Our research program aims to understand how a voltage-gated potassium (Kv) channel is made. This is a complicated multi-step process that requires acquisition of local secondary, tertiary, and quaternary structures, either sequentially or as coupled events. How this happens is, for the most part, unknown. Yet the impact of these steps is profound, often with pathological consequences. We focus our attention on the human tetrameric Kv1.3 channel, particularly its early-stage folding, and the determinants that regulate these folding events in the exit tunnel of the ribosome and the ER membrane. Three Aims comprise this grant proposal. The ability of a protein to form helices is a fundamental prerequisite for protein folding and function in all proteomes. However, this process has not been defined in the confined and heterogeneous microenvironment of the ribosome exit tunnel where a protein is first made. Helicity must occur at the right time and place during translation. Failure to meet this requirement impairs peptide targeting, chaperone association, efficient bilayer insertion, and oligomerization. In Aim 1, we specifically ask when, where, how, and why these critical secondary structures arise in the Kv1.3 nascent peptide. We will determine the molecular mechanisms that delay or initiate helix formation and identify the underlying peptide-tunnel interactions that are responsible. To do this, we use biochemical approaches and cryo-EM single particle reconstruction of peptide-ribosome complexes. In Aim 2, we explore an exciting new field of fundamental importance to how proteins are made, namely, how a peptide's sequence generates piconewtons of force that fine tune Kv peptide's rate of elongation and folding efficiency. We use experimental approaches and molecular dynamics simulations to identify the type of peptide-tunnel interactions giving rise to force, the nature of the force, and its consequences as the peptide is elongated. Given that human Kv1.3 is expressed in neuronal and immune cells, and impaired expression produces chronic inflammatory disease and autoimmune disorders, it is compelling to ask whether human disease-linked variants of the KCNA3, the gene that encodes Kv1.3, introduce folding/assembly/trafficking defects. In Aim 3, we address this question using the recently developed “genome- first” approach to determine the clinical consequences of specific KCNA3 rare variants and biophysical determinations of Kv1.3 folding and function to identify the molecular defects. Our overall vision of Kv folding includes complex coupled events between intrapeptide segments, the ribosome exit tunnel, and the ER membrane. We now expand this view by introducing two new concepts for further investigation: 1) repressor/activator activity acts as a molecular switch to govern the time and tunnel location of Kv helix initiation, and 2) cotranslational force generation modulates translation rates and folding. Both concepts represent paradigm shifts th...