Voltage-gated potassium (Kv) channels are heteromultimeric complexes consisting of pore- forming subunits and of accessory ß subunits. Several ß subunits, identified so far, interact with specific subunits to either modify their levels of expression, or some of their kinetic properties. The present study was aimed at isolating accessory proteins for KCNA10, a novel Kv channel a subunit, functionally related to both voltage-gated potassium and cyclic nucleotide gated cation channels. Since one of KCNA10 distinguishing features is a putative cyclic nucleotide binding domain located at the carboxy (C) terminus, the entire C terminal region was used to probe a human cardiac cDNA library using the yeast two-hybrid system. Interacting clones were then rescreened in a functional assay by co-injection with KCNA10 in Xenopus oocytes. One of these clones (KCNA4B), when injected alone in oocytes produced no detectable current. However, when co-injected with KCNA10 it increased KCNA10 current expression by nearly 3-fold. In addition, the current became more sentitive to activation by cAMP. KCNA4B can be co-immunoprecipitated with both the C terminus and full-length KCNA10. It encodes a soluble protein (141 amino acids) with no amino acid homology to known ß subunits, but with limited structural similarity to the NAD(P)H-dependent oxireductase superfamily. KCNA4B is located on Chromosome 13, spans approximately 16 Kb, and its coding region is made up of five exons. In conclusion, KCNA4B represents the first member of a new class of accessory proteins that modify the properties of Kv channels.