EVIDENCE FOR INTERACTION BETWEEN TRANSMEMBRANE SEGMENTS IN ASSEMBLY OF KV1.3

Previously, we showed that the N-terminal recognition domain (T1) of K v1.3 was not required for assembly of functional channels [Tu et al. ( 1996) J. Biol. Chem. 271, 18904-18911]. Moreover, specific Kv1.3 pepti de fragments including regions of the central core are able to inhibit expression of current produced from a channel lacking the T1 domain, Kv1.3(T1(-)). To elucidate the mechanism whereby Kv1.3 peptide fragmen ts suppress Kv1.3(T1(-)) current, we have studied the ability of pepti de fragments containing the transmembrane segments S1, S1-S2, or S1-S2 -S3 to physically associate with the Kv1.3(T1(-)) polypeptide subunit in vitro in microsomal membranes. Using c-myc (9E10) epitope-labeled p eptide fragments and anti-myc antibody as well as antisera to the Kv1. 3 C-terminus, we now demonstrate specific association of these peptide fragments with Kv1.3(T1(-)). Association of peptide fragments with Kv 1.3(T1(-)) was correlated with integration of both proteins into the m embrane. Furthermore, the relative strength and kinetics of this assoc iation directly correlated with the ability of fragments to suppress K v1.3(T1(-)) current. The rate-limiting step in the sequential synthesi s, integration, and formation of a complex was the association of inte grated polypeptides within the plane of the lipid bilayer. These resul ts strongly suggest that the physical association of transmembrane seg ments provides the basis for suppression of K+ channel function by Kchannel peptide fragments in vivo. Moreover, the S1-S2-S3 peptide frag ment potently suppressed full-length Kv1.3, thus implicating a role fo r the S1-S2-S3 region of Kv1.3 in the assembly of the Kv1.3 channel, W e refer to these putative association sites as IMA (intramembrane asso ciation) sites.