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The Journal of Neuroscience, June 7, 2006, 26(23):6386-6395; doi:10.1523/JNEUROSCI.1215-06.2006

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 Previous Article

Cellular/Molecular
Rapid Turnover of Stereocilia Membrane Proteins: Evidence from the Trafficking and Mobility of Plasma Membrane Ca²⁺-ATPase 2

M’hamed Grati,¹ Mark E. Schneider,² Karen Lipkow,³ Emanuel E. Strehler,⁴ Robert J. Wenthold,¹ and Bechara Kachar²

Laboratories of ¹Neurochemistry and ²Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, 20892, ³Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge CB2 3DY, United Kingdom, and ⁴Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905

Correspondence should be addressed to Dr. Bechara Kachar, Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 50 South Drive, Room 4249, Bethesda, MD 20892-8027. Email: kacharb{at}nidcd.nih.gov

We studied the spatial distribution, mobility, and trafficking of plasma membrane Ca²⁺ATPase-2 (PMCA2), a protein enriched in the hair cell apical membrane and essential for hair cell function. Using immunofluorescence, we determined that PMCA2 is enriched in the stereocilia and present at a relatively low concentration in the kinocilium and in the remaining apical membrane. Using an antibody to the extracellular domain of PMCA2 as a probe, we observed that PMCA2 diffuses laterally from the stereocilia membrane and is internalized at the apical cell border maintaining an estimated half-life of residency in the stereocilia of 5–7 h. A computer simulation of our data indicates that PMCA2 has an estimated global diffusion coefficient of 0.01–0.005 µm²/s. Using a green fluorescent protein tag, we observed that PMCA2 is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface. Fluorescence recovery after photobleaching experiments show that 60% of PMCA2 in the stereocilia exhibit high mobility with a diffusion coefficient of 0.1–0.2 µm²/s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal.

Key words: PMCA2; hair cells; endocytosis; hearing; membrane protein mobility; stereocilia

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Received Jan. 30, 2006; revised April 21, 2006; accepted April 23, 2006.

Correspondence should be addressed to Dr. Bechara Kachar, Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 50 South Drive, Room 4249, Bethesda, MD 20892-8027. Email: kacharb{at}nidcd.nih.gov

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