A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca2+ regulation of Kv4 channels

Abstract

Edited by Roger J. ColbranThe Kv4 family of A-type voltage-gated K ؉ channels regulates the excitability in hippocampal pyramidal neuron dendrites and are key determinants of dendritic integration, spike timing-dependent plasticity, long-term potentiation, and learning. Kv4.2 channel expression is down-regulated following hippocampal seizures and in epilepsy, suggesting A-type currents as therapeutic targets. In addition to pore-forming Kv4 subunits, modulatory auxiliary subunits called K ؉ channel-interacting proteins (KChIPs) modulate Kv4 expression and activity and are required to recapitulate native hippocampal A-type currents in heterologous expression systems. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. As members of the EF-hand domain-containing neuronal Ca 2؉ sensor protein family, KChIP auxiliary proteins may convey Ca 2؉ sensitivity upon Kv4 channels; however, to what degree intracellular Ca 2؉ regulates KChIP-Kv4.2 complexes is unclear. To answer this question, we expressed KChIP2 with Kv4.2 in HEK293T cells, and, with whole-cell patch-clamp electrophysiology, measured an ϳ1.5-fold increase in Kv4.2 current density in the presence of elevated intracellular Ca 2؉ . Intriguingly, the Ca 2؉ regulation of Kv4 current was specific to KChIP2b and KChIP2c splice isoforms that lack a putative polybasic domain that is present in longer KChIP2a1 and KChIP2a isoforms. Site-directed acidification of the basic residues within the polybasic motif of KChIP2a1 rescued Ca 2؉ -mediated regulation of Kv4 current density. These results support divergent Ca 2؉ regulation of Kv4 channels mediated by alternative splicing of KChIP2 isoforms. They suggest that distinct KChIP-Kv4 interactions may differentially control excitability and function of hippocampal dendrites.

Modulation of Kv4.2/KChIP3 interaction by the ceroid lipofuscinosis neuronal 3 protein CLN3

“…Because all current parameters were measured within a time range between tens of seconds and a few minutes after establishing the whole-cell configuration, the observed Ca 21 effects can be classified as acute. Acute Ca 21 effects on the surface expression of Kv4.2/KChIP complexes in the whole-cell patch-clamp configuration have been studied previously by Murphy and Hoffman (37), who found increased Kv4.2 current densities with elevated Ca 21 in the pipette solution for certain KChIP isoforms including the KChIP3a splice variant used in the present study. The authors suggested a post-ER/Golgi pool of Kv4/KChIP channel complexes recruited to the plasma membrane in a Ca 21 -dependent manner.…”

R-type voltage-gated Ca2+channels mediate A-type K+current regulation of synaptic input in hippocampal dendrites

“…We and others have reported that Kv4.x current density is increased when intracellular free Ca 2+ is increased to the μM range (42, 71). Nanoscale binding of Cav2.3 and Kv4.2 suggests that Cav2.3 could act as a local μM-source of free Ca 2+ .…”

“…Variant KChIPs have been categorized based on the tendency for the N-terminus to mediate membrane interactions (33). A subset of “cytoplasmic” KChIP isoforms confer Ca 2+ regulation onto Kv4.2 channel complexes, N- myristoylated isoforms do not, and reversibly palmitoylated isoforms have mixed Ca 2+ sensitivity (42). Members of a fourth transmembrane class of KChIPs retain Kv4.2 complexes within the ER and reduce Kv4.2 surface expression when compared with other KChIP isoforms (108).…”

“…Interestingly, Ca 2+ binding to purified KChIP induces global structural changes throughout the protein that may regulate oligomerization and Kv4 interactions (35)(36)(37)(38)(39). The effects of Ca 2+ on the Kv4-KChIP complex has been studied using EF-hand mutations or by altering intracellular free Ca 2+ concentrations (27,31,(40)(41)(42). However, while EF-hand mutations potently reduce KChIP regulation of Kv4 channel trafficking and function, they are likely to disrupt the tertiary structure of the protein independent of Ca 2+ (43).…”

“…Furthermore, patch clamp studies of Kv4.x channel function in various neuronal and non- neuronal cell types by addition of intracellular free Ca 2+ or Ca 2+ chelators in the patch pipette have produced variable results that are difficult to interpret (41, 44–46). In a similar effort to test the role of elevated intracellular Ca 2+ on Kv4.x-KChIP function, we recently reported an increase in Kv4.2 current density using a low affinity Ca 2+ chelator (HEDTA) in the patch pipette to hold intracellular free Ca 2+ at ∼10 μM in HEK293 cells (42). This effect was unique to a subset of KChIP isoforms suggesting that cell-type specific KChIP expression may be an under- appreciated aspect of Ca 2+ regulation of Kv4.x-KChIP complexes.…”