Increased basal K_ATP activity in both hetDel and hetT, del channels.

<p>⁸⁶Rb⁺ 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

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

Decreased K_ATP activity in both homDel and homT, del channels.

<p>Representative ⁸⁶Rb⁺ 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

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

Heterozygous S225T, deletion channels display higher channel open probability, assessed by the ‘PIP₂’ 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 PIP₂ 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

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_1/2 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

Altered B cell compartment in C1858T healthy controls and patients.

<p>Previously frozen PBMC from control C/T and C/C subjects and from C/T T1D and C/C patients were stained with antibodies to CD19, CD27, CD24 and CD38 and analyzed by FACS to determine the relative frequencies of transitional (<b>A</b>) and memory B cells (<b>B</b>) under basal conditions (day 0). In both, <b>A</b> and <b>B</b> graphs, horizontal lines represent the mean frequency and each symbol represents an individual.</p

Clinical characteristics of T1D patients.

<p>AT: autoimmune thyroiditis; CD: celiac disease; AG: autoimmune gastritis.</p><p>Statistical analysis of basal transitional B cell percentages in C/C and C/T patients: KS-test p>0.10; unpaired T-test with Welch’s correction p = 0.03.</p><p>Clinical characteristics of T1D patients.</p

B cell phenotype after 4 days of CpG stimulation.

<p>Ratio of proliferation of CpG stimulated over unstimulated CMFDA-labeled CD19⁺ cells in the four different categories of subjects (<b>A</b>). Ratio of mature B cell percentages (<b>B</b>) in unstimulated compared to CpG-stimulated PBMC, ratio of switched memory B cell (<b>C</b>) and of IgM⁺ memory B cell percentages (<b>D</b>), calculated as CpG-stimulated over unstimulated PBMC, in T1D patients carrying or non carrying the C/T <i>PTPN22</i> variant and in non-carrier C/C and C/T healthy controls. Percentage of plasma cells from IgM⁺ (<b>E</b>) and IgM⁻ (<b>F</b>) memory B cells in the PBMC of individuals heterozygous for the C/T <i>PTPN22</i> variant compared to non-carriers.</p

B cell phenotype after 7 days of CpG stimulation.

<p>Ratio of proliferation of CpG stimulated CMFDA-labeled CD19⁺ cells over unstimulated cells among the four different categories of subjects included in our study (<b>A</b>) Ratio of mature B cell percentages (<b>B</b>), calculated as unstimulated over CpG-stimulated PBMC, in T1D patients carrying or non carrying the C/T <i>PTPN22</i> variant and in non-carrier C/C and C/T healthy controls. Percentages of plasma cells from IgM⁺ memory B cells (<b>C</b>) in individuals carrying the C/T <i>PTPN22</i> variant compared to the C/C subjects.</p