Craig A Aspinwall

PMID: 17522836;Abstract:

Aims/hypothesis: The pancreatic beta cell ATP-sensitive potassium (K ATP) channel, composed of the pore-forming α subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory β subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate KATP channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. Materials and methods: Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. Results: Oocytes expressing Kir6.2ΔC26 K332A repeatedly generated K⁺currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 μmol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2ΔC26), Kir6.2ΔC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2ΔC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type KATP channel and Kir6.2ΔC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. Conclusions/interpretation: These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region. © 2007 Springer-Verlag.

Abstract:

A highly efficient contrast agent for magnetic resonance imaging was developed by encapsulating gadolinium within a stabilized porous liposome. The highly porous membrane leads to a high relaxivity of the encapsulated Gd. The stability of the liposome was improved by forming a polymer network within the bilayer membrane. © 2014 The Royal Society of Chemistry.

A porous phospholipid nanoshell (PPN) sensor functionalized with a specific aptamer sensor agent was prepared for rapid detection of Hg(2+) in human urine with minimal sample preparation. Aptamer sensors provide an important class of optical transducers that can be readily and reproducibly synthesized. A key limitation of aptamer sensors, and many other optical sensors, is the potential of biofouling or biodegradation when used in complex biological matrices such as serum or urine, particularly when high levels of nucleases are present. We prepared Hg(2+)-responsive, PPN-encapsulated aptamer sensors that overcome these limitations. PPNs provide a protective barrier to encapsulate the aptamer sensor in an aqueous environment free of diffusional restrictions encountered with many polymer nanomaterials. The unique porous properties of the PPN membrane enable ready and rapid transfer of small molecular weight ions and molecules into the sensor interior while minimizing the macromolecular interactions between the transducer and degradants or interferents in the exterior milieu. Using Hg(2+)-responsive, PPN-encapsulated aptamer sensors, we were able to detect sub-100 ppb (chronic threshold limit from urine test) Hg(2+) in human urine with no sample preparation, whereas free aptamer sensors yielded inaccurate results due to interferences from the matrix. The PPN architecture provides a new platform for construction of aptamer-functionalized sensors that target low molecular weight species in complex matrices, beyond the Hg(2+) demonstrated here.

PMID: 9395458;Abstract:

The effects of extracellular Zn²⁺ and pH and intravesicular pH on insulin and 5-hydroxytryptamine (5-HT) secretion from pancreatic beta cells were investigated. Insulin and 5-HT secretion from single cells was detected by amperometry as a series of current spikes corresponding to detection of multimolecular packets secreted by exocytosis. Spike width was used as a measure of the kinetics of clearance from the cell and the area of spikes as a measure of amount released. Changes in extracellular pH from 6.9 to 7.9 caused insulin spikes to become narrower with no change in area, whereas the same treatments had no effect on 5-HT secretion. Treatment of cells with Bafilomycin A1 or N-ethylmaleimide, both of which are expected to increase intravesicular pH by inhibiting V-type H⁺-ATPase, had no effect on 5-HT secretion but caused insulin spikes to become more narrow. These results indicate that exposure to high pH, whether intravesicular or extracellular, accelerates release of insulin during exocytosis without affecting the amount of insulin released. Increasing extracellular Zn²⁺ concentration from 0 to 25 μM increased the width and decreased the area of insulin spikes without affecting 5-HT secretion. Zn²⁺ effects were likely exerted through a common-ion effect on Zn²⁺-insulin dissociation. It was concluded that intravesicular storage conditions and extracellular ions can affect free insulin concentration in the vicinity of beta cells during secretion.