A mechanism for ATP-sensitive potassium channel diversity: Functional coassembly of two pore-forming subunits

Abstract

ATP-sensitive potassium channels are an octomeric complex of four pore-forming subunits of the Kir 6.0 family and four sulfonylurea receptors. The Kir 6.0 family consists of two known members, Kir 6.1 and Kir 6.2, with distinct functional properties. The tetrameric structure of the pore-forming domain leads to the possibility that mixed heteromultimers may form. In this study, we examine this by using biochemical and electrophysiological techniques after heterologous expression of these subunits in HEK293 cells. After the coexpression of Kir 6.1 and Kir 6.2, Kir 6.1 can be coimmunoprecipitated with isoform-specific Kir 6.2 antisera and vice versa. Coexpression of SUR2B and Kir 6.2 with Kir 6.1 dominant negatives at a 1:1 expression ratio and vice versa led to a potent suppression of current. Kir 6.1, and Kir 6.2 dominant negative mutants were without effect on an inwardly rectifying potassium channel from a different family, Kir 2.1. Single-channel analysis, after coexpression of SUR2B, Kir 6.1, and Kir 6.2, revealed the existence of five distinct populations with differing single-channel current amplitudes. All channel populations were inhibited by glibenclamide. A dimeric Kir 6.1-Kir 6.2 construct expressed with SUR2B had a single-channel conductance intermediate between that of either Kir 6.2 or Kir 6.1 expressed with SUR2B. In conclusion, Kir 6.1 and Kir 6.2 readily coassemble to produce functional channels, and such phenomena may contribute to the diversity of nucleotide-regulated potassium currents seen in native tissues.

Functional Expression of Inward Rectifier Potassium Channels in Cultured Human Pulmonary Smooth Muscle Cells: Evidence for a Major Role of Kir2.4 Subunits

“…HEK-293 cells were transfected with Kir2.1 and Kir2.4 subcloned into in pcDNA3 vectors (Invitrogen) using LipofectAMINE (Life Technologies, Inc) according to the manufacturer's instructions (Cui et al, 2001). Stable cell lines were established using the appropriate antibiotic selection (G418, for Kir2.1; Zeocin for Kir2.4) as detailed previously (Giblin et al, 1999).…”

Functional involvement of sulphonylurea receptor (SUR) type 1 and 2B in the activity of pig urethral ATP‐sensitive K+ channels

“…The channel conductance of this K ATP channel is intermediate between those of Kir6.1 and Kir6.2 (Babenko et al, 1998). In functional expression experiments, Cui et al (2001) have recently reported that heteromultimerization readily occurs between Kir6.1 and Kir6.2, producing functional glibenclamide-sensitive K ATP channels. Cui et al (2001) also suggest a possibility that K ATP channels in pig urethra may possess a heteromeric Kir6.1/ Kir6.2 structure.…”

“…In functional expression experiments, Cui et al (2001) have recently reported that heteromultimerization readily occurs between Kir6.1 and Kir6.2, producing functional glibenclamide-sensitive K ATP channels. Cui et al (2001) also suggest a possibility that K ATP channels in pig urethra may possess a heteromeric Kir6.1/ Kir6.2 structure. Further study is certainly necessary in order to elucidate the interaction of Kir subunits in pig urethral K ATP channels.…”

Sulfonylurea Receptors Type 1 and 2A Randomly Assemble to Form Heteromeric KATPChannels of Mixed Subunit Composition

“…Kir6.1 and Kir6.2, being 64% identical, display overlapping tissue expressions and form heteromeric complexes when coexpressed (Babenko et al, 2000;Cui et al, 2001;Pountney et al, 2001;van Bever et al, 2004). On the contrary, based on the study from Giblin et al, it is generally accepted that SUR1 cannot coassemble with SUR2 to form K ATP channels (Giblin et al, 2002;Babenko, 2005;Neagoe and Schwappach, 2005;Tricarico et al, 2006), even though SUR1 and SUR2 share ‫%56ف‬ identity and are coexpressed in many cell types (Baron et al, 1999;Liss et al, 1999;Shi et al, 2005;Tricarico et al, 2006).…”