Spontaneous action potential activity is crucial for mammalian sensory system development. In the auditory system, patterned firing activity has been observed in immature spiral ganglion cells and brain-stem neurons and is likely to depend on cochlear inner hair cell (IHC) action potentials. It remains uncertain whether spiking activity is intrinsic to developing IHCs and whether it shows patterning. We found that action potentials are intrinsically generated by immature IHCs of altricial rodents and that apical IHCs exhibit bursting activity as opposed to more sustained firing in basal cells. We show that the efferent neurotransmitter ACh, by fine-tuning the IHC’s resting membrane potential (Vm), is crucial for the bursting pattern in apical cells. Endogenous extracellular ATP also contributes to the Vm of apical and basal IHCs by activating SK2 channels. We hypothesize that the difference in firing pattern along the cochlea instructs the tonotopic differentiation of IHCs and auditory pathway.

1 Block of the human two-pore domain potassium (2-PK) channel TREK-1 by fluoxetine (Prozac R ) and its active metabolite, norfluoxetine, was investigated using whole-cell patch-clamp recording of currents through recombinant channels in tsA 201 cells. 2 Fluoxetine produced a concentration-dependent inhibition of TREK-1 current that was reversible on wash. The IC 50 for block was 19 mM. Block by fluoxetine was voltage-independent. Fluoxetine (100 mM) produced an 84% inhibition of TREK-1 currents, but only a 31% block of currents through a related 2-PK channel, TASK-3. 3 Norfluoxetine was a more potent inhibitor of TREK-1 currents with an IC 50 of 9 mM. Block by norfluoxetine was also voltage-independent. 4 Truncation of the C-terminus of TREK-1 (D89) resulted in a loss of channel function, which could be restored by intracellular acidification or the mutation E306A. The mutation E306A alone increased basal TREK-1 current and resulted in a loss of the slow phase of TREK-1 activation. 5 Progressive deletion of the C-terminus of TREK-1 had no effect on the inhibition of the channel by fluoxetine. The E306A mutation, on the other hand, reduced the magnitude of fluoxetine inhibition, with 100 mM producing only a 40% inhibition. 6 It is concluded that fluoxetine and norfluoxetine are potent inhibitors of TREK-1. Block of TREK-1 by fluoxetine may have important consequences when the drug is used clinically in the treatment of depression.

A homology model has been generated for the pore-forming domain of Kir6.2, a component of an ATP-sensitive K channel, based on the x-ray structure of the bacterial channel KcsA. Analysis of the lipid-exposed and pore-lining surfaces of the model reveals them to be compatible with the known features of membrane proteins and Kir channels, respectively. The Kir6.2 homology model was used as the starting point for nanosecond-duration molecular dynamics simulations in a solvated phospholipid bilayer. The overall drift from the model structure was comparable to that seen for KcsA in previous similar simulations. Preliminary analysis of the interactions of the Kir6.2 channel model with K(+) ions and water molecules during these simulations suggests that concerted single-file motion of K(+) ions and water through the selectivity filter occurs. This is similar to such motion observed in simulations of KcsA. This suggests that a single-filing mechanism is conserved between different K channel structures and may be robust to changes in simulation details. Comparison of Kir6.2 and KcsA suggests some degree of flexibility in the filter, thus complicating models of ion selectivity based upon a rigid filter.

KcsA is a bacterial K+ channel that is gated by pH. Continuum dielectric calculations on the crystal structure of the channel protein embedded in a low dielectric slab suggest that side chains E71 and D80 of each subunit, which lie adjacent to the selectivity filter region of the channel, form a proton-sharing pair in which E71 is neutral (protonated) and D80 is negatively charged at pH 7. When K+ ions are introduced into the system at their crystallographic positions the pattern of proton sharing is altered. The largest perturbation is for a K+ ion at site S3, i.e., interacting with the carbonyls of T75 and V76. The presence of multiple K+ ions in the filter increases the probability of E71 being ionized and of D80 remaining neutral (i.e., protonated). The ionization states of the protein side chains influence the potential energy profile experienced by a K+ ion as it is translated along the pore axis. In particular, the ionization state of the E71-D80 proton-sharing pair modulates the shape of the potential profile in the vicinity of the selectivity filter. Such reciprocal effects of ion occupancy on side-chain ionization states, and of side-chain ionization states on ion potential energy profiles will complicate molecular dynamics simulations and related studies designed to calculate ion permeation energetics.

In the adult auditory organ, mechanoelectrical transducer (MET) channels are essential for transducing acoustic stimuli into electrical signals. In the absence of incoming sound, a fraction of the MET channels on top of the sensory hair cells are open, resulting in a sustained depolarizing current. By genetically manipulating the in vivo expression of molecular components of the MET apparatus, we show that during pre-hearing stages the MET current is essential for establishing the electrophysiological properties of mature inner hair cells (IHCs). If the MET current is abolished in adult IHCs, they revert into cells showing electrical and morphological features characteristic of pre-hearing IHCs, including the re-establishment of cholinergic efferent innervation. The MET current is thus critical for the maintenance of the functional properties of adult IHCs, implying a degree of plasticity in the mature auditory system in response to the absence of normal transduction of acoustic signals.