Activation of conventional kinesin motors in clusters by shaw voltage-gated potassium channels

Abstract

SummaryThe conventional kinesin motor transports many different cargos to specific locations in neurons. How cargos regulate motor function remains unclear. Here we focus on KIF5, the heavy chain of conventional kinesin, and report that the Kv3 (Shaw) voltage-gated K + channel, the only known tetrameric KIF5-binding protein, clusters and activates KIF5 motors during axonal transport. Endogenous KIF5 often forms clusters along axons, suggesting a potential role of KIF5-binding proteins. Our biochemical assays reveal that the highaffinity multimeric binding between the Kv3.1 T1 domain and KIF5B requires three basic residues in the KIF5B tail. Kv3.1 T1 competes with the motor domain and microtubules, but not with kinesin light chain 1 (KLC1), for binding to the KIF5B tail. Live-cell imaging assays show that four KIF5-binding proteins, Kv3.1, KLC1 and two synaptic proteins SNAP25 and VAMP2, differ in how they regulate KIF5B distribution. Only Kv3.1 markedly increases the frequency and number of KIF5B-YFP anterograde puncta. Deletion of Kv3.1 channels reduces KIF5 clusters in mouse cerebellar neurons. Therefore, clustering and activation of KIF5 motors by Kv3 regulate the motor number in carrier vesicles containing the channel proteins, contributing not only to the specificity of Kv3 channel transport, but also to the cargo-mediated regulation of motor function.

Ankyrin-G Directly Binds to Kinesin-1 to Transport Voltage-Gated Na+ Channels into Axons

“…Consistent with the binding result that YFP-AR1 but not YFP-AR2 binds to the KIF5 tail domain (Figure 2C,D), CFP-AR1 but not CFP-AR2 markedly increased the anterograde transport frequency of KIF5B-YFP (Figure 6A,B). Binding of CFP-AR1 activated KIF5B-YFP likely by releasing autoinhibition of the motor protein, similar to other KIF5-binding proteins (Barry et al, 2013), but unlike Kv3.1, CFP-AR1 did not induce clusters of KIF5B-YFP. We further performed the two-color timelapse imaging.…”

“…For instance, the T63 region (aa 758–820) binds to KLC and the T70 region (aa 865–934) binds to Kv3.1 T1 domain, which we identified recently (Barry et al, 2013; Xu et al, 2010). Interestingly, the AnkG binding site in the KIF5 motor is the T70 region (Figure 2E), the exact same binding site for Kv3.1 T1 domain.…”

“…In fact, Kv3.1 channels may compete with AnkG for binding to KIF5 motors. The binding affinities of His-31T1 (K D : 6.0 ± 1.4 ×10 −8 M) and His-MB (K D : 2.4 ± 0.9 ×10 −9 M) to GST-Tail are comparable (Figure 2F) (Barry et al, 2013). It is important to note that although the binding affinities were measured using purified domains that can independently fold and function, these values might be different from those of full-length proteins.…”

“…Interestingly, GST-T70 (aa 865–934 in KIF5B tail, containing the Kv3.1 T1-binding site), but not GST-T63 (aa 758–820 in KIF5B tail, containing the KLC1-binding site), nor GST-T70 RKR (with the three basic residues (R 892 K 893 R 894 ) mutated to three Ds, no longer binding to Kv3.1 T1) (Barry et al, 2013), pulled down His-MB (Figure 2E). Thus, the AnkG MB domain binds to the Kv3.1 T1-binding site in KIF5B tail.…”

Physiological and Pathological Functions of Mechanosensitive Ion Channels

“…For instance, what are the channel parts that detect mechanical stimuli and are these channel parts conserved? Since many ion channels are linked to intracellular cytoskeletons via adaptor proteins and/or linked to the extracellular matrix via extracellular domains and sugar chains [52, 68–70], should their channel activities generally be influenced by mechanical forces? Existing evidence does suggest that cell membrane compression, expansion, bending and tension caused by mechanical forces applied to cell membrane intracellularly and/or extracellularly can lead to changes in the structural configuration of these ion channels, thereby modulating their opening and membrane conductance ([2] for review).…”