Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating

Abstract

Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C-terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D which inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C-terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.

Neurite outgrowth is driven by actin polymerization even in the presence of actin polymerization inhibitors

“…Indeed, branched organization of these actin structures poorly correlates with the idea that they are nondepolymerizable actin aggregates because branched actin networks are typically highly dynamic and quickly disassemble in cells if their depolymerization is not balanced by polymerization. Of note, CytoD-resistant actin filaments were also observed by others at plasma membrane sites that are normally occupied by branched actin networks (Forscher and Smith, 1988; Zhang et al. , 2016).…”

Mechanisms underlying auditory processing deficits in Fragile X syndrome

“…A surprisingly high proportion of those that do bind FMRP are, however, expressed in neurons of the auditory system. Potassium channels that play major roles in regulating the intrinsic excitability of auditory brainstem neurons and whose mRNAs are targets of FMRP include the Kv3.1, Kv3.3, Kv1.2, Kv11.3, and K Na 1.1 channels . The sodium channel Nav1.6 and the calcium channels Cav2.1, Cav2.2, and Cav 2.3 are also expressed in MNTB and their mRNAs are targets of FMRP .…”

“…Potassium channels that play major roles in regulating the intrinsic excitability of auditory brainstem neurons and whose mRNAs are targets of FMRP include the Kv3.1, Kv3.3, Kv1.2, Kv11.3, and K Na 1.1 channels. [67][68][69][70][71][72][73][74][75] The sodium channel Nav1.6 76 and the calcium channels Cav2.1, Cav2.2, and Cav 2.3 77 are also expressed in MNTB and their mRNAs are targets of FMRP. 16 These links between FMRP and its channel targets that directly determine intrinsic excitability have allowed analyses of how loss of FMRP contributes to changes in firing patterns such as those of Figure 3.…”

Cell type-specific CLIP reveals that NOVA regulates cytoskeleton interactions in motoneurons

“…The increased utilization of E3a in MNs positively correlated with a dramatically enhanced NOVA binding site in MN 121 nt downstream of E3a (4-fold increase, FDR = 4.02 × 10 −7 ) (Figure 4D). Interestingly, inclusion of E3a would lead to a Kv3.3 isoform with an extended C-terminal proline-rich domain, which has been shown to modulate channel inactivation through triggering actin nucleation at the plasma membrane [43]. Taken together, these observations suggest that unique NOVA binding patterns around alternative exons in MN contributes to MN-specific biology, particularly in shaping the cytoskeleton and regulating cytoskeleton interactions in unique ways within spinal cord MNs.…”