Extracellular chloride regulation of Kv2.1, contributor to the major outward Kv current in mammalian outer hair cells

Abstract

Outer hair cells (OHC) function as both receptors and effectors in providing a boost to auditory reception. Amplification is driven by the motor protein prestin, which is under anionic control. Interestingly, we now find that the major, 4-AP-sensitive, outward K+ current of the OHC ( IK) is also sensitive to Cl−, although, in contrast to prestin, extracellularly. IK is inhibited by reducing extracellular Cl− levels, with a linear dependence of 0.4%/mM. Other voltage-dependent K+ (Kv) channel conductances in supporting cells, such as Hensen and Deiters' cells, are not affected by reduced extracellular Cl−. To elucidate the molecular basis of this Cl−-sensitive IK, we looked at potential molecular candidates based on Cl− sensitivity and/or similarities in kinetics. For IK, we identified three different Ca2+-independent components of IK based on the time constant of inactivation: a fast, transient outward current, a rapidly activating, slowly inactivating current ( Ik1), and a slowly inactivating current ( Ik2). Extracellular Cl− differentially affects these components. Because the inactivation time constants of Ik1 and Ik2 are similar to those of Kv1.5 and Kv2.1, we transiently transfected these constructs into CHO cells and found that low extracellular Cl− inhibited both channels with linear current reductions of 0.38%/mM and 0.49%/mM, respectively. We also tested heterologously expressed Slick and Slack conductances, two intracellularly Cl−-sensitive K+ channels, but found no extracellular Cl− sensitivity. The Cl− sensitivity of Kv2.1 and its robust expression within OHCs verified by single-cell RT-PCR indicate that these channels underlie the OHC's extracellular Cl− sensitivity.

Protection from Noise-Induced Hearing Loss by Kv2.2 Potassium Currents in the Central Medial Olivocochlear System

“…Cochlear sections from WT and Kv2.2KO animals were treated with an identical protocol and then Kv2.2 and Kv2.1 immunohistochemistry was performed. Kv2.1 immunoreactivity was observed, consistent with previous reports (Li et al, 2012), but no specific Kv2.2 staining (Fig.3c, 3d) was observed in the hair cells or in the spiral ganglion (not shown). In conjunction with the positive control from the brainstem, we conclude that there is no Kv2.2 expressed in the cochlea and hence any changes in hearing associated with the absence of Kv2.2, must reflect a central rather than a peripheral location of this channel.…”

“…The Kv2 potassium channel family has two members: Kv2.1 and Kv2.2; the former is highly expressed in the hippocampus and neocortex (Du et al, 2000; Guan et al, 2007) and hair cells (Li et al, 2012) where Kv2.1 containing channels are considered the dominant delayed rectifiers and responsible for action potential repolarization. Kv2.2 has a much more restricted expression profile, and in the auditory brainstem is expressed exclusively in the initial segment of the MNTB neurons and in VNTB neurons.…”

Cl− as a bona fide signaling ion

“…The 4-Ap-sensitive outward rectifying K ϩ channel (K v ) is one of the two major K ϩ channels in the outer hair cell (OHC) and has several kinetic components when activated by a depolarizing pulse (59). The channel is activated by [Cl Ϫ ] out with a shallow linear dependence on [Cl Ϫ ] out between 5 and 150 mM (Fig.…”

“…The channel is activated by [Cl Ϫ ] out with a shallow linear dependence on [Cl Ϫ ] out between 5 and 150 mM (Fig. 2B), with [Cl Ϫ ] out differentially affecting each of its kinetic components (59). Topical expression of various outward rectifying channels has shown that K v 2.1 and K v 1.5 are activated by [Cl Ϫ ] out with the properties of K v 2.1 most resembling the K ϩ channel of OHC and indicating that K v 2.1 accounts for the [Cl Ϫ ] out sensitivity of OHC (59).…”

Chloride Sensing by WNK1 Involves Inhibition of Autophosphorylation

“…Although the chloride-mediated regulation of a few soluble proteins has been defined at both a biochemical and structural level, for example α-amylase and hemoglobin (13, 15), how changes in chloride concentration regulate other proteins including those in cellular membranes is unknown. For example, chloride regulates the voltage-gated potassium channel KCNB1, increasing the K + current as a function of chloride in patch-clamp studies (16). The chloride-dependent Na + /H + exchanger (Cl− NHE) is activated by chloride, as determined by intracellular pH measurements on exchanger-transfected cells (17).…”