Nicotine evoked efferent transmitter release onto immature cochlear inner hair cells.

Olivocochlear neurons make temporary cholinergic synapses on inner hair cells of the rodent cochlea in the first 2 to 3 wk after birth. Repetitive stimulation of these efferent neurons causes facilitation of evoked release and increased spontaneous release that continues for seconds to minutes. Presynaptic nicotinic acetylcholine receptors (nAChRs) are known to modulate neurotransmitter release from brain neurons. The present study explores the hypothesis that presynaptic nAChRs help to increase spontaneous release from efferent terminals on cochlear hair cells. Direct application of nicotine (which does not activate the hair cells' α9α10-containing nAChRs) produces sustained efferent transmitter release, implicating presynaptic nAChRs in this response. The effect of nicotine was reduced by application of ryanodine that reduces release of calcium from intraterminal stores.NEW & NOTEWORTHY Sensory organs exhibit spontaneous activity before the onset of response to external stimuli. Such activity in the cochlea is subject to modulation by cholinergic efferent neurons that directly inhibit sensory hair cells (inner hair cells). Those efferent neurons are themselves subject to various modulatory mechanisms. One such mechanism is positive feedback by released acetylcholine onto presynaptic nicotinic acetylcholine receptors causing further release of acetylcholine.

Synaptic studies inform the functional diversity of cochlear afferents.

Type I and type II cochlear afferents differ markedly in number, morphology and innervation pattern. The predominant type I afferents transmit the elemental features of acoustic information to the central nervous system. Excitation of these large diameter myelinated neurons occurs at a single ribbon synapse of a single inner hair cell. This solitary transmission point depends on efficient vesicular release that can produce large, rapid, suprathreshold excitatory postsynaptic potentials. In contrast, the many fewer, thinner, unmyelinated type II afferents cross the tunnel of Corti, turning basally for hundreds of microns to form contacts with ten or more outer hair cells. Although each type II afferent is postsynaptic to many outer hair cells, transmission from each occurs by the infrequent release of single vesicles, producing receptor potentials of only a few millivolts. Analysis of membrane properties and the site of spike initiation suggest that the type II afferent could be activated only if all its presynaptic outer hair cells were maximally stimulated. Thus, the details of synaptic transfer inform the functional distinctions between type I and type II afferents. High efficiency transmission across the inner hair cell's ribbon synapse supports detailed analyses of the acoustic world. The much sparser transfer from outer hair cells to type II afferents implies that these could respond only to the loudest, sustained sounds, consistent with previous reports from in vivo recordings. However, type II afferents could be excited additionally by ATP released during acoustic stress of cochlear tissues.

The alpha9/alpha10-containing nicotinic ACh receptor is directly modulated by opioid peptides, endomorphin-1, and dynorphin B, proposed efferent cotransmitters in the inner ear.

UNLABELLED: Opioid peptides have been detected in the auditory and vestibular efferent neurons where they colocalize with the major neurotransmitter, acetylcholine. We investigated the function of opioids to modulate neurotransmission mediated by hair cell's alpha9/alpha10-containing nicotinic acetylcholine receptors (alpha9/alpha10nAChRs). The endogenous opioid peptides, endomorphin-1 (mu agonist) and dynorphin B (kappa agonist), but not a delta agonist [D-Pen2,D-Pen-5]enkephalin, inhibited the acetylcholine-evoked currents in frog saccular hair cells and rat inner hair cells. This inhibition was noncompetitive, voltage-independent, and was accompanied by an acceleration of the rate of current decay. Selective mu- and kappa-opioid receptor antagonists did not block the inhibition, although partial reduction by naloxone was observed. All opioid antagonists tested also reduced the acetylcholine response. Endomorphin-1 and dynorphin B inhibited the acetylcholine-evoked currents in alpha9/alpha10-expressing Xenopus oocytes. Because oocytes lack opioid receptors, it provides strong evidence for the direct interaction of opioid peptides with alpha9/alpha10nAChR. CONCLUSION: alpha9/alpha10nAChR is a target for modulation by endomorphin-1 and dynorphin B, efferent cotransmitters in the inner ear.

Inflammatory mediators potentiate ATP-gated channels through the P2X(3) subunit.

The P2X(3) receptor is an ATP-gated ion channel predominantly expressed in nociceptive neurons from the dorsal root ganglion. P2X(3) receptor channels are highly expressed in sensory neurons and probably contribute to the sensation of pain. Kinetics of P2X(3) currents are characterized by rapid desensitization (<100 ms) and slow recovery (>20 s). Thus, any mechanism modulating rate of desensitization and/or recovery may have profound effect on susceptibility of nociceptive neurons expressing P2X(3) to ATP. Here we show that currents mediated by P2X(3) receptor channels and the heteromeric channel P2X(2/3) composed of P2X(2) and P2X(3) subunits are potentiated by the neuropeptides substance P and bradykinin, which are known to modulate pain perception. The effect is mediated by the respective neuropeptide receptors, can be mimicked by phorbol ester and blocked by inhibitors of protein kinases. Together with data from site-directed mutagenesis our results suggest that inflammatory mediators sensitize nociceptors through phosphorylation of P2X(3) and P2X(2/3) ion channels or associated proteins.

Cholinergic synaptic inhibition of inner hair cells in the neonatal mammalian cochlea.

Efferent feedback onto sensory organs provides a means to modulate input to the central nervous system. In the developing mammalian cochlea, inner hair cells are transiently innervated by efferent fibers, even before sensory function begins. Here, we show that neonatal inner hair cells are inhibited by cholinergic synaptic input before the onset of hearing. The synaptic currents, as well as the inner hair cell's response to acetylcholine, are mediated by a nicotinic (alpha9-containing) receptor and result in the activation of small-conductance calcium-dependent potassium channels.

Mechanically and ATP-induced currents of mouse outer hair cells are independent and differentially blocked by d-tubocurarine.

Mechano-electrical transducer channels (MET) and ATP-gated ion channels (P2X receptors) of hair cells have several properties in common: they share the same location at the apex of the cell, both channels are non-selective for cations and blocked by aminoglycosides and pyrazinecarboxamides (amiloride-related compounds). In this study, we test the relationship and possible identity of these two channel types. Using whole-cell patch-clamp recordings of outer hair cells (OHCs) of the cultured neonatal mouse cochlea and a fluid jet to stimulate their hair bundles mechanically, we show that d-tubocurarine, a blocker of P2X2 receptors, blocks MET channels with a half-blocking concentration of 2.3 microM. In contrast, the KD for the P2X2 receptors was 90 microM and 84 microM measured in hair cells and Xenopus oocytes, respectively. When hair bundles of OHCs were simultaneously stimulated with saturating mechanical stimuli and superfused by 100-300 microM ATP, transducer currents and ATP-activated currents were elicited simultaneously. Their amplitudes were additive, however. We conclude that MET- and ATP-activated currents are mediated by two distinct channel populations in hair cells.

Desensitization of the P2X(2) receptor controlled by alternative splicing.

P2X receptors are ion channels gated by extracellular ATP. We report here cloning of a P2X(2) receptor splice variant (P2X(2-2)) carrying a 207 bp deletion in the intracellular C-terminus and the analysis of the corresponding genomic structure of the P2X(2) gene. P2X(2-2) is as highly expressed as the original P2X(2) sequence in various tissues. ATP-activated currents mediated by heterologous expressed P2X(2) or P2X(2-2) receptors showed significant differences in desensitization time constants and steady-state currents in the continuous presence of ATP. These results imply functional differences between cells differentially expressing these P2X(2) isoforms.

Analysis of gene expression in the organ of Corti revealed by single-cell RT-PCR.

Many genes encoding proteins which are expressed in the auditory periphery have been identified in the last years. With single-cell reverse transcription-polymerase chain reaction (RT-PCR), the molecular analysis of gene expression can be done on the single-cell level. Furthermore a single-cell RT-PCR experiment can be combined with the electrophysiological characterization of an individual cell. The combination of these two methods will lead to a better understanding of how functional properties of neurons are controlled by the expression of complex proteins.

Cell-specific expression of the alpha 9 n-ACh receptor subunit in auditory hair cells revealed by single-cell RT-PCR.

Single-cell reverse transcription polymerase chain reaction was carried out in three different cell types from the organ of Corti of the four-day old rat. For this purpose, pieces of the organ of Corti were mounted under a differential-interference contrast video microscope. Two different mounting configurations were used to allow imaging of cells from two almost orthogonal angles. This method afforded unequivocal recognition of various cell types in the vital tissue, and extraction of nucleus and cytoplasm of specified individual cells with a patch pipette. Messenger RNA encoding the alpha 9 acetylcholine (ACh) receptor subunit was detected and sequenced from individual outer hair cells and inner hair cells, but was not found in Deiters' cells. The identical Deiters' cells were positive for a P2x receptor subunit. This indicates cell-specific expression of the alpha 9 subunit in inner hair cells and outer hair cells and supports the hypothesis that this subunit contributes to calcium (Ca2+) permeable ionotropic ACh receptors (ACh-R). ACh-dependent Ca2+ concentration increase has been observed in both outer hair cells and inner hair cells.

Subunit-dependent assembly of inward-rectifier K+ channels.

Inward-rectifier, G-protein-regulated and ATP-dependent K+ channels form a novel gene family of related proteins which share two transmembrane segments as a common structural feature. These K+ channels are only distantly related to the voltage-gated Shaker-type K+ channels comprising six transmembrane segments. Although the quaternary structure of voltage-gated K+ channels has been extensively studied in the past, little is known about subunit assembly of inward-rectifier K+ channels. Differential sensitivity of inward-rectifier K+ channels to voltage-dependent pore block by spermine was used to analyse subunit assembly. It is shown that inward-rectifier K+ channel proteins are composed of four subunits whose assembly obeys the rules of a binomial distribution. 'Strong' and 'mild' inward-rectifier K+ channel subunits (BIR10 and ROMK1) which are co-expressed in individual auditory hair cells form hetero-tetramers. Distribution of these hetero-tetramers, however, is not binomial. Hetero- and homo-oligomeric channels form with similar probabilities resulting in independent channel populations with distinct functional properties.

Strong voltage-dependent inward rectification of inward rectifier K+ channels is caused by intracellular spermine.

Inward rectifier K+ channels mediate the K+ conductance at resting potential in many types of cell. Since these K+ channels do not pass outward currents (inward rectification) when the cell membrane is depolarized beyond a trigger threshold, they play an important role in controlling excitability. Both a highly voltage-dependent block by intracellular Mg2+ and an endogenous gating process are presently assumed to underly inward rectification. It is shown that strong voltage dependence of rectification found under physiological conditions is predominantly due to the effect of intracellular spermine. Physiological concentrations of free spermine mediate strong rectification of IRK1 inward rectifier K+ channels even in the absence of free Mg2+ and in IRK1 mutant channels that have no endogenous rectification.

Kir2.1 inward rectifier K+ channels are regulated independently by protein kinases and ATP hydrolysis.

Second messenger regulation of IRK1 (Kir2.1) inward rectifier K+ channels was investigated in giant inside-out patches from Xenopus oocytes. Kir2.1-mediated currents that run down completely within minutes upon excision of the patches could be partly restored by application of Mg-ATP together with > 10 microM free Mg2+ to the cytoplasmic side of the patch. As restoration could not be induced by the ATP analogs AMP-PNP or ATP gamma S, this suggests an ATPase-like mechanism. In addition to ATP, the catalytic subunit of cAMP-dependent protein kinase (PKA) induced an increase in current amplitude, which could, however, only be observed if channels were previously or subsequently stimulated by Mg-ATP and free Mg2+. This indicates that functional activity of Kir2.1 channels requires both phosphorylation by PKA and ATP hydrolysis. Moreover, currents could be down-regulated by N-heptyl-5-chloro-1-naphthalenesulfonamide, a specific stimulator of protein kinase C (PKC), suggesting that PKA and PKC mediate inverse effects on Kir2.1 channels. Regulation of Kir2.1 channels described here may be an important mechanism for regulation of excitability.

A structural determinant of differential sensitivity of cloned inward rectifier K+ channels to intracellular spermine.

Large subtype-specific differences in the sensitivity of cloned inward-rectifier K+ channels of the IRK1, BIR10 and ROMK1 subtype to being blocked by intracellular spermine (SPM) are described. It is shown, by site-directed mutagenesis, that the four orders of magnitude larger SPM sensitivity of BIR10 channels compared to ROMK1 channels may be explained by a difference in a single amino acid in the putative transmembrane segment TMII. This residue, a negatively charged glutamate in BIR10, is homologous to the residue in IRK1 and ROMK1 which has previously been shown to change gating properties and Mg2+ sensitivity. Differential block by physiological SPM concentrations is suggested as a major functional difference between subtypes of inward-rectifier K+ channels.