Role of Adam23 and Lgi proteins in functional organisation of Kv1 channels in myelinated axons

Abstract

Strategic organisation of ion channels with defined biophysical properties plays a key role in regulating excitability of neuronal membranes. In myelinated axons, ion channel distribution is governed by interactions between the axon and the associated glial cell. However, the molecular mechanisms that drive their specific localisation in the axonal membrane differ between each ion channel family. Given the enormous diversity in the repertoire of ion channels, many of these mechanisms remain poorly understood. Here, I try to elucidate this process focusing on a particular family of channels – Shaker-type voltage-gated potassium channels (Kv1). These are expressed predominantly in axons, in particular at the axon initial segment (AIS) and the juxtaparanode (JXP), a region of myelinated axons flanking the paranodes on each side of the node of Ranvier. Kv1 channels at the JXP are localised under the myelin sheath, from where they are thought to regulate the membrane potential, protecting the axon from hyper-excitability.

Mutations in genes encoding Kv1 subunits and autoimmunity against the Kv1 channels have been implicated in several neurological diseases characterised by disrupted neuronal signalling, including epilepsy, neuromyotonia, ataxia, and neuropathic pain. Moreover, Kv1 dissociation from the JXP is an early sign of axonal demyelination and often proceeds worsening of symptoms in demyelinating conditions. It is therefore essential that we understand the molecular processes that govern the assembly and maintenance of Kv1 at the JXP.

Previous research showed that the juxtaparanodal organisation of Kv1 channels is related to their association with cell adhesion molecules Caspr2 and Tag-1, as well as the adaptor protein 4.1B. However, later studies disproved the initial model of this molecular mechanism and suggested that additional proteins are involved in this process. It was subsequently identified that Kv1 channels associate with yet another protein, called ADAM23, which is also expressed at the JXP. Members of the ADAM (a disintegrin and metalloprotease) family are known to interact with LGI (leucine-rich gliomainactivated) proteins in a receptor-ligand fashion. Having identified the presence of Lgi2 and Lgi3 at the JXP, along with Adam23, I set out to find the role of these proteins and their potential interactions in functional organisation and maintenance of the Kv1 complex in myelinated axons.

To do so, I employed mouse models to knock out the proteins of interest and assess their importance in clustering and maintenance of Kv1 complexes, as well as their regeneration after axonal injury. I showed that the expression of Adam23 in the axonal membrane within the JXP domain is a pre-requisite for the initial organisation and maintenance of not only Kv1 channels but also all previously identified members of the JXP, including Caspr2. In the absence of Lgi2 and Lgi3, Kv1 channels also did not cluster at the JXP, leading us to conclude that the Adam23-Lgi2/3 interaction was indeed required for this process. Interestingly, the overall Kv1 levels in axons were not affected by the absence of Adam23, suggesting that Adam23 does not play a role in the transport of these complexes to the axon but potentially governs their clustering in the JXP membrane and their maintenance thereafter. Moreover, through electrophysiological experiments using the genetic mouse models, I was able to analyse the effect of faulty organisation of Kv1 channels on the axonal physiology. Having identified the role of Adam23 and the LGI proteins in the functional Kv1 organisation, I set out to examine the specific molecular interactions which might be taking place within the JXP. I carried out an invitro investigation using the novel BioID proximity labelling approach to identify proteins present in the JXP complex which Adam23 could associate with. Altogether, my data gathered from the genetic mouse models and the cell culture experiments greatly improved our understanding of the Kv1 complexes and introduced significant changes to the previously accepted model of their organisation at the JXP of myelinated axons.