Elsevier

Hearing Research

Volume 345, March 2017, Pages 57-68
Hearing Research

Research paper
Genetic perturbations suggest a role of the resting potential in regulating the expression of the ion channels of the KCNA and HCN families in octopus cells of the ventral cochlear nucleus

https://doi.org/10.1016/j.heares.2017.01.001Get rights and content

Highlights

  • The large size of gh and gKL in octopus cells allowed us to resolve and measure Ih and IKL at the resting potential.

  • Perturbing the magnitude and subunit composition of these conductances genetically did not affect average resting potentials.

  • IKL balanced Ih at rest in all octopus cells, regardless of mouse strain and individual resting potential.

  • Instead of gh and gKL regulating the resting potential, it seems that the resting potential regulates gh and gKL.

Abstract

Low-voltage-activated K+ (gKL) and hyperpolarization-activated mixed cation conductances (gh) mediate currents, IKL and Ih, through channels of the Kv1 (KCNA) and HCN families respectively and give auditory neurons the temporal precision required for signaling information about the onset, fine structure, and time of arrival of sounds. Being partially activated at rest, gKL and gh contribute to the resting potential and shape responses to even small subthreshold synaptic currents. Resting gKL and gh also affect the coupling of somatic depolarization with the generation of action potentials. To learn how these important conductances are regulated we have investigated how genetic perturbations affect their expression in octopus cells of the ventral cochlear nucleus (VCN). We report five new findings: First, the magnitude of gh and gKL varied over more than two-fold between wild type strains of mice. Second, average resting potentials are not different in different strains of mice even in the face of large differences in average gKL and gh. Third, IKL has two components, one being α-dendrotoxin (α-DTX)-sensitive and partially inactivating and the other being α-DTX-insensitive, tetraethylammonium (TEA)-sensitive, and non-inactivating. Fourth, the loss of Kv1.1 results in diminution of the α-DTX-sensitive IKL, and compensatory increased expression of an α-DTX-insensitive, tetraethylammonium (TEA)-sensitive IKL. Fifth, Ih and IKL are balanced at the resting potential in all wild type and mutant octopus cells even when resting potentials vary in individual cells over nearly 10 mV, indicating that the resting potential influences the expression of gh and gKL. The independence of resting potentials on gKL and gh shows that gKL and gh do not, over days or weeks, determine the resting potential but rather that the resting potential plays a role in regulating the magnitude of either or both gKL and gh.

Introduction

Acoustic information is carried in the timing as well as the rate of firing of brain stem auditory neurons. The interplay between depolarization-activated, low-voltage-activated K+ conductances (gKL) mediated by ion channels of the Kv1 family and hyperpolarization-activated conductances (gh) mediated through HCN channels gives auditory neurons the ability to receive and convey precisely timed electrical signals, as has been demonstrated in spiral ganglion cells (Mo and Davis, 1997, Liu et al., 2014, Mo et al., 2002, Kim and Holt, 2013), bushy and octopus cells in the VCN (Oertel, 1983, Manis and Marx, 1991, Rusznak et al., 1996, Cao et al., 2007), neurons in the medial nucleus of the trapezoid body (Brew and Forsythe, 1995, Cuttle et al., 2001), medial superior olive (Scott et al., 2005, Khurana et al., 2011), lateral superior olive (Barnes-Davies et al., 2004), and the ventral nucleus of the lateral lemniscus (Wu, 1999, Berger et al., 2014). In octopus cells gKL and gh are especially large (Bal and Oertel, 2000, Bal and Oertel, 2001, Ferragamo and Oertel, 2002, Cao and Oertel, 2011, McGinley et al., 2012). These conductances have opposite voltage dependence of activation and at rest the currents flow in opposite directions, IKL flowing outward and Ih flowing inward, so that perturbations of the resting potential in either direction draw the voltage back toward rest.

The presence of gh and gKL enhances the timing of signaling in neurons in three ways. First, the partial activation of both conductances lowers the resting input resistance and speeds the rise and fall of voltage changes. Second, the voltage sensitivity around rest and the rapidity of the activation of gKL enables gKL to shorten and sharpen synaptic potentials. Third, the rapidity of the activation of gKL makes the firing of neurons that have this conductance sensitive to the rate at which they are depolarized, and thus makes them effective coincidence detectors. Since the rate of depolarization depends on the temporal scatter of coincident subthreshold inputs, the sensitivity to the rate of depolarization sharpens the window over which octopus cells detect coincident firing in their auditory nerve inputs (Ferragamo and Oertel, 2002, McGinley and Oertel, 2006, Golding and Oertel, 2012).

gKL is mediated through tetrameric, voltage-sensitive K+ channels of the Kv1 family; four α subunits associate with four β subunits that modulate functional properties. Kv1.1 α subunits are present on the somatic and dendritic membranes of octopus cells as well as at perinodes of auditory nerve axons and octopus cell axons (Oertel et al., 2008, Rusznak et al., 2008, Robbins and Tempel, 2012). Immunohistochemical visualization of Kv1.1 (Oertel et al., 2008, Robbins and Tempel, 2012), Kv1.2 (Rusznak et al., 2008), Kv1.4 (Fonseca et al., 1998, Lujan et al., 2003), and Kv1.6 (Rusznak et al., 2008) subunits suggests that these subunits are expressed in octopus cells. The sensitivity of gKL to DTX K, a blocker that is specific for Kv1.1 containing channels (Robertson et al., 1996), confirms the role of Kv1.1 in gKL (Bal and Oertel, 2001). To be expressed at the membrane, Kv1.1 subunits require coassembly with other subunits, most often with Kv1.2 and Kv1.4 (Hopkins et al., 1994, Manganas and Trimmer, 2000, Ovsepian et al., 2016). The sensitivity of gKL to α-DTX and to tityustoxin, selective for channels containing Kv1.2 subunits (Werkman et al., 1993, Robertson et al., 1996, Owen et al., 1997, Wang et al., 1999a, Wang et al., 1999b, Hopkins, 1998), functionally confirms the role of Kv1.2 subunits in octopus cells (Bal and Oertel, 2001).

gh is mediated through tetrameric voltage-sensitive channels that are composed of HCN1-4 α subunits (Ludwig et al., 1998, Robinson and Siegelbaum, 2003). HCN1, HCN2 and HCN4 are expressed in the VCN (Moosmang et al., 1999; Koch et al., 2004; Notomi and Shigemoto, 2004) and HCN1 and HCN2 have been shown specifically to be expressed in octopus cells (Koch et al., 2004). Homomeric HCN1 channels have the most rapid kinetics, HCN2 channels are slower, and HCN4 are the slowest; heteromeric channels have intermediate properties (Santoro et al., 1998, Moosmang et al., 1999, Ulens and Tytgat, 2001, Altomare et al., 2003, Whitaker et al., 2007). ZD7288 blocks gh in many neurons, including in octopus cells (Harris and Constanti, 1995, Khakh and Henderson, 1998, Luthi and McCormick, 1998, Maccaferri and McBain, 1996, Bal and Oertel, 2000). In spiral ganglion cells gh is formed from HCN1, HCN2, and HCN4 subunits (Kim and Holt, 2013, Liu et al., 2014). Many other auditory neurons also express gh (Banks et al., 1993, Brew and Forsythe, 1995, Rusznak et al., 1996, Wu, 1999, Cuttle et al., 2001, Dodson et al., 2002, Mo et al., 2002, Svirskis et al., 2004, Cao et al., 2007, Khurana et al., 2011). The HCN channels that mediate Ih are permeable to both Na+ and K+ and have a reversal potential of −40 mV so that Ih is inward at the resting potential in octopus and many other types of neurons (Banks et al., 1993, Bal and Oertel, 2000).

HCN1 and Kv1.1 are colocalized in the somatic and dendritic octopus cell membrane (Oertel et al., 2008, Rusznak et al., 2008, Robbins and Tempel, 2012). In an earlier study we examined mice in which HCN1 was eliminated to alter gh (Cao and Oertel, 2011). We found that in octopus cells of HCN1 null mutants gKL is concomitantly reduced with gh leading us to conclude that gh governs gKL (Cao and Oertel, 2011). Here we test the generality of that conclusion by addressing the complementary question, whether removing Kv1.1, a subunit that is present in many gKL channels, alters IKL or Ih or both. The present results support the earlier conclusion that gh governs the expression of gKL because we find that gKL in Kv1.1 null mutants at the steady state is unchanged, that the loss of Kv1.1 is compensated by other subunits.

The large size of gh and gKL in octopus cells allows us to resolve and compare the expression of these conductances at the resting potential in different strains of mice. We found that at the resting potential, the magnitude of Ih equals the magnitude of the sum of an α-DTX-sensitive and an α-DTX-insensitive, TEA-sensitive IKL in each of five strains of mutant and wild type mice even when resting potentials varied between cells over nearly 10 mV and gh varied over a five-fold range. It has been suggested by many investigators that gh and gKL set the resting potential. That is true in the short term but the present experiments show that in the long term it is the resting potential that regulates the gKL so that IKL balances Ih at rest.

Section snippets

Materials and methods

This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the School of Medicine and Public Health at the University of Wisconsin-Madison (M005303).

Influence of gh and gKL on octopus cells

Octopus cells were identified by their electrophysiological characteristics in recordings in current clamp: their low input resistance, the small size of somatic action potentials, and by strong rectification in both the depolarizing and hyperpolarizing directions (Golding et al., 1995, Bal and Oertel, 2000) (Fig. 1A, left panel).

One way to demonstrate the presence of gKL is to test the cell's sensitivity to its blockers (Bal and Oertel, 2001). α-DTX, at 50 nM, blocks K+ channels that have

Discussion

In auditory neurons, the importance of interactions between gh and gKL for signaling has long been appreciated (Rothman and Manis, 2003b, Golding and Oertel, 2012). The relatively sluggish gh helps give auditory neurons a low input resistance that makes voltage changes rapid while the speedy gKL repolarizes not only suprathreshold but also subthreshold synaptic potentials (Oertel, 1983, Manis and Marx, 1991, Brew and Forsythe, 1995, Golding et al., 1995, Rathouz and Trussell, 1998, Dodson

Acknowledgements

We are particularly grateful to Bruce Tempel who generously supplied us with the Kv1.1 mutant mice. We are also grateful for the colleagues who have made this work possible, especially Rebecca Welch and her effective office staff and Ravi Kochhar who kept our computers humming. We also thank Lin Lin for interesting discussion and for her thoughtful comments on multiple version of the manuscript. Steven Wiesner generously commented on one of the early versions of the manuscript. This work was

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