Please use this identifier to cite or link to this item: http://hdl.handle.net/2381/29943
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dc.contributor.authorWykes, Robert C. E.en
dc.date.accessioned2014-12-15T10:34:48Z-
dc.date.available2014-12-15T10:34:48Z-
dc.date.issued2004en
dc.identifier.urihttp://hdl.handle.net/2381/29943-
dc.description.abstractCalmodulin is a molecule implicated in regulating voltage-gated calcium channels (VGCCs) and the exocytotic machinery to fine tune neurotransmitter release. I have investigated the role this molecule plays in stimulus-secretion coupling in bovine adrenal chromaffin cells by over-expressing either wild-type (CaMwt), or a mutated calmodulin (CaM1234), rendered incapable of binding calcium by adenoviral infection. Stimulus-evoked secretion was monitored by combined measurements of membrane capacitance (DeltaC m) and voltage-clamp recording of calcium currents in single cells. Cells were clamped in either the perforated patch or whole-cell configuration and calcium-dependent exocytosis evoked by single depolarising voltage steps or trains of depolarisations. I show that the exocytotic efficiency, derived by dividing DeltaCm by the intergral of the calcium current, is reduced for N-type channels compared to P/Q-type channels. Cation substitution experiments revealed that pharmacologically isolated N-type channels displayed the most profound sensitivity to calcium-dependent inactivation. Studies aimed at eluding the molecular mechanisms underlying calcium-dependent inactivation show that inhibiting calcinuerin by 20 mins preincubation with 1muM cyclosporine A or by introducing 30muM calmodulin inhibitory peptides through the patch pipette did not significantly reduce the level of whole cell calcium-dependent inactivation. In contrast, adenoviral mediated expression of a mutant calmodulin deficient in calcium binding resulted in a highly significant reduction in N-type, but not P/Q-type channel inactivation. This is the first time that calmodulin has been shown to regulate endogenously expressed N-type calcium channels. These results are consistent with calmodulin acting directly to control N-type channel inactivation and therefore limit this channel's ability to couple to exocytosis during prolonged stimulation. Ca2+/calmodulin was also found to interact with the secretory machinery. Expression of CaM 1234 significantly reduced the exocytotic efficiency of brief depolarisations (≤ 100ms), however, the exocytotic efficiency to longer depolarisations (≥ 200ms) was not significantly different between cells expressing CaM 1234 and CaMwt. This suggests that Ca2+ -Calmodulin is required for filling and/or release from a rapidly releasable pool of vesicles which is easily depleted, but not from the slowly releasable pool which dominates exocytotic responses measured with prolonged responses.en
dc.language.isoenen
dc.rightsCopyright © the author. All rights reserved.en
dc.sourceProQuesten
dc.titleCalmodulin regulation of calcium channels and neurotransmitter release in bovine adrenal chromaffin cellsen
dc.typeThesisen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhDen
dc.date.award2004en
dc.publisher.departmentCell Physiology and Pharmacologyen
dc.publisher.institutionUniversity of Leicesteren
dc.identifier.proquestU186303en
dc.identifier.cataloguecontrola720382en
Appears in Collections:Theses, Dept. of Cell Physiology and Pharmacology
Leicester Theses

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