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 .
Each of the sur2 splice variants transiently expressed with the inward rectifier kir 6.2 formed functional k ( atp ) channels in hek 293 cells as assessed either by changes in dibac(4) (3) fluorescence responses or glyburide-sensitive whole cell currents .
To determine the presence of kir and surs subunits , rt-pcr was performed using kir6.1 , kir6.2 , sur1 , sur2a , and sur2b gene-specific primers .
Reverse transcriptase-polymerase chain reaction identified k ( atp ) channel component gene expression including regulatory sulfonylurea receptors ( sur ) sur1 and sur2b but not sur2a and pore-forming subunits ( kir ) kir6.1 and kir6.2 .
Rt-pcr demonstrated the presence of kir 6.2 and sur2b transcripts in colonic smooth muscle cells ; transcripts for kir 6.1 , sur1 , and sur2a were not detected .
We have used the baculovirus transduction system , bacmam , to demonstrate transient expression of multi-subunit katp channels in cho-k1 and hek-293 ebna cells using sulfonylurea receptor 1 ( sur ) , sur2a , sur2b , and kir 6.2 genes. [3h] -glyburide binding , patch clamp , and dibac4(3) measurements of membrane potential changes were used to monitor channel expression .
Polyclonal antibodies raised against certain fragments of known sulphonylurea receptor subunits , sur1 and sur2 , and against different epitopes of k+ inward rectifier subunits kir 6.1 and kir 6.2 of the atp-sensitive k+ channel of the plasma membrane ( cellkatp ) , were employed to detect similar subunits in brain mitochondria .
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 .
The k-cell lines also express relatively low levels of kir 6.1 , kir 6.2 , sur1 , and sur2 suggesting secretion is independent of k ( atp ) channels .
Here , we demonstrated for the first time that kir 6.1 , kir 6.2 and sur2 subunits of k ( atp ) channels are functionally expressed in hacat cells and both non-selective k ( atp ) channel opener pinacidil and mitok ( atp ) ( mitochondrial k ( atp ) ) channel opener diazoxide attenuated uv-induced keratinocytes cell death .
Thus decreased motility of the colon during inflammation may be associated with changes in the transcriptional regulation of kir 6.1 and sur2b gene expression .
The atp-sensitive k(+) channel ( k ( atp ) ) is a complex composed of an inwardly rectifying , pore-forming subunit ( kir 61 and kir 62 ) and the sulfonylurea receptor ( sur1 and sur2 ) .
Quantitative pcr showed that kir 6.1 gene expression was upregulated by almost 22-fold , whereas sur2b was downregulated by threefold after inflammation .
The interaction of glibenclamide ( but not opener ) with mutant sur2b is modified by coexpression with kir6.x in a manner depending on the kir subtype and on the integrity of the cell .
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 .
We adopted an rt-pcr strategy to identify , in aec , cdna transcripts for kir channels ( kir61 or 62 ) and sulfonylurea receptors ( sur1 , 2a , or 2b ) forming k ( atp ) channels .
Kcnq1-kcnq3 ) and nine kir channel alpha-subunits ( kir11 , kir22 , kir31-kir34 , kir41 , kir61 , kir6 2 ) were found .
Kcnq1-kcnq3 ) and for five different kir channel alpha-subunits ( kir11 , kir23 , kir32 , kir33 , kir62 ) were found .
For example , native katp channels appear to be a complex of a regulatory protein containing the su-binding site [sulfonylurea receptor ( sur ) ] and an inward-rectifying k+ channel ( kir ) serving as a pore-forming subunit .
Atp-regulated ( k ( atp ) ) channels are formed by an inward rectifier pore-forming subunit ( kir ) and a sulfonylurea ( glibenclamide ) -binding protein , a member of the atp binding cassette family ( sulfonylurea receptor ( sur ) or cystic fibrosis transmembrane conductance regulator ) .
Other types of kir channel ( kir11 , kir21 and kir41 ) were also irreversibly blocked by dids , suggesting that these channels may share common binding sites for these stilbene disulphonates .
It remains to be determined what are the molecular connections between the sur and kir subunits that enable this unique complex to work as a functional katp channel .
We recently engineered a series of transgenic ( tg ) mice overexpressing an atp-insensitive inward rectifying k(+) channel protein ( kir ) 6.2 mutant ( kir6.2[deltan30 , k185q] ) or the accessory sulfonylurea receptor ( sur ) 2a ( flag-sur2a ) or sur1 ( flag-sur1 ) subunits of the k ( atp ) channel , under transcriptional control of the alpha-myosin heavy chain promoter .
The kir 6.0 family consists of two known members , kir 6.1 and kir 6.2 , with distinct functional properties .
The sulfonylurea receptor ( sur ) subunits are targets for drugs that are inhibitors or openers of the katp channels , while the inwardly rectifying k+ ( kir ) subunits form the ion channel .
Kir 6.1 was localized to the plasma membrane , whereas kir 6.2 was mainly detected in the cytosol by immunohistochemistry .
The transgene expression of the k ( atp ) channel was examined by reverse transcription-polymerase chain reaction ( rt-pcr ) in rats transfected with cdna of kir 6.1 and 6.2 .
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 .
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 .
These channels are heteromultimers composed with a 4 : 4 stoichiometry of an inwardly rectifying k+ channel ( kir ) subunit 6.x plus a sulfonylurea receptor ( sur ) .
To study the specificity of the taurine sensitivity , intracellular taurine was tested on several members of the kir family expressed in xenopus oocytes. k(+) currents induced by kir1.1a , kir2.1 , kir3.2 , kir4.1 , or kir5.1 were insensitive to taurine , but all tested combinations of kir6.x with or without the sur subunit were significantly inhibited by taurine .
It seems that these nbfs play an essential role in conferring the mgadp and kco sensitivity to the channel , whereas the kir channel subunit itself possesses the atp-sensing mechanism as an intrinsic property .
Atp-sensitive potassium channels are an octomeric complex of four pore-forming subunits of the kir 6.0 family and four sulfonylurea receptors .
Atp-sensitive potassium channels ( k ( atp ) ) , unlike other inwardly rectifying potassium ( kir ) channels , require two structurally diverse subunits to form functional channels :
The analysis of the structure of the genes encoding k ( atp ) channels that are made of four kir subunits ( forming the ionic pore ) and four regulatory sur subunits ( that contain the binding site for antidiabetic sulfonylureas ) allowed to several subclasses of those ionic channels to be described :
Vasodilation to increased external k+ involves kir channels .
The expression of mrna for voltage-dependent ( kv ) and inward-rectifying k channels ( kir ) was studied in clonal rat somato-mammotroph cells ( gh3/b6 cells ) and rat pituitary using reverse transcription-polymerase chain reaction ( rt-pcr ) .
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 .
In the absence of mg(2+) , the stilbene disulphonate , dids , irreversibly inhibits k ( atp ) channels by binding to the kir subunit .
These results suggest a novel activity of aprindine to enhance k ( atp ) currents by inhibiting proteasomal degradation of kir 6.2 channels , which may be beneficial in the setting of cardiac ischemia .
The purpose of this study was to investigate the subunit type of k ( atp ) channel , that is , the combinations of the kir subunit and the sur subunit in the human corporal smooth muscle and determine whether the electrophysiological kinetics and pharmacological properties of k ( atp ) channels meet the subunit characteristics of the ion channel .
Additional pkc consensus sites exist on both kir and the larger sulfonylurea receptor ( sur ) subunits .
By precisely comparing the functional properties of the sur2a/kir6.2 and the sur2b/kir6.1 channels , we shall show that the single-channel characteristics and pharmacological properties of sur/kir6.0 channels are determined by kir and sur subunits , respectively , while responses to intracellular nucleotides are determined by both sur and kir subunits .
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 .
Voltage-activated k+ channels ( kv ) are encoded by the kv gene family , ca(2+) -activated k+ channels ( bkca ) are encoded by the slo gene , inward rectifiers ( kir ) by kir2.0 , and atp-sensitive k+ channels ( katp ) by kir6.0 and sulphonylurea receptor genes .
Mrna for both kir 6.1 and 6.2 were detected by rt-pcr .
Four regulatory sulfonylurea receptor ( sur ) embracing four poreforming inwardly rectifying potassium channel ( kir ) .