An artificial tetramerization domain restores efficient assembly of functional Shaker channels lacking T1

摘要

One feature shared by all Shaker-type voItage-gated K+ channels is a highly conserved domain (T1) located in the cytoplasmic N terminus. The T1 domain is a key determinant of which subtypes can form heteromultimeric channels, suggesting that T1 functions during channel assembly. To better define the role of T1 during channel assembly and separate this function from potential con- tributions to channeI permeation and gating. we replaced the T1 domain (residues 96--183) of ShakerB with a coiIed-coil sequence (GCN4-Ll) that forms parallel tetramers. Deleting T1 dramatically. but not completely, abolished channel formation under most expression conditions. Channels lacking T1 are functional and K+-selective. although they activate at more hyperpolarized mem- brane potentials and, inactivate less completely. Insertion of the artificial tetramerization domain (GCN4-Ll) restored efficient chan- nel formation. suggesting that tetramerization of the cytoplasmic T1 domain promotes transmembrane channel assembly by increas- ing the effective local subunit concentration for T1 compatible subunits. We propose that T1 tetramerization promotes subfamily- specific assembly through kinetic partitioning of the assembly process. but is not required for subsequent steps in channel assembly and folding.