Two Distinct Ca²⁺-Dependent Signaling Pathways Regulate the Motor Output of Cochlear Outer Hair Cells

Abstract

The outer hair cells (OHCs) of the cochlea have an electromotility mechanism, based on conformational changes of voltage-sensitive "motor" proteins in the lateral plasma membrane. The translocation of electrical charges across the membrane that accompanies electromotility imparts a voltage dependency to the membrane capacitance. We used capacitance measurements to investigate whether electromotility may be influenced by different manipulations known to affect intracellular Ca(2+) or Ca(2+)-dependent protein phosphorylation. Application of acetylcholine (ACh) to the synaptic pole of isolated OHCs evoked a Ca(2+)-activated apamin-sensitive outward K(+) current. It also enhanced electromotility, probably because of a phosphorylation-dependent decrease of the cell's axial stiffness. However, ACh did not change the voltage-dependent capacitance either in conventional whole-cell experiments or under perforated-patch conditions. The effects produced by the Ca(2+) ionophore ionomycin mimicked those produced by ACh. Hyperpolarizing shifts of the voltage dependence of capacitance and electromotility were induced by okadaic acid, a promoter of protein phosphorylation, whereas trifluoperazine and W-7, antagonists of calmodulin, caused opposite depolarizing shifts. Components of the protein phosphorylation cascade-IP(3) receptors and calmodulin-dependent protein kinase type IV-were immunolocalized to the lateral wall of the OHC. Our results suggest that two different Ca(2+)-dependent pathways may control the OHC motor output. The first pathway modulates cytoskeletal stiffness and can be activated by ACh. The second pathway shifts the voltage sensitivity of the OHC electromotile mechanism and may be activated by the release of Ca(2+) from intracellular stores located in the proximity of the lateral plasma membrane.

Slow motility in hair cells of the frog amphibian papilla: Myosin light chain-mediated shape change

“…5C), suggest a common substrate. Antagonistic effects of W-7 and okadaic acid have been observed by Frolenkov et al (2000) using the cell membrane nonlinear capacitance measurement in an OHC preparation. These investigators however concluded that the primary substrate for this phosphorylation/dephosphorylation process was the electromotile mechanism.…”

“…As yet there is no report demonstrating the presence of either MLCK or any CaMK in amphibian hair cells. However, immunological studies have suggested the presence of myosin light chain kinase in mammalian OHCs (Knipper et al, 1995), and have localized the Ca 2+ /CaM-dependent protein kinase IV in the OHC cytoplasm (Koyama et al, 1999;Frolenkov et al, 2000). ML-7 is a specific MLCK inhibitor (Saitoh et al, 1987;Bain et al, 2003), whose effects on the slow motility in OHCs have been previously reported (Puschner and Schacht, 1997).…”

“…Furthermore, acetylcholine is known to induce a small shortening both in OHCs (Szonyi et al, 1999a;Frolenkov et al, 2000) and RAPHCs (Farahbakhsh and Narins, 2004). We have also observed that the effects of acetylcholine could be mimicked by two other cholinergic ligands, carbachol and DMPP.…”

“…More recent studies suggest that up to 54% of the global stiffness is contributed by the prestin-containing plasma membrane, which is also modulated by ACh (He et al, 2003a). Frolenkov et al, (2000) suggested that intracellular calcium ([Ca 2+ ] i ) both modulates the OHCs' cytoskeletal stiffness, and shifts the voltage sensitivity of the electromotile mechanism. The effects of the [Ca 2+ ] i increase on electromotility could be inhibited by the calmodulin antagonists, W-7 ) and trifluoperazine, as well as by , an inhibitor of the multifunctional Ca 2+ /calmodulin-dependent kinases ).…”

ATP activates P2x receptors and requires extracellular Ca++ participation to modify outer hair cell nonlinear capacitance

“…5 and 7), indicating that extracellular Ca ++ plays a key role in the effect of ATP on OHC electromotility. It has been hypothesized that Ca ++ modulates OHC electromotility via two pathways: modifying cytoskeletal stiffness [32] and shifting the voltage sensitivity of OHC electromotility [33]. The effect of ATP on OHC electromotility may follow the same concept, activating the P2x receptors allowing Ca ++ influxing to modify OHC electromotility.…”