Chapter 13 - Insights into the Pore of the Hair Cell Transducer Channel from Experiments with Permeant Blockers

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This chapter presents recent experiments designed to infer the properties of the ion-conducting pore of the mechanoelectrical transducer channel of sensory hair cells using permeant blockers. By combining results from experiments with three classes of large cationic permeant blockers, the fluorescent dye FM1-43, the aminoglycoside antibiotics, and the potassium-sparing diuretic amiloride, information has been obtained on the free energy profile along the transducer channel's pore as sensed by these blocker molecules. These energy profiles provide information about the position of the negatively charged binding site for the blockers as well as about positively charged barriers near the extracellular and intracellular faces of the channel that impede the blockers' permeation. The extracellular barrier is relatively modest and allows almost diffusion-limited entry of blockers from the extracellular side. A larger intracellular energy barrier effectively prevents exit of the blocking molecules from the intracellular side, trapping the blockers inside the hair cells. A putative geometrical model of the transducer channel pore is presented that draws on results from all the three classes of permeant blockers. The pore contains a large vestibule that is easily accessible from the extracellular side.

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OVERVIEW

This chapter considers recent experiments designed to infer properties of the ion‐conducting pore of the mechanoelectrical transducer channel of sensory hair cells using permeant blockers. By combining results from experiments with three classes of large cationic permeant blockers, the fluorescent dye FM1‐43, the aminoglycoside antibiotics, and the potassium‐sparing diuretic amiloride, information has been obtained on the free energy profile along the transducer channel's pore as sensed by

INTRODUCTION

The molecular identity of the hair cell transducer channel is, despite intensive research efforts, still in question at the time of writing (Corey, 2006). Nevertheless, recent pharmacological characterization is beginning to yield information about key properties of the channel that may help toward its eventual molecular identification by comparison with other known channel types. Historically, the pharmacology of the transducer channel has been studied to try and find the mode of action of

IONIC SELECTIVITY OF THE TRANSDUCER CHANNEL

The hair cell transducer channel is a nonselective cation channel with very similar permeabilities for the alkali cations and a much higher permeability for divalent metal ions, the highest for Ca2+ (Ohmori, 1985, Jorgensen and Kroese, 1995). The permeability sequence of the alkali metal ions corresponds to Eisenman sequence XI, pointing to a high negative charge density of the selectivity filter (Hille, 2001). The high affinity of the Ca2+ ions for the selectivity filter has the effect that Ca

Evidence for Permeation of FM1‐43 Through the Hair Cell Transducer Channel

FM1‐43 (Fig. 1) is a fluorescent styryl dye with a divalent cationic head group related to TEA and a long lipophilic tail which enables it to partition reversibly into the outer leaflet of the cell membrane when present in the extracellular solution (Betz et al., 1992). On incorporation into the membrane, its fluorescence increases by two orders of magnitude. It cannot cross the lipid bilayer so that when it becomes internalized into cells by endocytosis, it remains trapped in the inner leaflet

Evidence for Permeation of Aminoglycoside Antibiotics Through the Transducer Channel

The studies of Ohmori, 1985, Kroese et al., 1989 showed that extracellularly applied aminoglycoside antibiotics reversibly blocked transducer currents at negative but not at positive potentials. Kroese et al. (1989) also found that intracellularly applied aminoglycosides did not block the transducer currents even at concentrations of 500 μM, one to two orders of magnitude higher than the concentrations with which they achieved block from the extracellular side. The KD for extracellularly applied

Amiloride and Amiloride Derivatives as Permeant Transducer Channel Blockers: A Reinterpretation

The synthetic drug amiloride and related compounds find clinical application as potassium‐sparing diuretics, thanks to their high‐affinity blocking action (at submicromolar concentrations) on epithelial Na+ channels in the distal and collecting tubules of the kidney (Kleyman and Cragoe, 1988). Amiloride has been found to reversibly inhibit the hair cell transducer current, but at higher concentrations (KD around 50 μM; Jorgensen and Ohmori, 1988, Rüsch et al., 1994), which is probably why no

CONCLUSIONS

The results discussed in the preceding sections may be summarized by constructing a putative geometrical model of the transducer channel with a specific charge distribution lining the pore (Fig. 6). The interactions of the channel with the alkali metal cations, divalent cations (in particular Ca2+), the permeant cationic blocker molecules discussed in this chapter as well as other polycationic blocker molecules that have been investigated (Farris et al., 2004), all support the conclusion that

Acknowledgments

Supported by the Netherlands Organisation for Scientific Research (S.M.v.N) and the MRC (C.J.K.). The authors thank Dr. Cécil J. W. Meulenberg for his comments on an early version of this chapter and his help with the preparation of Fig. 1.

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