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The Journal of Neuroscience, June 7, 2006, 26(23):6172-6180; doi:10.1523/JNEUROSCI.0447-06.2006

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Cellular/Molecular
Splice-Site A Choice Targets Plasma-Membrane Ca²⁺-ATPase Isoform 2 to Hair Bundles

Jennifer K. Hill,¹ Diane E. Williams,¹ Meredith LeMasurier,¹ Rachel A. Dumont,¹ Emanuel E. Strehler,² and Peter G. Gillespie¹

¹Oregon Hearing Research Center and Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, and ²Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905

Correspondence should be addressed to Dr. Peter G. Gillespie, L335A Auditory Neuroscience, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239. Email: gillespp{at}ohsu.edu

Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca²⁺-ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites (A and C) to hair-cell targeting of PMCA2. When PMCA2 isoforms were immunoprecipitated from purified hair bundles of rat utricle, 2w was the only site A variant detected; moreover, immunocytochemistry for 2w in rat vestibular and cochlear tissues indicated that this splice form was located solely in bundles. To demonstrate the necessity of the 2w sequence, we transfected hair cells with PMCA2 containing different variants at splice sites A and C. Although native hair bundles exclusively use the 2a form at splice-site C, epitope-tagged PMCA2w/a and PMCA2w/b were both concentrated in bundles, indicating that site C is not involved in bundle targeting. In contrast, PMCA2z/a was excluded from bundles and was instead targeted to the basolateral plasma membrane. Bundle-specific targeting of PMCA2w/a tagged with green fluorescent protein (GFP) was diminished, suggesting that GFP interfered with splice-site A. Together, these data demonstrate that PMCA2w/a is the hair-bundle isoform of PMCA in rat hair cells and that 2w targets PMCA2 to bundles. The 2w sequence is thus the first targeting signal identified for a hair-bundle membrane protein; moreover, the striking distribution of inner-ear PMCA isoforms dictated by selective targeting suggests a critical functional role for segregated pathways of Ca²⁺ transport.

Key words: hair cells; calcium; calcium pumps; stereocilia; apical targeting; alternative splicing; gene-gun

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Received Jan. 31, 2006; revised April 20, 2006; accepted April 24, 2006.

Correspondence should be addressed to Dr. Peter G. Gillespie, L335A Auditory Neuroscience, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239. Email: gillespp{at}ohsu.edu

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