Prestin in HEK cells is an obligate tetramer

Richard J. Hallworth, Michael G. Nichols

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

The unusual membrane motor protein prestin is essential for mammalian hearing and for the survival of cochlear outer hair cells. While prestin has been demonstrated to be a homooligomer, by Western blot and FRET analyses, the stoichiom-etry of self association is unclear. Prestin, coupled to the enhanced green fluorescent protein, was synthesized and membrane targeted in human embryonic kidney cells by plasmid transfection. Fragments of membrane containing immobilized fluorescent molecules were isolated by osmotic lysis. Diffraction-limited fluorescent spots consistent in size with single molecules were observed. Under continuous excitation, the spots bleached to background in sequential and approximately equal-amplitude steps. The average step count to background levels was 2.7. A binomial model of prestin oligomerization indicated that prestin was most likely a tetramer, and that a fraction of the green fluorescent protein molecules was dark. As a positive control, the same procedure was applied to cells transfected with plasmids coding for the human cyclic nucleotide-gated ion channel A3 subunit (again coupled to the enhanced green fluorescent protein), which is an obligate tetramer. The average step count for this molecule was also 2.7. This result implies that in cell membranes prestin oligomerizes to a tetramer.

Original languageEnglish
Pages (from-to)5-11
Number of pages7
JournalJournal of Neurophysiology
Volume107
Issue number1
DOIs
StatePublished - Jan 2012

Fingerprint

Outer Auditory Hair Cells
Plasmids
Cyclic Nucleotide-Gated Cation Channels
Membranes
Statistical Models
Green Fluorescent Proteins
Hearing
Transfection
Membrane Proteins
Western Blotting
Cell Membrane
Kidney
enhanced green fluorescent protein

All Science Journal Classification (ASJC) codes

  • Physiology
  • Neuroscience(all)

Cite this

Prestin in HEK cells is an obligate tetramer. / Hallworth, Richard J.; Nichols, Michael G.

In: Journal of Neurophysiology, Vol. 107, No. 1, 01.2012, p. 5-11.

Research output: Contribution to journalArticle

Hallworth, Richard J. ; Nichols, Michael G. / Prestin in HEK cells is an obligate tetramer. In: Journal of Neurophysiology. 2012 ; Vol. 107, No. 1. pp. 5-11.
@article{8b5fc8c6d3c8401daf7c75f699c18e28,
title = "Prestin in HEK cells is an obligate tetramer",
abstract = "The unusual membrane motor protein prestin is essential for mammalian hearing and for the survival of cochlear outer hair cells. While prestin has been demonstrated to be a homooligomer, by Western blot and FRET analyses, the stoichiom-etry of self association is unclear. Prestin, coupled to the enhanced green fluorescent protein, was synthesized and membrane targeted in human embryonic kidney cells by plasmid transfection. Fragments of membrane containing immobilized fluorescent molecules were isolated by osmotic lysis. Diffraction-limited fluorescent spots consistent in size with single molecules were observed. Under continuous excitation, the spots bleached to background in sequential and approximately equal-amplitude steps. The average step count to background levels was 2.7. A binomial model of prestin oligomerization indicated that prestin was most likely a tetramer, and that a fraction of the green fluorescent protein molecules was dark. As a positive control, the same procedure was applied to cells transfected with plasmids coding for the human cyclic nucleotide-gated ion channel A3 subunit (again coupled to the enhanced green fluorescent protein), which is an obligate tetramer. The average step count for this molecule was also 2.7. This result implies that in cell membranes prestin oligomerizes to a tetramer.",
author = "Hallworth, {Richard J.} and Nichols, {Michael G.}",
year = "2012",
month = "1",
doi = "10.1152/jn.00728.2011",
language = "English",
volume = "107",
pages = "5--11",
journal = "Journal of Neurophysiology",
issn = "0022-3077",
publisher = "American Physiological Society",
number = "1",

}

TY - JOUR

T1 - Prestin in HEK cells is an obligate tetramer

AU - Hallworth, Richard J.

AU - Nichols, Michael G.

PY - 2012/1

Y1 - 2012/1

N2 - The unusual membrane motor protein prestin is essential for mammalian hearing and for the survival of cochlear outer hair cells. While prestin has been demonstrated to be a homooligomer, by Western blot and FRET analyses, the stoichiom-etry of self association is unclear. Prestin, coupled to the enhanced green fluorescent protein, was synthesized and membrane targeted in human embryonic kidney cells by plasmid transfection. Fragments of membrane containing immobilized fluorescent molecules were isolated by osmotic lysis. Diffraction-limited fluorescent spots consistent in size with single molecules were observed. Under continuous excitation, the spots bleached to background in sequential and approximately equal-amplitude steps. The average step count to background levels was 2.7. A binomial model of prestin oligomerization indicated that prestin was most likely a tetramer, and that a fraction of the green fluorescent protein molecules was dark. As a positive control, the same procedure was applied to cells transfected with plasmids coding for the human cyclic nucleotide-gated ion channel A3 subunit (again coupled to the enhanced green fluorescent protein), which is an obligate tetramer. The average step count for this molecule was also 2.7. This result implies that in cell membranes prestin oligomerizes to a tetramer.

AB - The unusual membrane motor protein prestin is essential for mammalian hearing and for the survival of cochlear outer hair cells. While prestin has been demonstrated to be a homooligomer, by Western blot and FRET analyses, the stoichiom-etry of self association is unclear. Prestin, coupled to the enhanced green fluorescent protein, was synthesized and membrane targeted in human embryonic kidney cells by plasmid transfection. Fragments of membrane containing immobilized fluorescent molecules were isolated by osmotic lysis. Diffraction-limited fluorescent spots consistent in size with single molecules were observed. Under continuous excitation, the spots bleached to background in sequential and approximately equal-amplitude steps. The average step count to background levels was 2.7. A binomial model of prestin oligomerization indicated that prestin was most likely a tetramer, and that a fraction of the green fluorescent protein molecules was dark. As a positive control, the same procedure was applied to cells transfected with plasmids coding for the human cyclic nucleotide-gated ion channel A3 subunit (again coupled to the enhanced green fluorescent protein), which is an obligate tetramer. The average step count for this molecule was also 2.7. This result implies that in cell membranes prestin oligomerizes to a tetramer.

UR - http://www.scopus.com/inward/record.url?scp=84255182677&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84255182677&partnerID=8YFLogxK

U2 - 10.1152/jn.00728.2011

DO - 10.1152/jn.00728.2011

M3 - Article

C2 - 21975444

AN - SCOPUS:84255182677

VL - 107

SP - 5

EP - 11

JO - Journal of Neurophysiology

JF - Journal of Neurophysiology

SN - 0022-3077

IS - 1

ER -