Sodium entry pathways in isolated goldfish hair cells

Abstract

Hair cells are the exquisitely-sensitive mechanosensory transducers of the inner ear. While the electrophysiology of hair cells has been extensively studied, little is known about the vital background processes that maintain the steady-state intracellular ionic environment. This study explored Na-coupled ion transport processes in isolated goldfish hair cells both in the steady state and in response to perturbations. Intracellular Na⁺ concentration ([Na⁺]i) and pH (pHi) were measured using the fluorescent probes SBFI and BCECF, respectively. The total steady-state Na⁺ entry, determined by measuring the initial rate of increase in [Na⁺]i during inhibition of Na⁺,K⁺-ATPase, was 9 mM/min, a rate higher than in many other epithelial cells involved in rapid ion transport. To uncover the entry pathways for such a high Na⁺ influx, pharmacological agents and manipulations of extracellular composition were used to dissect apart the contributions of various transporters or channels. A combination of Na⁺ entry via transduction channels, the Na⁺/H⁺ exchanger, and the Na⁺/Ca²⁺ exchanger accounted for 3/4 of the total steady-state Na⁺ entry. Consistent with a significant component via the Na⁺/Ca²⁺ exchanger, nifedipine, a blocker of L-type calcium channels, also reduced the Na⁺ entry rate. Since the Na⁺/H⁺ exchanger in goldfish hair cells, in contrast to many other cell types, displayed significant activity in the steady state, interactions between regulation of [Na⁺]i and pHi were examined during recovery from intracellular acid loads. The rate of Na+ entry or acid extrusion via the Na⁺/H⁺ exchanger increased by a factor of 6 during recovery from an acid load. In most cells, [Na⁺]i doubled after the acid load and subsequently recovered, even though the

(cont.) was not exogenously inhibited. These results indicate that intracellular acidification inhibits the Na⁺, K⁺-ATPase, and therefore poses a potential conflict for maintenance of intracellular Na⁺ homeostasis. Although hair cells were traditionally described as passive resistors, this thesis demonstrates that goldfish hair cells have a significant metabolic load, even in the steady state, arising from the activities of specific Na⁺-coupled transporters and channels. Under certain pathophysiological circumstances, such as ischemia or acoustic trauma, increased Na⁺ influx via these pathways may overwhelm the ion-homeostatic capabilities of hair cells.