A fundamental factor in controlling membrane excitability is the precise and dynamic expression of voltage-gated ion channels at the cell surface. Voltage-gated potassium channels act as potent modulators of neuronal excitatory events and effect the shape, size and duration of action potentials(1,2,3). These electrophysiological properties conferred on the cell are affected by both channel surface density and exact subunit composition of Kv1 channels. Previous studies have shown that regulation of export of Kv1 channels from the ER to the plasma membrane is highly complex and may serve as a means to regulate both channel density and the deliverance of physiologically appropriate channel complexes. We investigated the role of a single amino acid in the S1-S2 linker in the trafficking of Kv1.4 using immunofluorescence microscopy, patch-clamp and biochemical techniques. We demonstrated that a single threonine residue in the S1-S2 linker is necessary for the correct trafficking of Kv1.4 to the cell surface. Mutation of this residue to alanine or to a phosphomimetic aspartic acid prevented cell surface expression by retaining channels in the ER, a phenomenon rescued by mutating to a serine residue. Of note is that this is a highly conserved residue within all VGK families and mutations of this residue in both Kv2.1 and Kv3.1 revealed similar results. These results demonstrate a critical role for a threonine residue in the biogenesis and/or trafficking of voltage-gated potassium channels to the cell surface.