A. J. Hudspeth

Vanilloid Receptor–Related Osmotically Activated Channel (VR-OAC), a Candidate Vertebrate Osmoreceptor

The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nervous system, the channel is expressed in neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells.

Compliance of the hair bundle associated with gating of mechanoelectrical transduction channels in the Bullfrog's saccular hair cell

Mechanical stimuli are thought to open the transduction channels of a hair cell by tensing elastic components, the gating springs, that pull directly on the channels. To test this model, we measured the stiffness of hair bundles during mechanical stimulation. A bundles compliance increased by about 40% at the position where half of the channels opened. This we attribute to conformational changes of transduction channels as they open and close. The magnitude and displacement dependence of the gating compliance provide quantitative information about the molecular basis of mechanoelectrical transduction: the force required to open each channel, the number of transduction channels per hair cell, the stiffness of a gating spring, and the swing of a channels gate as it opens.

Kinetic analysis of voltage- and ion-dependent conductances in saccular hair cells of the bull-frog, Rana catesbeiana.

1. By the use of whole‐cell and excised‐patch tight‐seal recording techniques, we studied ionic conductances in voltage‐clamped solitary hair cells isolated from the bull‐frogs sacculus. As a basis for assessing their contributions to hair cell electrical resonance, we developed kinetic models describing voltage‐dependent Ca2+ and Ca2+‐dependent K+ conductances. 2. A transient K+ current (IA) was activated by steps to potentials positive to ‐50 mV from holding potentials more negative than ‐70 mV. In the steady state, the current was fully inactivated at the normal resting potential. Possibly due to the dissipation of a Donnan potential between the pipettes interior and the cell, the voltage dependence of IA inactivation slowly shifted in the negative direction during whole‐cell recording. 3. The voltage‐gated Ca2+ current (ICa) was isolated by blocking IA with 4‐aminopyridine (4‐AP) and Ca2+‐activated K+ current with tetraethylammonium (TEA). The ICa was activated at potentials more positive than ‐60 to ‐50 mV and was maximal at about ‐10 mV. Its magnitude was highly variable among cells, with an average value of ‐240 pA at ‐30 mV. Its activation could be fitted well by a third‐order (m3) gating scheme. 4. A Ca2+‐activated K+ current (IK(Ca)) was isolated as the component of membrane current blocked by TEA. This current was activated at potentials more positive than ‐60 to ‐50 mV and had an average value of 1.5 nA at ‐30 mV. The Ca2+‐activated K+ conductance (gK(Ca)) showed a high apparent voltage dependence, increasing e‐fold every 3 mV at potentials between ‐50 and ‐40 mV. 5. The Ca2+‐activated K+ current displayed rapid activation and deactivation kinetics. The current reached half‐maximal activation in 2‐4 ms at voltages between ‐50 and ‐30 mV, and the tail current decayed exponentially with a time constant of 1.0 ms at ‐70 mV. The activation rate and magnitude of IK(Ca) were reduced by lowering the extracellular Ca2+ concentration. 6. The open probability of Ca2+‐activated K+ channels was estimated by ensemble‐fluctuation analysis of whole‐cell currents evoked by voltage steps to ‐30 mV. The average open probability was estimated to be 0.8 at this potential. 7. K+‐selective channels with a high conductance (140‐200 pS) were examined in excised, inside‐out membrane patches. The activity of these channels depended on intracellular Ca2+ and membrane potential. These properties suggest that the channels underlie the whole‐cell Ca2+‐activated K+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

Making an Effort to Listen: Mechanical Amplification in the Ear

The inner ears performance is greatly enhanced by an active process defined by four features: amplification, frequency selectivity, compressive nonlinearity, and spontaneous otoacoustic emission. These characteristics emerge naturally if the mechanoelectrical transduction process operates near a dynamical instability, the Hopf bifurcation, whose mathematical properties account for specific aspects of our hearing. The active process of nonmammalian tetrapods depends upon active hair-bundle motility, which emerges from the interaction of negative hair-bundle stiffness and myosin-based adaptation motors. Taken together, these phenomena explain the four characteristics of the ears active process. In the high-frequency region of the mammalian cochlea, the active process is dominated instead by the phenomenon of electromotility, in which the cell bodies of outer hair cells extend and contract as the protein prestin alters its membrane surface area in response to changes in membrane potential.

RIM - binding proteins (RBPs) couple Rab3 - interacting molecules (RIMs) to voltage - gated Ca2+ channels

Ca(2+) influx through voltage-gated channels initiates the exocytotic fusion of synaptic vesicles to the plasma membrane. Here we show that RIM binding proteins (RBPs), which associate with Ca(2+) channels in hair cells, photoreceptors, and neurons, interact with alpha(1D) (L type) and alpha(1B) (N type) Ca(2+) channel subunits. RBPs contain three Src homology 3 domains that bind to proline-rich motifs in alpha(1) subunits and Rab3-interacting molecules (RIMs). Overexpression in PC12 cells of fusion proteins that suppress the interactions of RBPs with RIMs and alpha(1) augments the exocytosis triggered by depolarization. RBPs may regulate the strength of synaptic transmission by creating a functional link between the synaptic-vesicle tethering apparatus, which includes RIMs and Rab3, and the fusion machinery, which includes Ca(2+) channels and the SNARE complex.

A model for electrical resonance and frequency tuning in saccular hair cells of the bull‐frog, Rana catesbeiana.

1. Electrical resonance in solitary hair cells was examined under several experimental conditions using the tight‐seal recording technique in the whole‐cell current‐clamp mode. 2. Resonance was characterized by the frequency and quality factor of oscillations in membrane potential evoked by depolarizing current pulses. Oscillation frequency increased with depolarization, from about 90 Hz at the resting potential to a limiting value of about 250 Hz. The quality factor of the oscillations was a bell‐shaped function of membrane potential that reached a maximum of up to 12.6 at a potential slightly positive to the resting potential. 3. Pharmacological experiments were performed to assess which of three ionic currents participate in electrical resonance. Reduction of the voltage‐gated Ca2+ current (ICa) and the Ca2+‐activated K+ current (IK(Ca)) by lowering the extracellular Ca2+ concentration, or reduction of IK(Ca) with tetraethylammonium ion (TEA) degraded the resonance. In contrast, blockade of the transient K+ current (IA) with 4‐aminopyridine (4‐AP) had no significant effect. 4. To test the sufficiency of the Ca2+ and the Ca2+‐activated K+ currents to account for resonance, we developed a model using mathematical descriptions of the two currents derived in the preceding paper (Hudspeth & Lewis, 1988), with additional terms for leakage conductance and membrane capacitance. The model correctly predicts the oscillatory responses to applied current pulses, including the non‐linear dependences of oscillation frequency and quality factor on membrane potential. 5. Simulations of current‐clamp experiments in the presence of a reduced extracellular Ca2+ concentration or of TEA were generated respectively by decreasing the models values for the maximal Ca2+ or Ca2+‐activated K+ conductances. The models predictions of membrane‐potential oscillations under these conditions agree qualitatively with experimental results, providing further support for the model as a description of the resonance mechanism. 6. To identify the factors most important in determining the hair cells resonance properties, we systematically altered the values of selected parameters in the model. Frequency was most profoundly influenced by increasing the magnitude and activation rate of the Ca2+‐activated K+ conductance, whereas the quality factor was most sensitive to increases in the level of the Ca2+ conductance. 7. By including a term describing activation of the hair cells mechanically sensitive transduction conductance, we used the model to predict a tuning curve for responses to mechanical inputs of various frequencies.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca2+ current-driven nonlinear amplification by the mammalian cochlea in vitro.

An active process in the inner ear expends energy to enhance the sensitivity and frequency selectivity of hearing. Two mechanisms have been proposed to underlie this process in the mammalian cochlea: receptor potential–based electromotility and Ca2+-driven active hair-bundle motility. To link the phenomenology of the cochlear amplifier with these cellular mechanisms, we developed an in vitro cochlear preparation from Meriones unguiculatus that affords optical access to the sensory epithelium while mimicking its in vivo environment. Acoustic and electrical stimulation elicited microphonic potentials and electrically evoked hair-bundle movement, demonstrating intact forward and reverse mechanotransduction. The mechanical responses of hair bundles from inner hair cells revealed a characteristic resonance and a compressive nonlinearity diagnostic of the active process. Blocking transduction with amiloride abolished nonlinear amplification, whereas eliminating all but the Ca2+ component of the transduction current did not. These results suggest that the Ca2+ current drives the cochlear active process, and they support the hypothesis that active hair-bundle motility underlies cochlear amplification.

Mechanical amplification of stimuli by hair cells

Unlike any other known sensory receptor, the hair cell uses positive feedback to augment the stimulus to which it responds. In the internal ears of many vertebrates, hair cells amplify the inputs to their mechanosensitive hair bundles. Outer hair cells of the mammalian cochlea display a unique form of somatal motility that may underlie their contribution to amplification. In other receptor organs, hair cells may effect amplification by hair-bundle movements driven by the activity of myosin or of transduction channels. Recent work has demonstrated the presence of several myosin isozymes in hair bundles, confirmed that bundles display myosin ATPase activity, and shown that the work performed by myosin molecules could account for one aspect of the amplificatory process.

Distribution of Ca2+-Activated K+ Channel Isoforms along the Tonotopic Gradient of the Chicken's Cochlea

In some cochleae, the number and kinetic properties of Ca2+-activated K+ (KCa) channels partly determine the characteristic frequency of each hair cell and thus help establish a tonotopic map. In the chickens basilar papilla, we found numerous isoforms of KCa channels generated by alternative mRNA splicing at seven sites in a single gene, cSlo. In situ polymerase chain reactions demonstrated cSlo expression in hair cells and revealed differential distributions of KCa channel isoforms along the basilar papilla. Analysis of single hair cells by the reverse transcription polymerase chain reaction confirmed the differential expression of channel variants. Heterologously expressed cSlo variants differed in their sensitivities to Ca2+ and voltage, suggesting that the distinct spatial distributions of cSlo variants help determine the tonotopic map.

Identification of a 120 kd hair-bundle myosin located near stereociliary tips

By adapting to sustained stimuli, hair cells of the internal ear maintain their optimal sensitivity to minute displacements. Biophysical experiments have suggested that adaptation is mediated by a molecular motor, most likely a member of the myosin family. To provide direct evidence for the presence of myosin isozymes in hair bundles, we used photoaffinity labeling with vanadate-trapped uridine and adenine nucleotides to identify proteins of 120, 160, and 230 kd in a preparation of hair bundles purified from the bullfrogs sacculus. The photoaffinity labeling properties of these proteins, particularly the 120 kd protein, resembled those of other well-characterized myosins. A 120 kd hair-bundle protein was also recognized by a monoclonal antibody directed against a vertebrate myosin I isozyme. Immunofluorescence microscopy localized this protein near the beveled top edge of the hair bundle, the site of mechanoelectrical transduction and adaptation.