The role of positively charged arginines in the fourth transmembrane domain (S4) and a single negatively charged amino acid in the third transmembrane domain (S3) on channel biogenesis and gating of voltage-gated K⁺ channels (Kv) has been well established. Both intermediate (KCa3.1) and small (KCa2.x) conductance, Ca²⁺-activated K⁺ channels have two conserved arginines in S4 and a single conserved glutamic acid in S3, although these channels are voltage-independent. We demonstrate that mutation of any of these charged amino acids in KCa3.1 or KCa2.3 to alanine, glutamine or charge reversal mutations results in a rapid degradation (<30 min) of total protein; confirming the critical role of these amino acids in channel biogenesis. Mutation of the S4 arginine closest to the cytosolic side of KCa3.1 to histidine resulted in expression at the cell surface. Excised patch-clamp experiments revealed that this R/H mutation had a dramatically reduced open probability (P_o), relative to wild type channels. Additionally, we demonstrate, using a combination of shRNA, dominant negative and co-IP studies, that both KCa3.1 and KCa2.3 are translocated out of the ER associated with Derlin-1. These misfolded channels are poly-ubiquitylated, recognized by p97 and targeted for proteosomal degradation. Our results suggest that S3 and S4 charged amino acids play an evolutionarily conserved role in the biogenesis and gating of KCa channels. Further, these improperly folded K⁺ channels are translocated out of the ER in a Derlin-1- and p97-dependent fashion, poly-ubiquitylated and targeted for proteosomal degradation.