Evidence for Direct Physical Association between a K+ Channel (Kir6.2) and an ATP-Binding Cassette Protein (SUR1) Which Affects Cellular Distribution and Kinetic Behavior of an ATP-Sensitive K+ Channel

Potassium channels are the most diverse group of ion channels, with molecular cloning revealing a number of structurally distinct families, including the subfamily of inwardly rectifying K+ (Kir) channels (11, 27, 35). Channel diversity is increased by the ability of constitutive subunits to form not only homomeric but also heteromultimeric complexes with distinct functional and regulatory properties (8, 9, 15, 21, 27, 30, 39, 53). Present in most excitable tissues, ATP-sensitive K+ (KATP) channels belong to the Kir family and are involved in signaling networks that transduce cellular metabolic events into membrane potential changes (1, 9, 40). These channels are regulated by intracellular nucleotides and have been implicated in hormone secretion, cardioprotection, and neurotransmitter release, with their function best understood in the pancreatic β cell, where KATP channels are essential in glucose-mediated membrane depolarization and insulin secretion (7, 9, 14, 31, 34, 42, 44, 52). Structurally unique among K+ channels, KATP channel activity can be reconstituted by coexpressing two unrelated proteins: the Kir channel Kir6.2 and the ATP-binding cassette (ABC) protein SUR, specifically the SUR1 isoform for the pancreatic channel phenotype (2, 22, 38). Expression of Kir6.2 alone does not result in functional ion channels, suggesting an intimate and required interaction between Kir6.2 with SUR1 (1, 7, 40, 41). Actually, expression of Kir6.2-SUR1 fusion constructs indicates that a subunit stoichiometry of 1:1 is necessary for assembly of active KATP channels (10, 24). Furthermore, Kir6.2 and SUR1 genes are clustered on chromosome 11 (p15.1), separated by a short intergenic sequence of 4.3 kb, suggesting that these genes could be cotranscribed and cotranslated to form a functional heteromultimeric channel (1, 9, 22, 40). To date, evidence for physical association between Kir6.2 and SUR1 is based on photoaffinity labeling of both channel subunits by radioactive sulfonylurea (10). Labeling of Kir6.2 was dependent on coexpression of SUR1, suggesting close association between the two subunits (10). However, photoaffinity labeling is based primarily on proximity rather than physical interaction between proteins (18). Recent evidence indicates that K+ channels are tetramers of single subunits comprising the K+-selective pore (27). The measurement of KATP channel activity in cells expressing mutant carboxy-truncated Kir6.2 has been interpreted to mean that the presence of the carboxy terminus in Kir6.2 prevents functional expression of the channel in the absence of SUR (51). However, it is not known whether the distal carboxy terminus of Kir6.2 merely serves as a suppressor of channel activity or is also important in regulating physical interaction between Kir6.2 and SUR1. To determine whether Kir6.2 and SUR1 can physically associate with each other, and to investigate the role of the carboxy terminus of Kir6.2 in complex formation, we used a Kir6.2-specific antibody to coimmunoprecipitate and to immunostain channel subunits. We truncated the carboxy terminus of Kir6.2 polypeptide to yield functional channels in the absence of SUR1 (49, 51) and then used such mutants to measure single-channel properties when expressed alone or with SUR1. We demonstrate that Kir6.2 and SUR1 physically associate in functional complexes and that the carboxy terminus of Kir6.2 is not required for subunit association. Furthermore, we provide evidence that the intraburst behavior of KATP channels is defined by Kir6.2 alone, whereas burst channel behavior is modulated by association with SUR1.