MNK-eIF4E signalling is a highly conserved mechanism for sensory neuron axonal plasticity: evidence from Aplysia californica.

Injury to sensory neurons causes an increase in the excitability of these cells leading to enhanced action potential generation and a lowering of spike threshold. This type of sensory neuron plasticity occurs across vertebrate and invertebrate species and has been linked to the development of both acute and persistent pain. Injury-induced plasticity in sensory neurons relies on localized changes in gene expression that occur at the level of mRNA translation. Many different translation regulation signalling events have been defined and these signalling events are thought to selectively target subsets of mRNAs. Recent evidence from mice suggests that the key signalling event for nociceptor plasticity is mitogen-activated protein kinase-interacting kinase (MNK) -mediated phosphorylation of eukaryotic translation initiation factor (eIF) 4E. To test the degree to which this is conserved in other species, we used a previously described sensory neuron plasticity model in Aplysia californica. We find, using a variety of pharmacological tools, that MNK signalling is crucial for axonal hyperexcitability in sensory neurons from Aplysia. We propose that MNK-eIF4E signalling is a core, evolutionarily conserved, signalling module that controls nociceptor plasticity. This finding has important implications for the therapeutic potential of this target, and it provides interesting clues about the evolutionary origins of mechanisms important for pain-related plasticity. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

Functional stoichiometry underlying KChIP regulation of Kv4.2 functional expression.

K channel-interacting proteins (KChIPs) enhance functional expression of Kv4 channels by binding to an N-terminal regulatory region located in the first 40 amino acids of Kv4.2 that we call the functional expression regulating N-terminal (FERN) domain. Mutating two residues in the FERN domain to alanines, W8A and F11A, disrupts KChIP binding and regulation of Kv4.2 without eliminating the FERN domain's control of basal expression level or regulation by DPP6. When Kv4.2(W8A,F11A) is co-expressed with wild type Kv4.2 and KChIP3 subunits, a dominant negative effect is seen where the current expression is reduced to levels normally seen without KChIP addition. The dominant negative effect correlates with heteromultimeric channels remaining on intracellular membranes despite KChIP binding to non-mutant Kv4.2 subunits. In contrast, the deletion mutant Kv4.2(Δ1-40), eliminating both KChIP binding and the FERN domain, has no dominant negative effect even though the maximal conductance level is 5x lower than seen with KChIP3. The 5x increased expression seen with KChIP integration into the channel is fully apparent even when a reduced number of KChIP subunits are incorporated as long as all FERN domains are bound. Our results support the hypothesis that KChIPs enhances Kv4.2 functional expression by a 1 : 1 suppression of the N-terminal FERN domain and by producing additional positive regulatory effects on functional channel expression.

Long-lasting hyperexcitability induced by depolarization in the absence of detectable Ca2+ signals.

Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca(2+) signals produced by depolarization. Indeed, transient Ca(2+) signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca(2+)-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca(2+) transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca(2+) levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve-ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis-dependent hyperexcitability under conditions in which Ca(2+) entry is prevented (by bathing in nominally Ca(2+) -free solutions containing EGTA) and detectable Ca(2+) transients are eliminated (by adding BAPTA-AM). Disruption of Ca(2+) release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca(2+)-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.

Acceleration of K+ channel inactivation by MEK inhibitor U0126.

Voltage-dependent (Kv)4.2-encoded A-type K+ channels play an important role in controlling neuronal excitability and are subject to modulation by various protein kinases, including ERK. In studies of ERK modulation, the organic compound U0126 is often used to suppress the activity of MEK, which is a kinase immediately upstream from ERK. We have observed that the inactivation time constant of heterologously expressed Kv4.2 channels was accelerated by U0126 at 1-20 microM. This effect, however, was not Kv4 family specific, because U0126 also converted noninactivating K+ currents mediated by Kv1.1 subunits into transient ones. To determine whether U0126 exerted these effects through kinase inhibition, we tested U0125, a derivative of U0126 that is less potent in MEK inhibition. At the same concentrations, U0125 had effects similar to those of U0126 on channel inactivation. Finally, we expressed a mutant form of Kv4.2 in which three identified ERK phosphorylation sites (T602, T607, and S616) were replaced with alanines. The inactivation of K+ currents mediated by this mutant was still accelerated by U0126. Our data favor the conclusion that the increase in the rate of channel inactivation by U0126 is likely to be independent of protein kinase inhibition and instead represents a direct action on channel gating.

Multiprotein assembly of Kv4.2, KChIP3 and DPP10 produces ternary channel complexes with ISA-like properties.

Kv4 pore-forming subunits are the principal constituents of the voltage-gated K+ channel underlying somatodendritic subthreshold A-type currents (I(SA)) in neurones. Two structurally distinct types of Kv4 channel modulators, Kv channel-interacting proteins (KChIPs) and dipeptidyl-peptidase-like proteins (DPLs: DPP6 or DPPX, DPP10 or DPPY), enhance surface expression and modify functional properties. Since KChIP and DPL distributions overlap in the brain, we investigated the potential coassembly of Kv4.2, KChIP3 and DPL proteins, and the contribution of DPLs to ternary complex properties. Immunoprecipitation results show that KChIP3 and DPP10 associate simultaneously with Kv4.2 proteins in rat brain as well as heterologously expressing Xenopus oocytes, indicating Kv4.2 + KChIP3 + DPP10 multiprotein complexes. Consistent with ternary complex formation, coexpression of Kv4.2, KChIP3 and DPP10 in oocytes and CHO cells results in current waveforms distinct from the arithmetic sum of Kv4.2 + KChIP3 and Kv4.2 + DPP10 currents. Furthermore, the Kv4.2 + KChIP3 + DPP10 channels recover from inactivation very rapidly (tau(rec) approximately 18-26 ms), closely matching that of native I(SA) and significantly faster than the recovery of Kv4.2 + KChIP3 or Kv4.2 + DPP10 channels. For comparison, identical triple coexpression experiments were performed using DPP6 variants. While most results are similar, the Kv4.2 + KChIP3 + DPP6 channels exhibit inactivation that slows with increasing membrane potential, resulting in inactivation slower than that of Kv4.2 + KChIP3 + DPP10 channels at positive voltages. In conclusion, the native neuronal subthreshold A-type channel is probably a macromolecular complex formed from Kv4 and a combination of both KChIP and DPL proteins, with the precise composition of channel alpha and auxiliary subunits underlying tissue and regional variability in I(SA) properties.

KChIP3 rescues the functional expression of Shal channel tetramerization mutants.

KChIP proteins regulate Shal, Kv4.x, channel expression by binding to a conserved sequence at the N terminus of the subunit. The binding of KChIP facilitates a redistribution of Kv4 protein to the cell surface, producing a large increase in current along with significant changes in channel gating kinetics. Recently we have shown that mutants of Kv4.2 lacking the ability to bind an intersubunit Zn(2+) between their T1 domains fail to form functional channels because they are unable to assemble to tetramers and remain trapped in the endoplasmic reticulum. Here we find that KChIPs are capable of rescuing the function of Zn(2+) site mutants by driving the mutant subunits to assemble to tetramers. Thus, in addition to known trafficking effects, KChIPs play a direct role in subunit assembly by binding to monomeric subunits within the endoplasmic reticulum and promoting tetrameric channel assembly. Zn(2+)-less Kv4.2 channels expressed with KChIP3 demonstrate several distinct kinetic changes in channel gating, including a reduced time to peak and faster entry into the inactivated state as well as extending the time to recover from inactivation by 3-4 fold.

Structural insights into the functional interaction of KChIP1 with Shal-type K(+) channels.

Four Kv channel-interacting proteins (KChIP1 through KChIP4) interact directly with the N-terminal domain of three Shal-type voltage-gated potassium channels (Kv4.1, Kv4.2, and Kv4.3) to modulate cell surface expression and function of Kv4 channels. Here we report a 2.0 Angstrom crystal structure of the core domain of KChIP1 (KChIP1*) in complex with the N-terminal fragment of Kv4.2 (Kv4.2N30). The complex reveals a clam-shaped dimeric assembly. Four EF-hands from each KChIP1 form each shell of the clam. The N-terminal end of Kv4.2 forming an alpha helix (alpha1) and the C-terminal alpha helix (H10) of KChIP1 are enclosed nearly coaxially by these shells. As a result, the H10 of KChIP1 and alpha1 of Kv4.2 mediate interactions between these two molecules, structurally reminiscent of the interactions between calmodulin and its target peptides. Site-specific mutagenesis combined with functional characterization shows that those interactions mediated by alpha1 and H10 are essential to the modulation of Kv4.2 by KChIPs.

The role of Zn2+ in Shal voltage-gated potassium channel formation.

Voltage-gated potassium channels are formed by the tetramerization of their alpha subunits, in a process that is controlled by their conserved N-terminal T1 domains. The crystal structures of Shaker and Shaw T1 domains reveal interesting differences in structures that are contained within a highly conserved BTB/POZ domain fold. The most surprising difference is that the Shaw T1 domain contains an intersubunit Zn2+ ion that is lacking in the Shaker T1 domain. The Zn2+ coordination motif is conserved in other non-Shaker channels making this the most distinctive difference between these channels and Shaker. In this study we show that Zn2+ is an important co-factor for the tetramerization of isolated Shaw and Shal T1 domains. Addition of Zn2+ increases the amount of tetramer formed, whereas chelation of Zn2+ with phenanthroline blocks tetramerization and causes assembled tetramers to disassemble. Within an intact cell, full-length Shal subunits containing Zn2+ site mutations also fail to form functional channels, with the majority of the protein found to remain monomeric by size exclusion chromatography. Therefore, zinc-mediated tetramerization also is a physiologically important event for full-length functional channel formation.