Immunolocalization of P2Y4 and P2Y2 purinergic receptors in strial marginal cells and vestibular dark cells

K+ secretion by strial marginal cell and vestibular dark cell epithelia is regulated by UTP and ATP at both the apical and basolateral membranes, suggesting control by P2Y(2) and/or P2Y(4) purinergic receptors. Immunolocalization was used to determine the identity and distribution of these putative receptors. Membrane proteins from gerbil brain, gerbil vestibular labyrinth and gerbil stria vascularis were isolated and analyzed by Western blot. P2Y(2) antibody stained one band at 42 kDa for each tissue, whereas P2Y(4) antibody stained 3 bands on gerbil brain (75, 55 and 36 kDa), one band on gerbil stria vascularis (55 kDa) and two bands on vestibular labyrinth (42 and 56 kDa). All bands were absent when the antibodies were blocked with their respective antigenic peptide. P2Y(4) was immunolocalized by fluorescence confocal microscopy to only the apical membrane of strial marginal cells and vestibular dark cells and was similar to apical immunostaining of KCNE1 in the same cells. By contrast, P2Y(2) was observed on the basolateral but not the apical membrane of dark cells. Similarly, in the stria vascularis P2Y(2) was observed in the basolateral region but not the apical membrane of marginal cells. Additional staining was observed in the spiral ligament underlying the stria vascularis. These findings identify the molecular bases of the regulation of K+ secretion by apical and basolateral UTP in the inner car.