Large membrane domains in hair bundles specify spatially constricted radixin activation.

The plasma membrane of vertebrate hair bundles interacts intimately with the bundle cytoskeleton to support mechanotransduction and homeostasis. To determine the membrane composition of bundles, we used lipid mass spectrometry with purified chick vestibular bundles. While the bundle glycerophospholipids and acyl chains resemble those of other endomembranes, bundle ceramide and sphingomyelin nearly exclusively contain short-chain, saturated acyl chains. Confocal imaging of isolated bullfrog vestibular hair cells shows that the bundle membrane segregates spatially into at least three large structural and functional domains. One membrane domain, including the stereocilia basal tapers and ∼1 µm of the shaft, the location of the ankle links, is enriched in the lipid phosphatase PTPRQ (protein tyrosine phosphatase Q) and polysialylated gangliosides. The taper domain forms a sharp boundary with the shaft domain, which contains the plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]; moreover, a tip domain has elevated levels of cholesterol, PMCA2, and PI(4,5)P(2). Protein mass spectrometry shows that bundles from chick vestibular hair cells contain a complete set of proteins that transport, synthesize, and degrade PI(4,5)P(2). The membrane domains have functional significance; radixin, essential for hair-bundle stability, is activated at the taper-shaft boundary in a PI(4,5)P(2)-dependent manner, allowing assembly of protein complexes at that site. Membrane domains within stereocilia thus define regions within hair bundles that allow compartmentalization of Ca(2+) extrusion and assembly of protein complexes at discrete locations.

Splice-site A choice targets plasma-membrane Ca2+-ATPase isoform 2 to hair bundles.

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.