SEDLIN forms homodimers: characterisation of SEDLIN mutations and their interactions with transcription factors MBP1, PITX1 and SF1

BACKGROUND SEDLIN, a 140 amino acid subunit of the Transport Protein Particle (TRAPP) complex, is ubiquitously expressed and interacts with the transcription factors c-myc promoter-binding protein 1 (MBP1), pituitary homeobox 1 (PITX1) and steroidogenic factor 1 (SF1). SEDLIN mutations cause X-linked spondyloepiphyseal dysplasia tarda (SEDT). METHODOLOGY/PRINCIPAL FINDINGS We investigated the effects of 4 missense (Asp47Tyr, Ser73Leu, Phe83Ser and Val130Asp) and the most C-terminal nonsense (Gln131Stop) SEDT-associated mutations on interactions with MBP1, PITX1 and SF1 by expression in COS7 cells. Wild-type SEDLIN was present in the cytoplasm and nucleus and interacted with MBP1, PITX1 and SF1; the SEDLIN mutations did not alter these subcellular localizations or the interactions. However, SEDLIN was found to homodimerize, and the formation of dimers between wild-type and mutant SEDLIN would mask a loss in these interactions. A mammalian SEDLIN null cell-line is not available, and the interactions between SEDLIN and the transcription factors were therefore investigated in yeast, which does not endogenously express SEDLIN. This revealed that all the SEDT mutations, except Asp47Tyr, lead to a loss of interaction with MBP1, PITX1 and SF1. Three-dimensional modelling studies of SEDLIN revealed that Asp47 resides on the surface whereas all the other mutant residues lie within the hydrophobic core of the protein, and hence are likely to affect the correct folding of SEDLIN and thereby disrupt protein-protein interactions. CONCLUSIONS/SIGNIFICANCE Our studies demonstrate that SEDLIN is present in the nucleus, forms homodimers and that SEDT-associated mutations cause a loss of interaction with the transcription factors MBP1, PITX1 and SF1.This work was supported by the Oliver Bird Fund (Studentship No. RHE/00029/G), The Nuffield Foundation (J.J.), Arthritis Research Campaign (Grant ID 16438) (M.A.N. and R.V.T.), European Community Framework 7 programme grant TREAT-OA (HEALTH-F2-2008-00) (M.A.N. and R.V.T.) and the Medical Research Council (J.J., M.A.N. and R.V.T.). J.J. was an Oliver Bird funded PhD student

Activation of Ca2+-Dependent K+ Channels Contributes to Rhythmic Firing of Action Potentials in Mouse Pancreatic β Cells

We have applied the perforated patch whole-cell technique to β cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K+ conductance. The current was dependent on Ca2+ influx but unaffected by apamin and charybdotoxin, two blockers of Ca2+-activated K+ channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K+ channels) but partially (>60%) blocked by high (10–20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca2+-activated K+ current plays an important role in the generation of oscillatory electrical activity in the β cell

Regulated Exocytosis of GABA-containing Synaptic-like Microvesicles in Pancreatic β-cells

We have explored whether γ-aminobutyric acid (GABA) is released by regulated exocytosis of GABA-containing synaptic-like microvesicles (SLMVs) in insulin-releasing rat pancreatic β-cells. To this end, β-cells were engineered to express GABAA-receptor Cl−-channels at high density using adenoviral infection. Electron microscopy indicated that the average diameter of the SLMVs is 90 nm, that every β-cell contains ∼3,500 such vesicles, and that insulin-containing large dense core vesicles exclude GABA. Quantal release of GABA, seen as rapidly activating and deactivating Cl−-currents, was observed during membrane depolarizations from −70 mV to voltages beyond −40 mV or when Ca2+ was dialysed into the cell interior. Depolarization-evoked GABA release was suppressed when Ca2+ entry was inhibited using Cd2+. Analysis of the kinetics of GABA release revealed that GABA-containing vesicles can be divided into a readily releasable pool and a reserve pool. Simultaneous measurements of GABA release and cell capacitance indicated that exocytosis of SLMVs contributes ∼1% of the capacitance signal. Mathematical analysis of the release events suggests that every SLMV contains 0.36 amol of GABA. We conclude that there are two parallel pathways of exocytosis in pancreatic β-cells and that release of GABA may accordingly be temporally and spatially separated from insulin secretion. This provides a basis for paracrine GABAergic signaling within the islet

Corelease and Differential Exit via the Fusion Pore of GABA, Serotonin, and ATP from LDCV in Rat Pancreatic β Cells

The release of γ-aminobutyric acid (GABA) and ATP from rat β cells was monitored using an electrophysiological assay based on overexpression GABA(A) or P(2)X(2) receptor ion channels. Exocytosis of LDCVs, detected by carbon fiber amperometry of serotonin, correlated strongly (∼80%) with ATP release. The increase in membrane capacitance per ATP release event was 3.4 fF, close to the expected capacitance of an individual LDCV with a diameter of 0.3 μm. ATP and GABA were coreleased with serotonin with the same probability. Immunogold electron microscopy revealed that ∼15% of the LDCVs contain GABA. Prespike “pedestals,” reflecting exit of granule constituents via the fusion pore, were less frequently observed for ATP than for serotonin or GABA and the relative amplitude (amplitude of foot compared to spike) was smaller: in some cases the ATP-dependent pedestal was missing entirely. An inward tonic current, not dependent on glucose and inhibited by the GABA(A) receptor antagonist SR95531, was observed in β cells in clusters of islet cells. Noise analysis indicated that it was due to the activity of individual channels with a conductance of 30 pS, the same as expected for individual GABA(A) Cl(−) channels with the ionic gradients used. We conclude that (a) LDCVs accumulate ATP and serotonin; (b) regulated release of GABA can be accounted for by exocytosis of a subset of insulin-containing LDCVs; (c) the fusion pore of LDCVs exhibits selectivity and compounds are differentially released depending on their chemical properties (including size); and (d) a glucose-independent nonvesicular form of GABA release exists in β cells

SUR1 Regulates PKA-independent cAMP-induced Granule Priming in Mouse Pancreatic B-cells.

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS–insensitive) component correlated with a rapid increase in membrane capacitance of ~80 fF that plateaued within ~200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the Kd values were 6 and 29 µM for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1-/- mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1-/- mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl- into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane KATP-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca2+-induced exocytosis

SEDLIN Forms Homodimers: Characterisation of SEDLIN Mutations and Their Interactions with Transcription Factors MBP1, PITX1 and SF1

BACKGROUND: SEDLIN, a 140 amino acid subunit of the Transport Protein Particle (TRAPP) complex, is ubiquitously expressed and interacts with the transcription factors c-myc promoter-binding protein 1 (MBP1), pituitary homeobox 1 (PITX1) and steroidogenic factor 1 (SF1). SEDLIN mutations cause X-linked spondyloepiphyseal dysplasia tarda (SEDT). METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effects of 4 missense (Asp47Tyr, Ser73Leu, Phe83Ser and Val130Asp) and the most C-terminal nonsense (Gln131Stop) SEDT-associated mutations on interactions with MBP1, PITX1 and SF1 by expression in COS7 cells. Wild-type SEDLIN was present in the cytoplasm and nucleus and interacted with MBP1, PITX1 and SF1; the SEDLIN mutations did not alter these subcellular localizations or the interactions. However, SEDLIN was found to homodimerize, and the formation of dimers between wild-type and mutant SEDLIN would mask a loss in these interactions. A mammalian SEDLIN null cell-line is not available, and the interactions between SEDLIN and the transcription factors were therefore investigated in yeast, which does not endogenously express SEDLIN. This revealed that all the SEDT mutations, except Asp47Tyr, lead to a loss of interaction with MBP1, PITX1 and SF1. Three-dimensional modelling studies of SEDLIN revealed that Asp47 resides on the surface whereas all the other mutant residues lie within the hydrophobic core of the protein, and hence are likely to affect the correct folding of SEDLIN and thereby disrupt protein-protein interactions. CONCLUSIONS/SIGNIFICANCE: Our studies demonstrate that SEDLIN is present in the nucleus, forms homodimers and that SEDT-associated mutations cause a loss of interaction with the transcription factors MBP1, PITX1 and SF1

Palmitate-Induced β-Cell Dysfunction Is Associated with Excessive NO Production and Is Reversed by Thiazolidinedione-Mediated Inhibition of GPR40 Transduction Mechanisms

BACKGROUND: Type 2 diabetes often displays hyperlipidemia. We examined palmitate effects on pancreatic islet function in relation to FFA receptor GPR40, NO generation, insulin release, and the PPARgamma agonistic thiazolidinedione, rosiglitazone. PRINCIPAL FINDINGS: Rosiglitazone suppressed acute palmitate-stimulated GPR40-transduced PI hydrolysis in HEK293 cells and insulin release from MIN6c cells and mouse islets. Culturing islets 24 h with palmitate at 5 mmol/l glucose induced beta-cell iNOS expression as revealed by confocal microscopy and increased the activities of ncNOS and iNOS associated with suppression of glucose-stimulated insulin response. Rosiglitazone reversed these effects. The expression of iNOS after high-glucose culturing was unaffected by rosiglitazone. Downregulation of GPR40 by antisense treatment abrogated GPR40 expression and suppressed palmitate-induced iNOS activity and insulin release. CONCLUSION: We conclude that, in addition to mediating acute FFA-stimulated insulin release, GPR40 is an important regulator of iNOS expression and dysfunctional insulin release during long-term exposure to FFA. The adverse effects of palmitate were counteracted by rosiglitazone at GPR40, suggesting that thiazolidinediones are beneficial for beta-cell function in hyperlipidemic type 2 diabetes

Low-frequency electromagnetic field effects on ion channels and calcium oscillations in cells

It is well established that many life forms, lower as well as higher, are capable of sensing electromagnetic fields and to use them in order to sustain their survival. Some of them, such as electric rays, utilise this feature to locate pray and display the capability to detect electric fields as low as 0.1 mV/m generated by normal bodily functions of pray fish. Epidemiological research indicates that the alternating magnetic fields within mT range can be a health risk factor. The results of numerous experimental investigations suggest that in a variety of biological systems, including humans, the electromagnetic fields may interfere with the functioning of cells through the calcium signalling pathways. Because of the electromagnetic properties of biological tissue, some of the most probable molecular sites for the field coupling with these pathways are ion channels located within various cellular membranes. In this study the interaction of low-frequency weak electromagnetic signals with ion channels and their effects on intracellular Ca2+ intracellular oscillations in human leukemic T cell line Jurkat have been investigated theoretically, by numerical simulations, and experimentally with the help of microfluorimetry and digital imaging. In T lymphocytes, similarly to many other types of a cell, an external stimulation may lead to oscillatory changes of intracellular Ca2+ concentration. It is generally accepted that both the oscillation frequency and amplitude may be involved in the transduction of the external signal to some intracellular target. In this study it is shown that poly-L-lysine, a highly positively charged peptide widely used to enhance the attachment of cells to supporting surface, under certain conditions forces Jurkat T cells into a state of sustained intracellular Ca2+ spiking for up to 3 hours. Such cells represent the cytosolic calcium oscillator, and they are used in this study to investigate the effects of 50 Hz alternating magnetic fields on calcium signalling in cells. Microfluorimetric recording of intracellular Ca2+ in oscillating cells demonstrated that the total spectral power of the cytosolic Ca2+ oscillator was reduced by exposure of the cells to an alternating magnetic field and that the effect increased in an explicit dose response manner. Many biochemical processes and subcellular components are involved in sustaining the oscillatory behaviour of intracellular Ca2+. The precise mechanisms and components involved in mediating the field effects on oscillation magnitude remain elusive. One of the main problems in understanding the biological effects of the low-frequency weak electromagnetic fields is the low energy of the perturbations inflicted on subcellular components by these fields. The amplification of primary signals within cells is necessary prior to inducement of any biological response. It is shown in this study theoretically and by numerical simulations that such amplification can be achieved by a system of identical ion channels embedded in a cell membrane. The amplification of the signal amplitude relative to the noise amplitude is found to be proportional to the square root of the number of channels modulated and inversely proportional to the square root of the sum of the channel dwell times. Further, the possibility that the cytosolic calcium oscillator is the subcellular system that responds to this amplified signal is explored. Using mathematical models of intracellular Ca2+ oscillations, it is shown by numerical simulations of the non-linear system of differential equations that the cytosolic calcium oscillator is a sensitive detector of external influencies. Its response to the external forcing signal exhibits a multitude of frequency and amplitude "windows". It also exhibits resonance behaviour. From these results it is concluded that forcing of the cytosolic calcium oscillator by external alternating magnetic and electric fields amplified by mechanisms available to cells may be one of the mechanisms of field interaction with biological systems

Exocytosis from pancreatic β-cells: mathematical modelling of the exit of low-molecular-weight granule content

Pancreatic β-cells use Ca2+-dependent exocytosis of large dense core vesicles to release insulin. Exocytosis in β-cells has been studied biochemically, biophysically and optically. We have previously developed a biophysical method to monitor release of endogenous intragranular constituents that are co-released with insulin. This technique involves the expression of ionotropic membrane receptors in the β-cell plasma membrane and enables measurements of exocytosis of individual vesicles with sub-millisecond resolution. Like carbon fibre amperometry, this method allows fine details of the release process, like the expansion of the fusion pore (the narrow connection between the granule lumen and the extracellular space), to be monitored. Here, we discuss experimental data obtained with this method within the framework of a simple mathematical model that describes the release of low-molecular constituents during exocytosis of the insulin granules. Our findings suggest that the fusion pore functions as a molecular sieve, allowing differential release of low- and high-molecular-weight granule constituents