Measuring calibration factors by imaging a dish of cells expressing different tandem constructs plasmids

Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors

The Journal of General Physiology ◽

10.1085/jgp.200409042 ◽

2004 ◽

Vol 123 (6) ◽

pp. 697-708 ◽

Cited By ~ 28

Author(s):

Paul J. Groot-Kormelink ◽

Steven D. Broadbent ◽

James P. Boorman ◽

Lucia G. Sivilotti

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Nicotinic Receptors ◽

Functional Characterization ◽

Functional Expression ◽

Neuronal Nicotinic Acetylcholine Receptors ◽

Neuronal Nicotinic Receptors ◽

Channel Mutation ◽

Tandem Constructs

Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking α (α2–α4, α6) and β (β2, β4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (β4_α2, β4_α3, β4_α4, and β2_α4) when coexpressed with the corresponding β monomer subunit. All tandems expressed when injected alone, except for β4_α3, which produced functional channels only together with β4 monomer and was chosen for further characterization. These channels produced from β4_α3 tandem constructs plus β4 monomer were identical with receptors expressed from monomer α3 and β4 constructs in acetylcholine sensitivity and in the number of α and β subunits incorporated in the channel gate. However, separately mutating the β subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of β subunits from the tandem and one from the β monomer construct, whereas the rest incorporated two or three β monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily.

Download Full-text

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

The Journal of General Physiology ◽

10.1085/jgp.200709894 ◽

2007 ◽

Vol 131 (1) ◽

pp. 43-58 ◽

Cited By ~ 18

Author(s):

Kim W. Chan ◽

Adam Wheeler ◽

László Csanády

Keyword(s):

Katp Channels ◽

Sulfonylurea Receptor ◽

Pharmacological Characterization ◽

Mixing Ratios ◽

Metabolic Stresses ◽

Inwardly Rectifying ◽

Global Fitting ◽

Tandem Constructs ◽

Heteromeric Channels

ATP-sensitive potassium (KATP) channels play important roles in regulating insulin secretion, controlling vascular tone, and protecting cells against metabolic stresses. KATP channels are heterooctamers of four pore-forming inwardly rectifying (Kir6.2) subunits and four sulfonylurea receptor (SUR) subunits. KATP channels containing SUR1 (e.g. pancreatic) and SUR2A (e.g. cardiac) display distinct metabolic sensitivities and pharmacological profiles. The reported expression of both SUR1 and SUR2 together with Kir6.2 in some cells raises the possibility that heteromeric channels containing both SUR subtypes might exist. To test whether SUR1 can coassemble with SUR2A to form functional KATP channels, we made tandem constructs by fusing SUR to either a wild-type (WT) or a mutant N160D Kir6.2 subunit. The latter mutation greatly increases the sensitivity of KATP channels to block by intracellular spermine. We expressed, individually and in combinations, tandem constructs SUR1-Kir6.2 (S1-WT), SUR1-Kir6.2[N160D] (S1-ND), and SUR2A-Kir6.2[N160D] (S2-ND) in Xenopus oocytes, and studied the voltage dependence of spermine block in inside-out macropatches over a range of spermine concentrations and RNA mixing ratios. Each tandem construct expressed alone supported macroscopic K+ currents with pharmacological properties indistinguishable from those of the respective native channel types. Spermine sensitivity was low for S1-WT but high for S1-ND and S2-ND. Coexpression of S1-WT and S1-ND generated current components with intermediate spermine sensitivities indicating the presence of channel populations containing both types of Kir subunits at all possible stoichiometries. The relative abundances of these populations, determined by global fitting over a range of conditions, followed binomial statistics, suggesting that WT and N160D Kir6.2 subunits coassemble indiscriminately. Coexpression of S1-WT with S2-ND also yielded current components with intermediate spermine sensitivities, suggesting that SUR1 and SUR2A randomly coassemble into functional KATP channels. Further pharmacological characterization confirmed coassembly of not only S1-WT and S2-ND, but also of coexpressed free SUR1, SUR2A, and Kir6.2 into functional heteromeric channels.

Download Full-text