Investigation of Prestin Membrane Topology by Substituted Cysteine Accessibility Method.

Prestin, an integral membrane motor protein in the outer hair cells of the inner ear, is proposed to be crucial for cochlear amplification. In support of this, in prestin knock out mice, there is a 40-60 dB loss of hearing sensitivity. Little is known about prestin’s structure-function relationship, and its membrane topology is highly debated. Two research groups have developed two contradicting topology models: the Dallos 12 transmembrane domain model, and the Santos-Sacchi 10 transmembrane domain model. These two models are grossly different, in that the extracellular loops in one model are intracellular loops in the other. I hypothesize that prestin topology is a combination of the two published topology models. A methodology that has not yet been used to study prestin’s topological map is substituted cysteine accessibility method. This method requires the introduction of cysteine residues into predicted extracellular or intracellular loops of prestin. Plasmids containing the prestin sequence with the appropriate sitedirected mutations were transiently transfected into a mammalian cell line. Upon reacting the transfected cells with a membrane impermeable thiol-reactive probe, and a fluorescent label, residues were identified as accessible (extracellular) or inaccessible (intracellular). The resulting fluorescence was quantified and analyzed. It was found that residue A170 is accessible, and that residues C124 and G366 are inaccessible. The double mutation, C124A/A170C, helped to confirm that residue C124 is inaccessible and A170 is accessible. The data suggests that the mutations A73C and C124A/G366C interfere with oligomerization, are misfolded, or are not properly plasma membrane inserted. This report supports the Dallos 12 transmembrane domain prestin topology model.

Grantor dc:publisher

Creighton University

Year dc:date.issued

2008

Author dc:creator

• Rossino, Danielle A.

Advisor dc:contributor.advisor

• Hallworth, Richard

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/10504/87

OAI identifier oai:identifier

oai:cdr.creighton.edu:10504/87