Optimized Tuning of Auditory Inner Hair Cells to Encode Complex Sound through Synergistic Activity of Six Independent K+ Current Entities.

Auditory inner hair cells (IHCs) convert sound vibrations into receptor potentials that drive synaptic transmission. For the precise encoding of sound qualities, receptor potentials are shaped by K+ conductances tuning the properties of the IHC membrane. Using patch-clamp and computational modeling, we unravel this membrane specialization showing that IHCs express an exclusive repertoire of six voltage-dependent K+ conductances mediated by Kv1.8, Kv7.4, Kv11.1, Kv12.1, and BKCa channels. All channels are active at rest but are triggered differentially during sound stimulation. This enables non-saturating tuning over a far larger potential range than in IHCs expressing fewer current entities. Each conductance contributes to optimizing responses, but the combined activity of all channels synergistically improves phase locking and the dynamic range of intensities that IHCs can encode. Conversely, hypothetical simpler IHCs appear limited to encode only certain aspects (frequency or intensity). The exclusive channel repertoire of IHCs thus constitutes an evolutionary adaptation to encode complex sound through multifaceted receptor potentials.

ß-Secretase BACE1 Is Required for Normal Cochlear Function.

Cleavage of amyloid precursor protein (APP) by ß-secretase BACE1 initiates the production and accumulation of neurotoxic amyloid-ß peptides, which is widely considered an essential pathogenic mechanism in Alzheimer's disease (AD). Here, we report that BACE1 is essential for normal auditory function. Compared with wild-type littermates, BACE1-/- mice of either sex exhibit significant hearing deficits, as indicated by increased thresholds and reduced amplitudes in auditory brainstem responses (ABRs) and decreased distortion product otoacoustic emissions (DPOAEs). Immunohistochemistry revealed aberrant synaptic organization in the cochlea and hypomyelination of auditory nerve fibers as predominant neuropathological substrates of hearing loss in BACE1-/- mice. In particular, we found that fibers of spiral ganglion neurons (SGN) close to the organ of Corti are disorganized and abnormally swollen. BACE1 deficiency also engenders organization defects in the postsynaptic compartment of SGN fibers with ectopic overexpression of PSD95 far outside the synaptic region. During postnatal development, auditory fiber myelination in BACE1-/- mice lags behind dramatically and remains incomplete into adulthood. We relate the marked hypomyelination to the impaired processing of Neuregulin-1 when BACE1 is absent. To determine whether the cochlea of adult wild-type mice is susceptible to AD treatment-like suppression of BACE1, we administered the established BACE1 inhibitor NB-360 for 6 weeks. The drug suppressed BACE1 activity in the brain, but did not impair hearing performance and, upon neuropathological examination, did not produce the characteristic cochlear abnormalities of BACE1-/- mice. Together, these data strongly suggest that the hearing loss of BACE1 knock-out mice represents a developmental phenotype.SIGNIFICANCE STATEMENT Given its crucial role in the pathogenesis of Alzheimer's disease (AD), BACE1 is a prime pharmacological target for AD prevention and therapy. However, the safe and long-term administration of BACE1-inhibitors as envisioned in AD requires a comprehensive understanding of the various physiological functions of BACE1. Here, we report that BACE1 is essential for the processing of auditory signals in the inner ear, as BACE1-deficient mice exhibit significant hearing loss. We relate this deficit to impaired myelination and aberrant synapse formation in the cochlea, which manifest during postnatal development. By contrast, prolonged pharmacological suppression of BACE1 activity in adult wild-type mice did not reproduce the hearing deficit or the cochlear abnormalities of BACE1 null mice.

Histidine at position 462 determines the low quinine sensitivity of ether-à-go-go channel superfamily member Kv 12.1.

BACKGROUND AND PURPOSE: The ether-à-go-go (Eag) Kv superfamily comprises closely related Kv 10, Kv 11, and Kv 12 subunits. Kv 11.1 (termed hERG in humans) gained much attention, as drug-induced inhibition of these channels is a frequent cause of sudden death in humans. The exclusive drug sensitivity of Kv 11.1 can be explained by central drug-binding pockets that are absent in most other channels. Currently, it is unknown whether Kv 12 channels are equipped with an analogous drug-binding pocket and whether drug-binding properties are conserved in all Eag superfamily members. EXPERIMENTAL APPROACH: We analysed sensitivity of recombinant Kv 12.1 channels to quinine, a substituted quinoline that blocks Kv 10.1 and Kv 11.1 at low micromolar concentrations. KEY RESULTS: Quinine inhibited Kv 12.1, but its affinity was 10-fold lower than for Kv 11.1. Contrary to Kv 11.1, quinine inhibited Kv 12.1 in a largely voltage-independent manner and induced channel opening at more depolarised potentials. Low sensitivity of Kv 12.1 and characteristics of quinine-dependent inhibition were determined by histidine 462, as site-directed mutagenesis of this residue into the homologous tyrosine of Kv 11.1 conferred Kv 11.1-like quinine block to Kv 12.1(H462Y). Molecular modelling demonstrated that the low affinity of Kv 12.1 was determined by only weak interactions of residues in the central cavity with quinine. In contrast, more favourable interactions can explain the higher quinine sensitivity of Kv 12.1(H462Y) and Kv 11.1 channels. CONCLUSIONS AND IMPLICATIONS: The quinoline-binding "motif" is not conserved within the Eag superfamily, although the overall architecture of these channels is apparently similar. Our findings highlight functional and pharmacological diversity in this group of evolutionary-conserved channels.

Kv12.1 channels are not sensitive to GqPCR-triggered activation of phospholipase Cß.

Kv12.1 K+ channels are expressed in several brain areas, but no physiological function could be attributed to these subunits so far. As genetically-modified animal models are not available, identification of native Kv12.1 currents must rely on characterization of distinct channel properties. Recently, it was shown in Xenopus laevis oocytes that Kv12.1 channels were modulated by membrane PI(4,5)P2. However, it is not known whether these channels are also sensitive to physiologically-relevant PI(4,5)P2 dynamics. We thus studied whether Kv12.1 channels were modulated by activation of phospholipase C ß (PLCß) and found that they were insensitive to receptor-triggered depletion of PI(4,5)P2. Thus, Kv12.1 channels add to the growing list of K+ channels that are insensitive to PLCß signaling, although modulated by PI(4,5)P2 in Xenopus laevis oocytes.

Inverse Modulation of Neuronal Kv12.1 and Kv11.1 Channels by 4-Aminopyridine and NS1643.

The three members of the ether-à-go-go-gene-like (Elk; Kv12.1-Kv12.3) family of voltage-gated K+ channels are predominantly expressed in neurons, but only little information is available on their physiological relevance. It was shown that Kv12.2 channels modulate excitability of hippocampal neurons, but no native current could be attributed to Kv12.1 and Kv12.3 subunits yet. This may appear somewhat surprising, given high expression of their mRNA transcripts in several brain areas. Native Kv12 currents may have been overlooked so far due to limited knowledge on their biophysical properties and lack of specific pharmacology. Except for Kv12.2, appropriate genetically modified mouse models have not been described; therefore, identification of Kv12-mediated currents in native cell types must rely on characterization of unique properties of the channels. We focused on recombinant human Kv12.1 to identify distinct properties of these channels. We found that Kv12.1 channels exhibited significant mode shift of activation, i.e., stabilization of the voltage sensor domain in a "relaxed" open state after prolonged channel activation. This mode shift manifested by a slowing of deactivation and, most prominently, a significant shift of voltage dependence to hyperpolarized potentials. In contrast to related Kv11.1, mode shift was not sensitive to extracellular Na+, which allowed for discrimination between these isoforms. Sensitivity of Kv12.1 and Kv11.1 to the broad-spectrum K+ antagonist 4-aminopyridine was similar. However, 4-AP strongly activated Kv12.1 channels, but it was an inhibitor of Kv11 channels. Interestingly, the agonist of Kv11 channels NS1643 also differentially modulated the activity of these channels, i.e., NS1643 activated Kv11.1, but strongly inhibited Kv12.1 channels. Thus, these closely related channels are distinguished by inverse pharmacological profiles. In summary, we identified unique biophysical and pharmacological properties of Kv12.1 channels and established straightforward experimental protocols to characterize Kv12.1-mediated currents. Identification of currents in native cell types with mode shift that are activated through 4-AP and inhibited by NS1643 can provide strong evidence for contribution of Kv12.1 to whole cell currents.

Direct modulation of TRPM4 and TRPM3 channels by the phospholipase C inhibitor U73122.

BACKGROUND AND PURPOSE: Signalling through phospholipase C (PLC) controls many cellular processes. Much information on the relevance of this important pathway has been derived from pharmacological inhibition of the enzymatic activity of PLC. We found that the most frequently employed PLC inhibitor, U73122, activates endogenous ionic currents in widely used cell lines. Given the extensive use of U73122 in research, we set out to identify these U73122-sensitive ion channels. EXPERIMENTAL APPROACH: We performed detailed biophysical analysis of the U73122-induced currents in frequently used cell lines. KEY RESULTS: At concentrations required to inhibit PLC, U73122 modulated the activity of transient receptor potential melastatin (TRPM) channels through covalent modification. U73122 was shown to be a potent agonist of ubiquitously expressed TRPM4 channels and activated endogenous TRPM4 channels in CHO cells independently of PLC and of the downstream second messengers PI(4,5)P2 and Ca(2+) . U73122 also potentiated Ca(2) (+) -dependent TRPM4 currents in human Jurkat T-cells, endogenous TRPM4 in HEK293T cells and recombinant human TRPM4. In contrast to TRPM4, TRPM3 channels were inhibited whereas the closely related TRPM5 channels were insensitive to U73122, showing that U73122 exhibits high specificity within the TRPM channel family. CONCLUSIONS AND IMPLICATIONS: Given the widespread expression of TRPM4 and TRPM3 channels, these actions of U73122 must be considered when interpreting its effects on cell function. U73122 may also be useful for identifying and characterizing TRPM channels in native tissue, thus facilitating the analysis of their physiology.

Electrical Wiring of the Aldehyde Oxidoreductase PaoABC with a Polymer Containing Osmium Redox Centers: Biosensors for Benzaldehyde and GABA.

Biosensors for the detection of benzaldehyde and γ-aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below -0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A "reagentless" biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10-150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer.