Intracellular loop between transmembrane segments IV and V of cystic fibrosis transmembrane conductance regulator is involved in regulation of chloride channel conductance state

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) contains two membrane-spanning domains; each consists of six transmembrane segments joined by three extracellular and two intracellular loops of different length. To examine the role of intracellular loops in CFTR channel function, we studied a deletion mutant of CFTR (Δ19 CFTR) in which 19 amino acids were removed from the intracellular loop joining transmembrane segments IV and V. This mutant protein was expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (~170 kDa) was immunoprecipitated from cells transfected with wildtype CFTR cDNA, while cells transfected with the mutant gene expressed only a core-glycosylated form (~ 140 kDa). The chloride efflux rate (measured by 6-methoxyl-N-(3-sulfopropyl) quinolinium SPQ fluorescence) from cells expressing wild-type CFTR increased 600% in response to forskolin. In contrast, Δ19 CFTR-expressing cells had no significant response to forskolin. Western blotting performed on subcellular membrane fractions showed that A19 CFTR was located in the same fractions as ΔF508 CFTR, a processing mutant of CFTR. These results suggest that Δ19 CFTR is located in the intracellular membranes, without reaching the cell surface. Upon reconstitution into lipid bilayer membranes, Δ19 CFTR formed a functional Cl- channel with gating properties nearly identical to those of the wild-type CFTR channel. However, Δ19 CFTR channels exhibited frequent transitions to a 6-pleosiemens subconductance state, whereas wild-type CFTR channels rarely exist in this subconductance state. These data suggest that the intracellular loop is involved in stabilizing the full conductance state of the CFTR Cl- channel.