NKCCs in the fibrocytes of the spiral ligament are silent on the unidirectional K+ transport that controls the electrochemical properties in the mammalian cochlea

Abstract

Unidirectional K(+) transport across the lateral cochlear wall contributes to the endocochlear potential (EP) of +80 mV in the endolymph, a property essential for hearing. The wall comprises two epithelial layers, the syncytium and the marginal cells. The basolateral surface of the former and the apical membranes of the latter face the perilymph and the endolymph, respectively. Intrastrial space (IS), an extracellular compartment between the two layers, exhibits low [K(+)] and a potential similar to the EP. This IS potential (ISP) dominates the EP and represents a K(+) diffusion potential elicited by a large K(+) gradient across the syncytial apical surface. The K(+) gradient depends on the unidirectional K(+) transport driven by Na(+),K(+)-ATPases on the basolateral surface of each layer and the concomitant Na(+),K(+),2Cl(-)-cotransporters (NKCCs) in the marginal cell layer. The NKCCs coexpressed with the Na(+),K(+)-ATPases in the syncytial layer also seem to participate in the K(+) transport. To test this hypothesis, we examined the electrochemical properties of the lateral wall with electrodes measuring [K(+)] and potential. Blocking NKCCs by perilymphatic perfusion of bumetanide suppressed the ISP. Unexpectedly and unlike the inhibition of the syncytial Na(+),K(+)-ATPases, the perfusion barely altered the electrochemical properties of the syncytium but markedly augmented [K(+)] of the IS. Consequently, the K(+) gradient decreased and the ISP declined. These observations resembled those when the marginal cells' Na(+),K(+)-ATPases or NKCCs were blocked with vascularly applied inhibitors. It is plausible that NKCCs in the marginal cells are affected by the perilymphatically perfused bumetanide, and these transporters, but not those in the syncytium, mediate the unidirectional K(+) transport.

The unique ion permeability profile of cochlear fibrocytes and its contribution to establishing their positive resting membrane potential

“…1b) [26]. In vivo, the membranes of fibrocytes continuously show a significantly positive potential of +5 to +12 mV relative to the perilymph [1,14,26,41]. If this RMP was negative, like the RMPs of neurons and glial cells, then the ISP and EP would be markedly low.…”

“…Electrophysiological assays with the cochleae of live guinea pigs were performed using procedures similar to those of our previous studies [1,26,41]. Double-barreled K + -selective microelectrodes and single-barreled microelectrodes, both of which were prepared as described above, were used to measure the electrochemical properties of the lateral wall and the EP, respectively.…”

“…Each barrel was connected via an Ag/AgCl electrode to a dual electrometer with high input-resistance (FD223a; WPI). Calibration of each K + -selective electrode was performed before and after the experiment using previously described methods [1,41]. In this study, ionic concentrations were calculated based on activity coefficients for K + of 0.727 [22].…”

“…Perfusates were applied at a rate of 10 μL/min using a syringe pump through a capillary tube inserted in the inlet hole as described previously [1,41]. Although the native perilymph likely contains 130-150 mM [Na + ] and ∼5 mM [K + ] [14,39], for the control artificial perilymph, a solution with the following ionic composition (in mM) was used: 100 Na + , 5 K + , 46 N-methyl-D-glucamine (NMDG), 1.2 Ca 2+ , 1.0 Mg 2+ , 131.4 Cl − , 24 HCO 3 − , 5.0 HEPES, and 4.0 glucose saturated with 5 % O 2 , 5 % CO 2 , and 90 % N 2 at pH 7.4 and 295 mOsm (for a justification, see below).…”

“…1b) [9,40,42]. Of these, Na + /K + ATPases appear to maintain high [K + ] in the syncytial layer by continuous K + uptake from the perilymph [1,41]. However, neither physiological nor pharmacological assays targeting the channels and transporters, including the ATPases, have revealed how cells establish the positive RMP.…”