 age- and sex-matched healthy controls (CTRL). The standard oral glucose tolerance test (oGTT) was performed in 61 patients. Twenty-two patients were classified as IR according to their values on fasting and after-load immunoreactive insulin concentrations. Serum and total erythrocyte Mg (tErMg) (atomic absorption spectro-photometry) and free erythrocyte Mg (fErMg) concentrations ((31) P NMR spectroscopy) were determined prior to and two hours after the glucose load. Ten out of 39 insulin-sensitive (IS) patients, but only one out of 22 insulin-resistant (IR) patients, had a low basal fErMg concentration (<162.2 micromol/l, chi2, p<0.01). IR patients had higher serum Mg, total erythrocyte Mg and bound erythrocyte Mg (bErMg) concentrations (both before and after glucose load) when compared with the IS group. Both groups responded to the glucose load with a significant decrease in serum Mg concentration (within the normal range), while the IR group also exhibited a decline in tErMg and bErMg. The mean sum of insulin needed to metabolize the same glucose load correlated positively with tErMg (r=0.545, p<0.01) and bErMg (r=0.560, p<0.01) in the IR patients. It is concluded that, at an early stage of renal dysfunction, IR is not associated with the decline in free erythrocyte Mg concentration, but the magnesium handling in red blood cells is altered.     

Role of terminal dipole charges in aggregation of α-helix pair in the voltage gated K+ channel

The voltage sensor domain (VSD) of the potassium ion channel KvAP is comprised of four (S1–S4) α-helix proteins, which are encompassed by several charged residues. Apart from these charges, each peptide α-helix having two inherent equal and opposite terminal dipolar charges behave like a macrodipole. The activity of voltage gated ion channel is electrostatic, where all the charges (charged residues and dipolar terminal charges) interact with each other and with the transmembrane potential. There are evidences that the role of the charged residues dominate the stabilization of the conformation and the gating process of the ion channel, but the role of the terminal dipolar charges are never considered in such analysis. Here, using electrostatic theory, we have studied the role of the dipolar terminal charges in aggregation of the S3b–S4 helix pair of KvAP in the absence of any external field (V = 0). A system attains stability, when its potential energy reaches minimum values. We have shown that the presence of terminal dipole charges (1) change the total potential energy of the charges on S3b–S4, affecting the stabilization of the α-helix pair within the bilayer lipid membrane and (2) the C- and the N-termini of the α-helices favor a different dielectric medium for enhanced stability. Thus, the dipolar terminal charges play a significant role in the aggregation of the two neighboring α-helices.     

Insulin secretion: Feed-forward control of insulin biosynthesis?

It has long been accepted wisdom that insulin secreted from islet beta cells has either no effect, or an inhibitory feedback effect, on insulin synthesis and secretion. Recent work suggests, instead, that secreted insulin acts directly on beta cells, via its own receptor, to enhance insulin production in an autocrine feed-forward loop.     

Is the Ca2+-sensitive K+ channel under metabolic control in human red cells?

It is widely known that a rise in internal Ca2+ leads to an increased K+ permeability of human red blood cells [1,2,3]. Binding of Ca2+ to some membrane receptors is required for the opening of the K+ channel [4]. This requirement, however, seems to alter after "ageing" red cells in vitro in acid-citrate-dextrose solutions. Thus, the free Ca2+ concentration producing half-maximal effect on K+ permeability ([Ca2+]K+-50) of 4-weeks stored cells is approx. 2.10(-4) M (calculated from ref. 3 using 50% free Ca2+ according to Schatzmann [5]); nearly ten times lower than that reported for fresh cells [6]. This observation suggests the possibility that the K+ channel may become more sensitive to Ca2+ on cold storage. The experiments described below support this idea.