A C-terminal domain directs Kv3.3 channels to dendrites

Abstract

Pyramidal neurons of the electrosensory lateral line lobe (ELL) of Apteronotus leptorhynchus express Kv3-type voltage-gated potassium channels that give rise to high-threshold currents at the somatic and dendritic levels. Two members of the Kv3 channel family, AptKv3.1 and AptKv3.3, are coexpressed in these neurons. AptKv3.3 channels are expressed at uniformly high levels in each of four ELL segments, whereas AptKv3.1 channels appear to be expressed in a graded manner with higher levels of expression in segments that process high-frequency electrosensory signals. Immunohistochemical and recombinant channel expression studies show a differential distribution of these two channels in the dendrites of ELL pyramidal neurons. AptKv3.1 is concentrated in somas and proximal dendrites, whereas AptKv3.3 is distributed throughout the full extent of the large dendritic tree. Recombinant channel expression of AptKv3 channels through in vivo viral injections allowed directed retargeting of AptKv3 subtypes over the somadendritic axis, revealing that the sequence responsible for targeting channels to distal dendrites lies within the C-terminal domain of the AptKv3.3 protein. The targeting domain includes a consensus sequence predicted to bind to a PDZ (postsynaptic density-95/Discs large/zona occludens-1)-type protein-protein interaction motif. These findings reveal that different functional roles for Kv3 potassium channels at the somatic and dendritic level of a sensory neuron are attained through specific targeting that selectively distributes Kv3.3 channels to the dendritic compartment.

Figure 1.

Characterization of the AptKv3.1 K⁺ channel. A, Alignment of the amino acid sequences for AptKv3.1 with the rat Kv3.1b channel. The six predicted transmembrane segments (S1–S6) and the predicted ion pore (P) are indicated by solid lines above the sequence. Amino acid identities are outlined in black. B, Phylogenetic comparison of AptKv3.1 to members of the mammalian Kv3 family. The sequence used for comparison is rat Kv3, except for human Kv3.4b. Analysis by the parsimony method was performed using the PROTPARS program in the Phylogeny Inference Package (Golding and Felsenstein, 1990). The Drosophila Shaw K⁺ channel was used as the outgroup. C, Outside-out voltage-clamp recordings of AptKv3.1 current expressed in CHO cells demonstrate an outwardly rectifying and high-threshold potassium channel. The command pulses were stepped in 10 mV increments (100 ms) from a holding potential of –70 to 50 mV and stepped back to –40 mV.

Figure 2.

AptKv3.1 and AptKv3.3 are coexpressed in the pyramidal neurons of the ELL. A, Transverse section of the Apteronotus hindbrain stained with cresyl violet. The two prominent cell layers in the ELL, pyramidal cell layer (PCL), and granule cell layer (GCL), are indicated. B, In situ hybridization analysis of AptKv3 mRNAs in the hindbrain neurons. Dashed lines indicate the boundaries between different segments of the ELL. AptKv3.1 mRNA is expressed most strongly in granule neurons of the cerebellum (CCb and EGp) and in the pyramidal neurons of the ELL. Granule neurons of the ELL exhibit low levels of signal. In the pyramidal neurons, the intensity of the signal varies among the segments such that LS/MS > CLS > CMS. AptKv3.3 mRNA is at high levels in pyramidal and granule neurons of all segments of the ELL. Expression of AptKv3.3 is present in only a subset of neurons in the CCb and EGp and only at very light levels in granule neurons of the cerebellum. C, AptKv3.1 channel protein appears to be expressed in a gradient, with LS/MS > CLS > CMS. ELL sections were stained withα-AptKv3.1 antibody and visualized using HRP histochemistry. Antibody labeling is restricted to the somatic and proximal dendrites. Scale bar: B, 300 μm; C, 25 μm.

Figure 3.

AptKv3.3, but not Apt Kv3.1, is localized to the distal apical dendrites of pyramidal neurons in the ELL. A, B, ELL pyramidal cell layer and overlying molecular layer reacted with antibody to AptKv3.1 (A) and AptKv3.3 (B). The immunolabel for AptKv3.3 but not AptKv3. 1 in the dendrites of the VML and DML is shown. Strong signals for both are observed in pyramidal cell somata. PCL, Pyramidal cell layer; DML, dorsal molecular layer; VML, ventral molecular layer. C–F, All images are of the dorsal molecular layer ∼300–500μm above the pyramidal cell layer. Sections stained with antibodies to AptKv3.1, AptKv3.3, MAP2, and SV2 are shown. Apical dendrites as identified by MAP2 (red) are strongly labeled for AptKv3.3 (green) in C but are not labeled for AptKv3.1 (green) in D. In E, AptKv3.3 (green) staining in the dendritic shaftis outlined by the immunolabel for the presynaptic vesicle marker SV2(red). In F, the immunolabel for Apt Kv3. 1 shows partial overlap with the SV2 label (red). Scale bars: (in B) A, B, 100μm; (in Fiii) C–F, 25μm.

Figure 4.

Schematic representation of the various GFP-AptKv3 channel proteins, including the recombinant chimeric or deletion proteins. Amino acid positions are indicated above each construct. For segment switches, CT and NT indicate C-terminal (C-term) and N-terminal (N-term) segments. ΔCT indicates the truncation of the C-terminal region. ΔSIL indicates the removal of the SIL sequence from the C terminus. The amino acid positions of EGFP fusion to AptKv3.3 are indicated by (+1) and (+19). The presence (+) or absence (–) of GFP fluorescence 300–500 μm above the pyramidal cell layer is indicated on the right.

Figure 5.

The C-terminal segment of AptKv3.3 is required for channel localization to distal dendrites of pyramidal neurons. GFP fluorescence was analyzed in 80 μm sections of ELL from fish injected with recombinant SFV viral particles in the dorsal molecular layer of ELL under stereotaxic control. Apical dendrites extend upward to the most dorsal edge of the ELL, shown as a dashed line. A, Viral expression of EGFP-F exclusively labels pyramidal neurons. The fluorescent signal extends dorsally to the full extent of the apical dendrites (black arrows) and ventrally to the basilar dendrite (white arrow). B, GFP-AptKv3.3(+19) channels localize throughout the full extent of the apical (black arrows) and the basilar (white arrow) dendrites. C, GFP-AptKv3.1 channels are restricted to the somas and proximal segments of the apical dendrites. D, Replacement of the C-terminal segment of GFP-AptKv3.3 with the corresponding segment from AptKv3.1 inhibits distal dendritic localization. E, Replacement of the C-terminal segment of GFP-Kv3.1 with the corresponding segment for AptKv3.3 promotes localization to the apical (black arrows) and basilar (white arrow) dendrites. F, Deletion of the C-terminal tripeptide from GFP-AptKv3.3 inhibits localization to distal apical dendrites.