Ca ²⁺ regulation of PMCA2 calcium pump expression in hair cells of Tmc1 deafness mutants

Mutations in transmembrane channel-like protein 1 (TMC1), the pore-forming component of the mechano-electrical transducer (MET) channel in auditory hair cells, cause cell death and deafness in 3-wk old mice. We studied mice with mutations Tmc1 p.T416K, p.M412K, p.D528N, and p.D569N, which possessed functional MET channels at postnatal day (P)6 but became deaf before P21. These Tmc1 mutants had channels with reduced Ca ²⁺ permeability and lower expression of the PMCA2 calcium pump in outer hair cell (OHC) stereocilia. The reduction in PMCA2 density was directly correlated with reduced Ca ²⁺ entry via the MET channel. Over the first postnatal week, PMCA2 expression decreased in cochlear cultures containing lowered extracellular Ca ²⁺ , but after P11, PMCA2 density was insensitive to Ca ²⁺ changes. The lower PMCA2 density in mutants was maintained into adulthood and may contribute to hair cell death. Before the onset of hearing, PMCA2 continuously turns over, and its insertion into stereocilia from an identified vesicular compartment is regulated by cytoplasmic [Ca ²⁺ ]. PMCA2 turnover was strongly inhibited by the endocytosis blocker, Pitstop2, and by PtdIns(4,5)P ₂ blockers like phenylarsine oxide (PAO), which was shown to elevate stereociliary [Ca ²⁺ ]. We argue the effects of PAO on mechanotransduction may be partly due to this Ca ²⁺ increase. OHC bundles also expressed Neuroplastin (NPTN), a PMCA2 accessory protein, whose development paralleled PMCA2, and we propose it stabilizes the pump complex in stereocilia. NPTN expression was delayed about 2 d relative to PMCA2 and was less Ca ²⁺ sensitive, implying it originates from a different internal pool of vesicles.