ABSTRACT
We report the cloning and characterization of rat alpha10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the alpha10 nAChR subunit gene is most similar to the rat alpha9 nAChR, and both alpha9 and alpha10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with alpha10 cRNA alone or in pairwise combinations with either alpha2-alpha6 or beta2-beta4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of alpha9 and alpha10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric alpha9 channels, the alpha9alpha10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular Ca(2+) ions. The pharmacological profiles of homomeric alpha9 and heteromeric alpha9alpha10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both alpha9 and alpha10 subunits.
Subject(s)
Hair Cells, Auditory/metabolism , Hair Cells, Vestibular/metabolism , Receptors, Nicotinic/physiology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Cochlea/cytology , Female , Gene Expression , Hair Cells, Auditory/physiology , Hair Cells, Vestibular/physiology , Humans , Mice , Molecular Sequence Data , Rats , Receptors, Nicotinic/genetics , Vestibule, Labyrinth/cytology , Xenopus laevisABSTRACT
In the present study, we report that the alpha9 nicotinic acetylcholine receptor (nAChR) expressed in Xenopus laevis oocytes is reversibly blocked by aminoglycoside antibiotics. The aminoglycosides tested blocked the alpha9 nAChR in a concentration-dependent manner with the following rank order of potency: neomycin>gentamicin>streptomycin>amikacin>kanamycin. The antagonistic effect of gentamicin was not overcome by increasing the concentration of acetylcholine (ACh), indicative of a non-competitive type of block. Blockage of ACh-evoked currents by gentamicin was found to be voltage-dependent, being more potent at hyperpolarized than at depolarized holding potentials. Furthermore, gentamicin blockage was dependent upon the extracellular Ca(2+) concentration, shown by the fact that increments in extracellular Ca(2+) significantly reduced the potency of this aminoglycoside to block the alpha9 nAChR. Possible mechanisms of blockage by the aminoglycosides are discussed. The present results suggest that the initial reversible actions of aminoglycosides at the organ of Corti, such as the elimination of the olivocochlear efferent function, are due in part to the interaction with the native alpha9-containing cholinergic receptor of the outer hair cells.
Subject(s)
Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/toxicity , Hearing Disorders/chemically induced , Nicotinic Antagonists/pharmacology , Receptors, Nicotinic/metabolism , Animals , Calcium/metabolism , Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Electric Stimulation , Electrophysiology , Gentamicins/pharmacology , Gentamicins/toxicity , Olivary Nucleus/drug effects , Oocytes/metabolism , Rats , XenopusABSTRACT
At the synapse between olivocochlear efferent fibers and outer hair cells (OHCs) of the cochlea, a non-classical ionotropic cholinergic receptor allows Ca(2+) entry into the hair cell, thus activating a Ca(2+)-sensitive K(+) current which hyperpolarizes the cell's membrane. In the mammalian ear, this leads to a reduction in basilar membrane motion, altering auditory nerve fiber activity and reducing the dynamic range of hearing. The alpha9 nicotinic acetylcholine receptor (nAChR) subunit mediates synaptic transmission between cholinergic olivocochlear fibers and OHCs. Given that Ca(2+) is a key player at this inhibitory synapse, we evaluated the permeability to Ca(2+) of the recombinant alpha9 receptor expressed in Xenopus laevis oocytes and the modulation of its activity by extracellular Ca(2+). Our results show that the alpha9 receptor is highly permeable to Ca(2+) and that this cation potently blocks monovalent currents through this channel (IC(50)=100 microM, at -70 mV) in a voltage-dependent manner. At a Ca(2+) concentration similar to that found in the perilymph bathing the base of the OHCs, approximately 90% of the Na(+) current through the alpha9 receptor is blocked, suggesting that one of the main functions of this channel could be to provide a pathway for Ca(2+) influx.
Subject(s)
Calcium/metabolism , Receptors, Nicotinic/metabolism , Animals , Base Sequence , Calcium/pharmacology , Calcium Signaling , Cochlear Nerve/metabolism , DNA Primers/genetics , Female , Hair Cells, Auditory, Outer/metabolism , In Vitro Techniques , Neurons, Efferent/metabolism , Oocytes/metabolism , Permeability , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sodium/metabolism , Xenopus laevisSubject(s)
Censuses , Housing , Socioeconomic Factors , Americas , Brazil , Demography , Developing Countries , Economics , Geography , Latin America , Population , Population Characteristics , Research , Residence Characteristics , Sanitation , South America , Water SupplySubject(s)
Income , Life Expectancy , Mortality , Sanitation , Water Supply , Americas , Brazil , Conservation of Natural Resources , Demography , Developing Countries , Economics , Environment , Health , Latin America , Longevity , Population , Population Dynamics , Public Health , Socioeconomic Factors , South America , Urban PopulationABSTRACT
PIP: The authors examine the relationship between mortality and the availability of public health facilities in two metropolitan regions in Brazil. Estimated life expectancies of children of different income groups are used to determine the extent to which the adequacy of water and sewage facilities affects mortality rates^ieng