Site-directed mutagenesis in the ATM region alters axon–dendrite targeting of Kv3.1 channels. Two point mutations (Kv3.1aHAIPR and Kv3.1aHAPP) in Kv3.1aHA and two (Kv3.1bHAKKK and Kv3.1bHAKKH) in Kv3.1bHA were constructed by Quickchange strategy. Hippocampal neurons were transfected at 5 DIV, fixed, and stained 2 d later. HA staining was performed under both nonpermeabilized (non-perm; left column) and permeabilized (perm; right column) conditions. The levels of Kv3.1aHAPP (B), but not of Kv3.1aHAIPR (A), in axons significantly increased compared with Kv3.1aHA. Compared with Kv3.1bHA (C), axonal targeting of both Kv3.1bHAKKK (D) and Kv3.1bHAKKH dramatically decreased. E, Summary of polarized targeting of Kv3.1 constructs. Kv3.1aHA: non-perm Faxon/Fdendrite, 0.34 ± 0.03 (n = 45); perm Faxon/Fdendrite, 0.12 ± 0.02 (n = 32). Kv3.1aHAIPR: non-perm Faxon/Fdendrite, 0.18 ± 0.02 (n = 28); perm Faxon/Fdendrite, 0.09 ± 0.01 (n = 32). Kv3.1aHAPP: non-perm Faxon/Fdendrite, 0.72 ± 0.05 (n = 31); perm Faxon/Fdendrite, 0.37 ± 0.05 (n = 30). Kv3.1bHA: non-perm Faxon/Fdendrite, 1.85 ± 0.09 (n = 43); perm Faxon/Fdendrite, 0.45 ± 0.03 (n = 25). Kv3.1bHAKKK: non-perm Faxon/Fdendrite, 0.29 ± 0.04 (n = 20); perm Faxon/Fdendrite, 0.11 ± 0.02 (n = 31). Kv3.1bHAKKH: non-perm Faxon/Fdendrite, 0.30 ± 0.04 (n = 21); perm Faxon/Fdendrite, 0.09 ± 0.01 (n = 24). Scale bars, 100 μm. Kv3.1aHA and Kv3.1bHA are the two controls for their mutants. One-way ANOVA followed by Dunnett's test. *p < 0.001; **p < 0.0001.