Functional effects of spinocerebellar ataxia type 13 mutations are conserved in zebrafish Kv3.3 channels

Abstract

BackgroundThe zebrafish has been suggested as a model system for studying human diseases that affect nervous system function and motor output. However, few of the ion channels that control neuronal activity in zebrafish have been characterized. Here, we have identified zebrafish orthologs of voltage-dependent Kv3 (KCNC) K+ channels. Kv3 channels have specialized gating properties that facilitate high-frequency, repetitive firing in fast-spiking neurons. Mutations in human Kv3.3 cause spinocerebellar ataxia type 13 (SCA13), an autosomal dominant genetic disease that exists in distinct neurodevelopmental and neurodegenerative forms. To assess the potential usefulness of the zebrafish as a model system for SCA13, we have characterized the functional properties of zebrafish Kv3.3 channels with and without mutations analogous to those that cause SCA13.ResultsThe zebrafish genome (release Zv8) contains six Kv3 family members including two Kv3.1 genes (kcnc1a and kcnc1b), one Kv3.2 gene (kcnc2), two Kv3.3 genes (kcnc3a and kcnc3b), and one Kv3.4 gene (kcnc4). Both Kv3.3 genes are expressed during early development. Zebrafish Kv3.3 channels exhibit strong functional and structural homology with mammalian Kv3.3 channels. Zebrafish Kv3.3 activates over a depolarized voltage range and deactivates rapidly. An amino-terminal extension mediates fast, N-type inactivation. The kcnc3a gene is alternatively spliced, generating variant carboxyl-terminal sequences. The R335H mutation in the S4 transmembrane segment, analogous to the SCA13 mutation R420H, eliminates functional expression. When co-expressed with wild type, R335H subunits suppress Kv3.3 activity by a dominant negative mechanism. The F363L mutation in the S5 transmembrane segment, analogous to the SCA13 mutation F448L, alters channel gating. F363L shifts the voltage range for activation in the hyperpolarized direction and dramatically slows deactivation.ConclusionsThe functional properties of zebrafish Kv3.3 channels are consistent with a role in facilitating fast, repetitive firing of action potentials in neurons. The functional effects of SCA13 mutations are well conserved between human and zebrafish Kv3.3 channels. The high degree of homology between human and zebrafish Kv3.3 channels suggests that the zebrafish will be a useful model system for studying pathogenic mechanisms in SCA13.

KCNC3R420H, a K+ channel mutation causative in spinocerebellar ataxia 13 displays aberrant intracellular trafficking

“…Analyses of the electrophysiological profile for KCNC3 R420H in a Xenopus oocyte heterologous system was consistent with a non-functional channel, whereas the KCNC3 F448L mutation altered the gating of KCNC3 thus prolonging the open state (Waters et al, 2006) with these biophysical profiles for these mutants duplicated in the zebra fish KCNC3 genes (Mock et al, 2010). Autosomal dominant inheritance in the Filipino family is characterized by cerebellar signs including gait and limb ataxia, brisk tendon reflexes and mild cognitive impairment (Subramony et al, 2013), as well as abnormal sound localization cueing (Middlebrooks et al, 2013).…”

“…Studies in Xenopus oocytes and zebrafish (Issa et al, 2011; Minassian et al, 2012; Mock et al, 2010; Zhao et al, 2013) have implicated dominant negative effects of KCNC3 R420H based on electrophysiological studies demonstrating the lack of channel activity in heterologous systems. However, evidence for the underlying molecular basis of disease is lacking.…”

“…The most pronounced gross pathological feature in both the Filipino and French kindred was severe, isolated cerebellar atrophy revealed by magnetic resonance (MR) imaging (Subramony et al, 2013; Waters et al, 2006). This pathological hallmark in SCA13, as well as other SCAs is in sharp contrast to the complete lack of alterations in brain anatomy and motor function in any single mouse gene ablation models (Espinosa et al, 2001; McMahon et al, 2004; Zagha et al, 2010) or studies in the zebrafish (Issa et al, 2011; Mock et al, 2010). …”

“…We therefore sought to understand the underlying mechanisms that might explain the dominant inheritance of the KCNC3 R420H allele in SCA13 patients, given that this channel mutation is electrophysiologically non-functional (Mock et al, 2010; Waters et al, 2006). Our studies reveal that KCNC3 R420H displays altered post-translational modifications and aberrant intracellular trafficking with associated changes in Golgi and cellular morphology and dominant phenotypes in the Drosophila wing and eye.…”

Spinocerebellar Ataxia Type 13 Mutant Potassium Channel Alters Neuronal Excitability and Causes Locomotor Deficits in Zebrafish

“…Anti-Kv3.3 antibodies were raised in rabbits by Covance Research Products (Denver, PA) against the peptide epitope CRIGADPDDPTASAHTAFK (Mock et al, 2010). The amino-terminal cysteine residue was added to couple the peptide to keyhole limpet hemocyanin.…”