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Marie Kelly

Publications and source records attributed to Marie Kelly.

5 recordsLinked to original sources
PubMed2004

Regulation of the water channel aquaporin-1: isolation and reconstitution of the regulatory complex.

Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes. The molecular regulation of AQP is unknown. Here we report the isolation, identification and reconstitution of the regulatory complex of AQP-1. AQP-1 and Galphai3 have been implicated in GTP-induced gating of water in zymogen granules (ZG), the secretory vesicles in exocrine pancreas. In the present study, detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, co-isolates AQP-1, PLA2, Galphai3, potassium channel IRK-8, and the chloride channel ClC-2. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO-RS-082, blocked GTP-induced ZG swelling. RBC known to possess AQP-1 at the plasma membrane, swell on exposure to the Galphai-agonist mastoparan, and respond similarly to ONO-RS-082 and glyburide, as ZGs. Liposomes reconstituted with the AQP-1 immunoisolated complex from solubilized ZG, also swell in response to GTP. Glyburide or ONO-RS-082 abolished the GTP effect. Immunoisolate-reconstituted planar lipid bilayers demonstrate conductance, which is sensitive to glyburide and an AQP-1 specific antibody. Our results demonstrate a Galphai3-PLA2 mediated pathway and potassium channel involvement in AQP-1 regulation.

Aminobenzoates↗
PubMed2004

Calcium drives fusion of SNARE-apposed bilayers.

N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) has been proposed to play a critical role in the membrane fusion process. The SNARE complex was suggested to be the minimal fusion machinery. However, there is mounting evidence for a major role of calcium in membrane fusion. Hence, the role of calcium in SNARE-induced membrane fusion was the focus of this study. It revealed that recombinant v-SNARE and t-SNARE, reconstituted into separate liposomes, interact to bring lipid vesicles into close proximity, enabling calcium to drive fusion of opposing bilayers. Exposure to calcium triggered vesicle fusion at both, high potency and efficacy. The half-time for calcium-induced fusion of SNARE-reconstituted vesicles was determined to be approximately 10 s, which is two orders of magnitude faster than in its absence. Calcium acts downstream of SNAREs, since the presence of SNAREs in bilayers increases the potency of calcium-induced vesicle fusion, without significantly influencing its efficacy. Hence, this study suggests that in the physiological state in cells, both SNAREs and calcium operate as the minimal fusion machinery.

Calcium↗
PubMed2003

Reconstituted fusion pore.

Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the pores. Transmission electron microscopy (TEM) further confirmed the presence of fusion pores, and immunoAFM, and immunochemical studies demonstrated t-SNAREs to localize at the base of the fusion pore. In the present study, the morphology, function, and composition of the immunoisolated fusion pore was investigated. TEM studies reveal in further detail the structure of the fusion pore. Immunoblot analysis of the immunoisolated fusion pore reveals the presence of several isoforms of the proteins, identified earlier in addition to the association of chloride channels. TEM and AFM micrographs of the immunoisolated fusion pore complex were superimposable, revealing its detail structure. Fusion pore reconstituted into liposomes and examined by TEM, revealed a cup-shaped basket-like morphology, and were functional, as demonstrated by their ability to fuse with isolated secretory vesicles.

Animals↗
PubMed2002

SNAREs in opposing bilayers interact in a circular array to form conducting pores.

The process of fusion at the nerve terminal is mediated via a specialized set of proteins in the synaptic vesicles and the presynaptic membrane. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion. The structure and arrangement of these SNAREs associated with lipid bilayers were examined using atomic force microscopy. A bilayer electrophysiological setup allowed for measurements of membrane conductance and capacitance. Here we demonstrate that the interaction of these proteins to form a fusion pore is dependent on the presence of t-SNAREs and v-SNARE in opposing bilayers. Addition of purified recombinant v-SNARE to a t-SNARE-reconstituted lipid membrane increased only the size of the globular t-SNARE oligomer without influencing the electrical properties of the membrane. However when t-SNARE vesicles were added to a v-SNARE membrane, SNAREs assembles in a ring pattern and a stepwise increase in capacitance, and increase in conductance were observed. Thus, t- and v-SNAREs are required to reside in opposing bilayers to allow appropriate t-/v-SNARE interactions leading to membrane fusion.

Aluminum Silicates↗