6 research outputs found
Increased basal K<sub>ATP</sub> activity in both hetDel and hetT, del channels.
<p><sup>86</sup>Rb<sup>+</sup> efflux of mutant Kir6.2 subunits coexpressed with WT subunits in 1∶1 DNA ratio, under metabolic inhibition (A) and in basal states (B). Data points indicate means ± SEM of n = 5. * indicates P<0.05 compared with WT by One-Way ANOVA analysis. NT  =  not transfected (no statistic given).</p
Decreased K<sub>ATP</sub> activity in both homDel and homT, del channels.
<p>Representative <sup>86</sup>Rb<sup>+</sup> efflux profile comparing untransfected COSm6 cells (Un) and cells transfected with WT, homS225T, homDel and homT, del channels in metabolic inhibition (A) and in basal conditions (B). Data points indicate means ± SEM of n = 4. NT  =  not transfected.</p
Homology modeling of Kir6.2 from Kir2.2 structure with PyMOL software.
<p>(A) S225T is colored in yellow and deleted amino acids 226–232 (-PEGEVVP-) are colored in orange. (B) E227 and E229 are colored and labeled in red while R192 and R314 are colored and labeled in blue. The structure model reveals the possible interaction between the deleted amino acid P232 (orange spheres) and V319 in the proposed Kir6.2 Ankyrin-B binding site (a.a. 316 to a.a. 323 –VPIVAEED- colored in magenta).</p
Expression of WT and homomeric mutant channels in patch clamp recordings.
<p>(A) representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT channels and various mutant channels, at +50 mV pipette potential. Patches were exposed to different concentrations of Mg-free ATP as indicated. Dashed line indicates zero current (WT and homS225T channels) or zero channel level (homDel and homT, del channels). (B) Western Blot of Flag-tagged SUR1 (fSUR1) from various constructs are indicated. The mature (cell surface) complex-glycosylated bands and immature core-glycosylated bands are indicated as upper bands (white arrow) and lower bands (black arrow) respectively. NT = not transfected.</p
Decreased ATP sensitivity in both hetDel and hetT, del channels.
<p>(A) Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT channels and hetT, del mutants. Patches were exposed to different concentrations of Mg-free ATP as indicated. (B) ATP dose-response relationships, fit by Hill equation as described in methods. Data points indicate means ± SEM of n = 5–7 patches. * indicates P<0.05 compared with WT by One-Way ANOVA analysis. The fitted K<sub>1/2</sub> for WT, homS225T, hetS225T, hetDel, and hetT, del channels are 13.75, 25.06, 14.8, 22.87 and 43.94 (in μM) and the Hill coefficients are 1.6, 1.3, 1.1, 1.28 and 1.2, respectively.</p
Heterozygous S225T, deletion channels display higher channel open probability, assessed by the ‘PIP<sub>2</sub>’ method.
<p>(A) Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT channels and hetT, del mutants. Patches were exposed to different concentrations of Mg-free ATP and PIP<sub>2</sub> as indicated. (B) Mean estimated Po for various channels: WT (0.53±0.04); homS225T (0.62±0.04); hetS225T (0.59±0.03); hetDel (0.64±0.04); hetT, del (0.66+0.02). * indicates statistically significant difference compared with WT (Student's t-test, p-value <0.05). n = 6–12.</p