Vestibular hair bundles control pH with (Na⁺, K ⁺)/H⁺ exchangers NHE6 and NHE9

In hair cells of the inner ear, robust Ca²⁺/H⁺ exchange mediated by plasma-membrane Ca²⁺-ATPase would rapidly acidify mechanically sensitive hair bundles without efficient removal of H ⁺. We found that, whereas the basolateral membrane of vestibular hair cells from the frog saccule extrudes H⁺ via an Na ⁺-dependent mechanism, bundles rapidly remove H⁺ in the absence of Na⁺ and HCO₃^-, even when the soma is acidified. K⁺ was fully effective and sufficient for H⁺ removal; in contrast, Rb⁺ failed to support pH recovery. Na ⁺/H⁺-exchanger isoform 1 (NHE1) was present on hair-cell soma membranes and was likely responsible for Na⁺-dependent H ⁺ extrusion. NHE6 and NHE9 are organellar isoforms that can appear transiently on plasma membranes and have been proposed to mediate K ⁺/H⁺ exchange. We identified NHE6 in a subset of hair bundles; NHE9 was present in all bundles. Heterologous expression of these isoforms in yeast strains lacking endogenous exchangers conferred pH-dependent tolerance to high levels of KCl and NaCl. NHE9 preferred cations in the order K⁺, Na⁺ ≫ Rb⁺, consistent with the relative efficacies of these ions in promoting pH recovery in hair bundles. Electroneutral K⁺/H⁺ exchange, which we propose is performed by NHE9 in hair bundles, exploits the high-K⁺ endolymph, responds only to pH imbalance across the bundle membrane, is unaffected by the +80 mV endocochlear potential, and uses mechanisms already present in the ear for K⁺ recycling. This mechanism allows the hair cell to remove H⁺ generated by Ca²⁺ pumping without ATP hydrolysis in the cell.