Reconstituted human cardiac KATP channels: functional identity with the native channels from the sarcolemma of human ventricular cells.
ATP-sensitive potassium (KATP) channels in striated myocytes are heteromultimers of KIR6.2, a weak potassium inward rectifier, plus SUR2A, a low-affinity sulfonylurea receptor. We have cloned human KIR6.2 (huKIR6.2) and a huSUR2A that corresponds to the major, full-length splice variant identified by polymerase chain reaction analysis of human cardiac poly A+ mRNA. ATP- and glibenclamide-sensitive K+ channels were produced when both subunits were coexpressed in COSm6 and Chinese hamster ovary cells lacking endogenous KATP channels, but not when huSUR2A or huKIR6.2 were transfected alone. Recombinant channels activated by metabolic inhibition in cell-attached configuration or in inside-out patches with ATP-free internal solution were compared with sarcolemmal KATP channels in human ventricular cells. The single-channel conductance of approximately 80 pS measured at -40 mV in quasi-symmetrical approximately 150 mmol/L K+ solutions, the intraburst kinetics that were dependent on K+ driving force, and the weak inward rectification were indistinguishable for both channels. Similar to the native channels, huSUR2A/huKIR6.2 recombinant channels were inhibited by ATP at quasi-physiological free Mg2+ ( approximately 0. 7 mmol/L) or in the absence of Mg2+, with an apparent IC50 of approximately 20 micromol/L and a pseudo-Hill coefficient of approximately 1. They were "refreshed" by MgATP and stimulated by ADP in the presence of Mg2+ when inhibited by ATP. The huSUR2A/huKIR6.2 channels were stimulated by cromakalim and pinacidil in the presence of ATP and Mg2+ but were insensitive to diazoxide. The results suggest that reconstituted huSUR2A/huKIR6.2 channels represent KATP channels in sarcolemma of human cardiomyocytes and are an adequate experimental model with which to examine structure-function relationships, molecular physiology, and pharmacology of these channels from human heart.
Babenko
A P
AP
Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, TX, USA. ababenko@bcm.tmc.edu
Gonzalez
G
G
Aguilar-Bryan
L
L
Bryan
J
J
D018528:ATP-Binding Cassette Transporters; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000293:Adolescent; D000328:Adult; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002648:Child; D003001:Cloning, Molecular; D006224:Cricetinae; D019806:Cromakalim / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D003981:Diazoxide / Q000494:pharmacology; D015723:Gene Library; D005905:Glyburide / Q000494:pharmacology; D006352:Heart Ventricles / Q000166:cytology / Q000648:ultrastructure; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008875:Middle Aged; D009206:Myocardium / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D020110:Pinacidil / Q000494:pharmacology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D051381:Rats; D011955:Receptors, Drug / Q000187:drug effects / Q000235:genetics; D012508:Sarcolemma / Q000737:chemistry / Q000378:metabolism; D064233:Sulfonylurea Receptors; D014044:Tolbutamide / Q000494:pharmacology; D014162:Transfection
C577510:ABCC9 protein, human; D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D019806:Cromakalim; D000244:Adenosine Diphosphate; D020110:Pinacidil; D000255:Adenosine Triphosphate; D014044:Tolbutamide; D008274:Magnesium; D003981:Diazoxide; D005905:Glyburide