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Functional Implications of SNARE Protein Interactions with N-type Calcium Channels in the xenopus Neuromuscular Juntion

Keith, Ryan K. (2004) Functional Implications of SNARE Protein Interactions with N-type Calcium Channels in the xenopus Neuromuscular Juntion. Master's Thesis, University of Pittsburgh. (Unpublished)

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Classical Neurotransmitter (NT) release is dependent upon the influx of calcium (Ca2+) through voltage gated Ca2+ channels. Once the local concentration of intracellular calcium increases to approximately 100 ƒÝM the calcium sensor detects the calcium ions and the process of vesicle fusion goes to completion. As such, transmitter release is very sensitive to alterations in the influx of Ca2+ into the presynaptic active zone. The objective of these experiments is to study how a known interaction between core complex proteins and presynaptic calcium channels might be important in the modulation of vesicle fusion. Syntaxin 1A (stx1A) is part of the minimal protein machinery necessary for vesicle fusion. Stx1A has also been shown to interact with Ca2+ channels in vitro. The functional significance of this interaction however is unclear. Work in expression systems supports the idea that stx1A functionally interacts with the N-type Ca2+ channel causing it to become inactivated. It has also been proposed that the Ca2+ channel- syntaxin interaction functions to co-localize the release machinery in the vicinity of the Ca2+ channel. Essentially, this would put the trigger for release next to the source of Ca2+ influx. To help discern whether this stx1A-N-type calcium channel interaction alters channel gating or simply serves to co-localize the two proteins, I employed a combination of molecular manipulations of syntaxin and the calcium channel. Electrophysiological recordings assaying NT release were then used to determine how these manipulations altered vesicle fusion. Data from two sets of experiments yielded the following information. 1) When the portion of the N-type Ca2+ channel that binds with syntaxin 1A was injected into Xenopus embryos, the strength of NT release onto the postsynaptic cell appeared to decrease. This was suggested by increases in both paired pulse facilitation and tetanic potentiation after injection of the competitive peptide. 2) Recordings of paired pulse facilitation and tetanic potentiation with injection of a mutant form of stx1A, which couples with the calcium channel but which has been shown not to modulate the channel, suggest an increase in synaptic strength as assayed through a tendency for both paired pulse and tetanic potentiation to decrease following injections of the mutant. Taken together, these results suggest that the interaction between stx1A and N-type calcium channels influences the level of NT release. They do not however, definitively distinguish between the interaction strictly being structural or modulatory. Instead, the data suggests that both may be occurring in vivo and a balance between these modulatory influences determines that what may be physiologically important.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Keith, Ryan
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairMeriney, Stephenmeriney@bns.pitt.eduMERINEY
Committee MemberJohnson, Jonjohnson@bns.pitt.eduJJOHNSON
Committee MemberLiu,
Date: 29 June 2004
Date Type: Completion
Defense Date: 24 February 2004
Approval Date: 29 June 2004
Submission Date: 12 March 2004
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Faculty of Arts and Sciences > Neuroscience
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: calcium; SNARE; synprint; syntaxin
Other ID:, etd-03122004-132357
Date Deposited: 10 Nov 2011 19:32
Last Modified: 15 Nov 2016 13:37


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