Putative subunits of the rat mesangial KATP: a type 2B sulfonylurea receptor and an inwardly rectifying K+ channel.
BACKGROUND
Sulfonylurea agents exert their physiological effects in many cell types via binding to specific sulfonylurea receptors (SUR). SUR couple to inwardly-rectifying K+ channel (Kir6.x) to form tetradimeric ATP-sensitive K+ channels (KATP). The SUR subunits confer ATP-sensitivity on KATP and also provide the binding sites for sulfonylureas and other pharmacological agents. Our previous work demonstrated that the exposure of mesangial cells (MC) to sulfonylureas generated profound effects on MC glucose uptake and matrix metabolism and induced heightened cell contractility in association with Ca2+ transients. Because these responses likely resulted from the binding of sulfonylurea to a mesangial SUR2, we subsequently documented [3H]-glibenclamide binding to MC and the gene expression of several mesangial SUR2 transcripts. From these data, we inferred that MC expressed the components of a mesangial KATP and sought to establish their presence in primary MC.


METHODS
To obtain mesangial SUR2 cDNA sequences, rapid amplification of cDNA ends (RACE) was utilized. DNA sequences were established by the fluorescent dye termination method. Gene expression of mesangial SUR2 and Kir6.1/2 was examined by reverse transcription polymerase chain reaction (RT-PCR) and Northern analysis. SUR2 proteins were identified by immunoblotting of mesangial proteins from membrane-enriched fractions with polyclonal antiserum directed against SUR2.


RESULTS
RACE cloning yielded two mesangial SUR2 cDNAs of 4.8 and 6.7 kbp whose open reading frames translated proteins of 964 and 1535 aa, respectively. Using probes specific to each cDNA, the presence of a unique, 5.5 kbp serum-regulated mesangial SUR2 splice variant was established. The sequence of this mesangial SUR2 (mcSUR2B) shares identity with the recently cloned rat SUR2B (rSUR2B), but, in comparison to rSUR2B, is truncated by 12 exons at the N-terminus where it contains a unique insert of 16 aa. Immunoblotting studies with anti-SUR2 antiserum demonstrated SUR2 proteins of 108 and 170 kD in membrane-enriched fractions of MC protein extracts. Complementary studies showed abundant gene expression of Kir6.1, thereby establishing gene expression of both components of KATP.


CONCLUSIONS
Based upon analogy to vascular smooth muscle cells (VSMC), there are at least two putative mesangial KATP that most likely represent hetero-octamers, comprised of either rSUR2B or mcSUR2 in complex with Kir6.1. Our results define the mesangial SUR2B as the possible first link in a chain of cellular events that culminates in MC contraction and altered extracellular matrix metabolism following exposure to sulfonylureas. In addition, our results serve as the basis for the future elucidation of the electrophysiologic characteristics of the mesangial KATP and the study of endogenous regulators of mesangial cell contractility.
Szamosfalvi
Balázs
B
Department of Medicine, Division of Nephrology and Hypertension, Henry Ford Hospital, Detroit, Michigan 48202, USA.
Cortes
Pedro
P
Alviani
Rebecca
R
Asano
Kenichiro
K
Riser
Bruce L
BL
Zasuwa
Gary
G
Yee
Jerry
J
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression / Q000502:physiology; D005920:Glomerular Mesangium / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics*; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics*; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D011955:Receptors, Drug / Q000737:chemistry / Q000235:genetics*; D015398:Signal Transduction / Q000502:physiology; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; C577512:Abcc9 protein, rat; D018076:DNA, Complementary; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D000255:Adenosine Triphosphate