Inward-rectifier K ؉ channels of the ROMK (Kir1.1) subtype are responsible for K ؉ secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other K ir channels. Such anomalous titration of this lysine residue (Lys-80 in K ir1.1) is accomplished by the tertiary structure of the Kir protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in K ir1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pK a into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pK a of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of K ir channels as well as for the channel defects found in patients with antenatal Bartter syndrome.

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Before new drugs for the treatment of inner ear disorders can be studied in controlled clinical trials, it is important that their pharmacokinetics be established in inner ear fluids. Microdialysis allows drug levels to be measured in perilymph without the volume disturbances and potential cerebrospinal fluid contamination associated with fluid sampling. The aims of this study were to show: (i) that despite low recovery rates from miniature dialysis probes, significant amounts of drug are removed from small fluid compartments, (ii) that dialysis sampling artifacts can be accounted for using computer simulations and (iii) that microdialysis allows quantification of the entry rates through the round window membrane (RWM) into scala tympani (ST). Initial experiments used microdialysis probes in small compartments in vitro containing sodium fluorescein. Stable concentrations were observed in large compartments (1000 μl) but significant concentration declines were observed in smaller compartments (100, 10 and 5.6 μl) comparable to the size of the inner ear. Computer simulations of these experiments closely approximated the experimental data. In in vivo experiments, sodium fluorescein 10 mg/ml and dexamethasone-dihydrogen-phosphate disodium salt 8 mg/ml were simultaneously applied to the RWM of guinea pigs. Perilymph concentration in the basal turn of ST was monitored using microdialysis. The fluorescein concentration reached after 200 min application (585 ± 527 μg/ml) was approximately twice that of dexamethasone phosphate (291 ± 369 μg/ml). Substantial variation in concentrations was found between animals by approximately a factor of 34 for fluorescein and at least 41 for dexamethasone phosphate. This is, to a large extent, thought to be the result of the RWM permeability varying in different animals. It was not caused by substance analysis variations, because two different analytic methods were used and the concentration ratio between the two substances remained nearly constant across the experiments and because differences were apparent for the repeated samples obtained in each animal. Interpretation of the results using computer simulations allowed RWM permeability to be quantified. It also demonstrated, however, that cochlear clearance values could not be reliably obtained with microdialysis because of the significant contribution of dialysis to clearance. The observed interanimal variation, e.g., in RWM permeability, is likely to be clinically relevant to the local application of drugs in patients.

Blocking cloned inward-rectifier potassium (Kir) channels from the cytoplasmic side was analyzed with a rapid application system exchanging the intracellular solution on giant inside-out patches from Xenopus oocytes in <2 ms. Dependence of the pore-block on interaction of the blocking molecule with permeant and impermeant ions on either side of the membrane was investigated in Kir1.1 (ROMK1) channels blocked by ammonium derivatives and in Kir4.1 (BIR10) channels blocked by spermine. The blocking reaction in both systems showed first-order kinetics and allowed separate determination of on- and off-rates. The off-rates of block were strongly dependent on the concentration of internal and external bulk ions, but almost independent of the ion species at the cytoplasmic side of the membrane. With K+ as the only cation on both sides of the membrane, off-rates exhibited strong coupling to the K+ reversal potential (E(K)) and increased and decreased with reduction in intra and extracellular K+ concentration, respectively. The on-rates showed significant dependence on concentration and species of internal bulk ions. This control of rate-constants by interaction of permeant and impermeant internal and external ions governs the steady-state current-voltage relation (I-V) of Kir channels and determines their physiological function under various conditions.

Hypothesis To determine whether intracochlearly applied dexamethasone will lead to better control of drug levels, higher peak concentrations and lower base-to apex concentration gradients in scala tympani (ST) of the guinea pig than after intratympanic (round window, RW) application. Background Local application of drugs to the RW results in substantial variation of intracochlear drug levels and significant base-to apex concentration gradients in ST. Methods Two μL of dexamethasone-phosphate (10 mg/mL) were injected into ST either through the RW membrane which was covered with 1% sodium hyaluronate gel or through a cochleostomy with a fluid tight seal of the micropipette. Perilymph was sequentially sampled from the apex at a single time point for each animal, at 20, 80, or 200 min after the injection ended. Results were mathematically interpreted by the means of an established computer model and compared with prior experiments performed by our group with the same experimental techniques but using intratympanic applications. Results Single intracochlear injections over 20 min resulted in approximately ten times higher peak concentrations (on average) than 2-3 hours of intratympanic application to the round window niche. Intracochlear drug levels were less variable and could be measured for at least up to 220 min. Concentration gradients along scala tympani were less pronounced. The remaining variability in intracochlear drug levels was attributable to perilymph and drug leak from the injection site. Conclusion With significantly higher, less variable drug levels and smaller base-to apex concentration gradients, intracochlear applications have advantages to intratympanic injections. For further development of this technique, it is of importance to control leaks of perilymph and drug from the injection site and to evaluate its clinical feasibility and associated risks.