The repertoire of Kv1 potassium channels expressed in presynaptic terminals of mammalian central neurons is shaped by intrinsic trafficking signals that determine surface-expression efficiencies of homomeric and heteromeric Kv1 channel complexes. Here, we show that a determinant controlling surface expression of Kv1 channels is localized to the highly conserved pore region. Pointmutation analysis revealed two residues as critical for channel trafficking, one in the extracellular ''turret'' domain and one in the region distal to the selectivity filter. Interestingly, these same residues also form the binding sites for polypeptide neurotoxins. Our findings demonstrate a previously uncharacterized function for the channel-pore domain as a regulator of channel trafficking. Shaker or mammalian Kv1 ␣-and Kv-subunits can assemble promiscuously into functional homo-and heterotetrameric complexes, resulting in biophysically and pharmacologically distinct ␣ 4  4 channel complexes (1-4). However, biochemical and immunohistochemical studies have demonstrated that specific Kv1 heteromeric complexes predominate in mammalian brain, and many other possible subunit combinations are not detected (5-9). Particularly noteworthy is the absence of Kv1.1 homotetramers. These observations suggest not only a functional importance for particular heteromeric channel complexes, but that cellular mechanisms exist to restrict surface expression to only those channels with appropriate subunit composition.Mammalian Kv1 channels are assembled in the endoplasmic reticulum (ER) (10); however, the mechanisms that regulate ER export, cell-surface expression and targeting of Kv1 channels in neurons are unknown. The rate-limiting step for trafficking͞ expression of most membrane proteins is ER export (11), and export competence can be determined by diverse mechanisms, including folding, assembly, and specific ER retention͞export signals (12). We have found that mammalian Kv1 ␣-subunits possess distinct trafficking and surface-expression properties when expressed in mammalian cells including cultured hippocampal neurons (13). Therefore, we constructed a number of chimeric Kv1 ␣-subunits between efficiently trafficked Kv1.4 and inefficiently trafficked Kv1.1 and compared their trafficking and surface-expression properties to wild-type subunits. Our results demonstrate that a Kv1 channel-trafficking regulator is localized to the highly conserved pore region. Point-mutation analyses revealed a correlation between residues responsible for trafficking and binding to polypeptide neurotoxins. These data suggest a previously uncharacterized role for the Kv1 pore as a potential quality control mediator. MethodsAntibodies. Antibodies generated against the cytoplasmic and extracellular domains of potassium channel ␣-subunits have been described (10,(13)(14)(15)(16)(17). Anti-vimentin (monoclonal, clone no. 9) antibody was purchased from Sigma.Transient Transfection of COS-1 Cells. Cells were transfected with mammalian expression vectors for rat Kv1.1 (RBK1) an...
Voltage-gated Kv1 potassium channels consist of poreforming ␣ subunits and cytoplasmic Kv subunits. The latter play diverse roles in modulating the gating, stability, and trafficking of Kv1 channels. The crystallographic structure of the Kv2 subunit revealed surprising structural homology with aldo-keto reductases, including a triosephosphate isomerase barrel structure, conservation of key catalytic residues, and a bound NADP ؉ cofactor (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell 90, 943-952). Each Kv1-associated Kv subunit (Kv1.1, Kv1.2, Kv2, and Kv3) shares striking amino acid conservation in key catalytic and cofactor binding residues. Here, by a combination of structural modeling and biochemical and cell biological analyses of structure-based mutations, we investigate the potential role for putative Kv subunit enzymatic activity in the trafficking of Kv1 channels. We found that all Kv subunits promote cell surface expression of coexpressed Kv1.2 ␣ subunits in transfected COS-1 cells. Kv1.1 and Kv2 point mutants lacking a key catalytic tyrosine residue found in the active site of all aldo-keto reductases have wild-type trafficking characteristics. However, mutations in residues within the NADP ؉ binding pocket eliminated effects on Kv1.2 trafficking. In cultured hippocampal neurons, Kv subunit coexpression led to axonal targeting of Kv1.2, recapitulating the Kv1.2 localization observed in many brain neurons. Similar to the trafficking results in COS-1 cells, mutations within the cofactor binding pocket reduced axonal targeting of Kv1.2, whereas those in the catalytic tyrosine did not. Together, these data suggest that NADP ؉ binding and/or the integrity of the binding pocket structure, but not catalytic activity, of Kv subunits is required for intracellular trafficking of Kv1 channel complexes in mammalian cells and for axonal targeting in neurons.
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