Literaturdatenbank
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Farris, H. E., LeBlanc, C. L., Goswami, J., & Ricci, A. J. (2004). Probing the pore of the auditory hair cell mechanotransducer channel in turtle. Journal of Physiology, 558(3), 769–792.
Added by: Sarina Wunderlich (18 Nov 2012 17:43:23 UTC) |
| Resource type: Journal Article DOI: 10.1113/jphysiol.2004.061267 BibTeX citation key: Farris2004 View all bibliographic details  |
Categories: General Keywords: akustische Kommunikation = acoustic communication, Emydidae, Schildkröten = turtles + tortoises, Trachemys, Trachemys scripta Creators: Farris, Goswami, LeBlanc, Ricci Collection: Journal of Physiology |
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| Abstract |
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Trachemys scripta elegans Hair cell mechano-electric transducer (MET) channels play a pivotal role in auditory and vestibular signal detection, yet few data exist regarding their molecular nature. Present work characterizes the MET channel pore, a region whose properties are thought to be intrinsically determined. Two approaches were used. First, the channel was probed with antagonists of candidate channel subtypes including: cyclic nucleotide-gated channels, transient receptor potential channels and gap-junctional channels. Eight new antagonists were identified. Most of the effective antagonists had a partially charged amine group predicted to penetrate the channel pore, antagonizing current flow, while the remainder of the molecule prevented further permeation of the compound through the pore. This blocking mechanism was tested using curare to demonstrate the open channel nature of the block and by identifying methylene blue as a permeant channel blocker. The second approach estimated dimensions of the channel pore with simple amine compounds. The narrowest diameter of the pore was calculated as 12.5 ± 0.8 Å and the location of a binding site ∼45% of the way through the membrane electric field was calculated. Channel length was estimated as ∼31 Å and the width of the pore mouth at < 17 Å. Each effective antagonist had a minimal diameter, measured about the penetrating amine, of less than the pore diameter, with a direct correlation between IC50 and minimal diameter. The IC50 was also directly related to the length of the amine side chains, further validating the proposed pore blocking mechanism. Data provided by these two approaches support a hypothesis regarding channel permeation and block that incorporates molecular dimensions and ion interactions within the pore. Hair cells are the mechano-sensory cells of both auditory and vestibular systems, imparting sensitivity through mechano-electric transducer (MET) ion channels located at the tops of actin-filled stereocilia (hair bundle) (Fettiplace et al. 2001). Deflection of the hair bundle toward its tall edge opens MET channels while deflection away from the tall edge closes channels (Shotwell et al. 1981). Molecular identification of this channel has been elusive due to problems arising from the scarcity of expressed channels and the limited number of hair cells per sensory organ. Many MET channel properties including activation and adaptation kinetics, displacement sensitivity and perhaps even mechanical sensitivity may not be intrinsic to the channel but imposed by accessory proteins linking the channel to the cytoskeleton and\or to extracellular proteins, implying that when the putative channel is identified the expressed properties may differ significantly from native properties. However, channel pore characteristics should be intrinsic to the channel protein and should not vary due to accessory structures, thus providing a useful target for identification. The present work characterizes MET channel pore properties to gain insight into the molecular identity and to create a template upon which future molecular characterizations can be based. The MET channel exhibits a non-specific cation conductance with a high calcium permeability (Ohmori, 1985; Crawford et al. 1991; Lumpkin & Hudspeth, 1995; Ricci & Fettiplace, 1998). MET channels pass large molecules such as tetraethyl ammonium and the membrane dye FM1-43 (Corey & Hudspeth, 1979; Gale et al. 2001). Single channel conductance estimates vary from 10 to 300 pS (Ohmori, 1985; Holton & Hudspeth, 1986; Crawford et al. 1991; Geleoc et al. 1997; Ricci et al. 2003). No voltage dependence of the channel (Ohmori, 1987; Crawford et al. 1991) has been reported. Biophysical characteristics suggest the channel is related to the broad class of non-specific cation channels that include the transient receptor potential channels (TRP), a subgroup of which are the vanniloid receptors (TRPV), cyclic nucleotide gated channels (CNG), mechanically gated channels, the nicotinic family of channels and even gap junctional hemi-channels. The presence of both CNG type and TRP type channels in hair cells (Kolesnikov et al. 1991; Liedtke et al. 2000; Drescher et al. 2002) make these channel classes prime candidates for the MET channel. With the identification of TRP-like channels regulating mechanosensation in Drosophila (Walker et al. 2000) and zebrafish (Sidi et al. 2003), a great deal of attention is being paid to the TRP family (Corey, 2003; Strassmaier & Gillespie, 2003). However, existing data cannot clearly classify the channel type. Pharmacological data regarding MET channels are limited in that the blockers were not specific to channel classes, nor do they provide insight into the properties of the channel pore. For example, aminoglycoside antibiotics that can antagonize calcium channels, are open MET channel blockers (Ohmori, 1985; Kroese et al. 1989; Kimitsuki & Ohmori, 1993; Ricci, 2002). Amiloride and its derivatives, which block some CNG channels as well as channels of the epithelial sodium channel (ENAC) family (Frings et al. 1992; Kellenberger & Schild, 2002) also antagonize MET channels of hair cells as well as oocytes (Jorgensen & Ohmori, 1988; Lane et al. 1993; Rusch et al. 1994; Ricci, 2002). Tubocurarine, a nicotinic antagonist, also blocks the MET channel (Glowatzki et al. 1997). Cisplatin is yet another antagonist of the MET channel (Kimitsuki et al. 1993). Present work creates a pharmacological profile of the MET channel pore to serve as a basis for comparisons with other known channel types and a template for the molecular identification and characterization of this channel. Eight new MET channel blockers were identified demonstrating the channel has broad similarities to other non-specific cation channels including the TRP and CNG channels. Furthermore, the molecular dimensions of the channel were estimated and support a novel open channel blocking hypothesis for charged amines.
Added by: Sarina Wunderlich |