PMC3012173_01
V290M pedigrees and clinical responses to therapy. A: Arrow indicates the V290M proband in each case; black, gray, and empty symbols indicate genetically homozygous, heterozygous, and unaffected individuals, respectively. B: Results of fluoro-DOPA scan of proband 2 indicates focal lesion (yellow arrowheads) in the body of the pancreas. C: Mean daily glucose levels and SD for final period on octreotide alone (8 mug/kg/day divided every 6 h), following switch to octreotide 8 mug/kg/day divided every 6 h plus amlodipine (0.1 mg/kg/day divided every 12 h). Gray area represents target range for alarm for CGMS (70-140 mg/dl glucose). mo, month. (A high-quality digital representation of this figure is available in the online issue.)
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F1
As shown in Fig. 1C, octreotide alone maintained average blood glucose levels in the desired range, but marked excursions were still present with episodes >300 mg/dl. There was only one episode of blood glucose <60 mg/dl in >3,000 sensor readings (Fig. 1B).
Decreased KATP activity in homomeric V290M channels (M/M) and intermediate activity with heteromeric WT and V290M channels (V/M) relative to wild-type (V/V) channels. A: Representative 86Rb+ efflux shown as cumulative counts relative to total counts as a function of time, under basal conditions, in the presence of MI, or the presence of KATP channel opener diazoxide. B: Data were fit to a single exponential, and rate constants were obtained from untransfected cells, k1, for non-KATP channel 86Rb+ leak and from transfected cells, k2, for KATP channel-mediated 86Rb+ current. Graphs show compiled data (mean +- SEM) from 4 to 6 experiments.
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F2
Homomeric Kir6.2[V290M] (M/M) channels show considerably reduced 86Rb efflux rates, with k2 reduced by ~80% under metabolically inhibited or diazoxide-stimulated conditions (Fig. 2). Cells expressing heteromeric WT plus V290M (V/M) channels show intermediate efflux rates (k2, reduced by ~50%) (Fig. 2B). Given that heteromeric V290M/WT channels exhibit ~50% reduction of KATP conductance (Fig. 2) and the anecdotal evidence of hypersensitivity to glucose exhibited by the proband's brother, we pursued OGTT in the mother (Table 1) and the brother (Table 2), both of whom are heterozygous for the V290M mutation.
Molecular basis of loss of KATP activity. A: Western blots of FLAG-tagged SUR1 (fSUR1) from COSm6 cells expressing fSUR1 alone, or co-expressing fSUR1 with either WT Kir6.2 or mutant Kir6.2[V290M]. The mature (cell surface) complex-glycosylated and immature core-glycosylated fSUR1 bands are indicated by upper and lower arrows, respectively. In the absence of Kir6.2, fSUR1 is exclusively core glycosylated. B: Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT or mutant V290M KATP channels at -50 mV membrane potential (+50 mV pipette). Patches were excised from the cell (arrow) into zero ATP solution, then subsequently exposed to 5 mmol/l ATP. C: Structural model of Kir6.2 tetramer indicates location of V290. R301 and E292 of adjacent Kir6.2 subunits form a salt bridge that is essential for channel function. V290 lies within seven angstroms of R301, and mutations at this site may destabilize the intersubunit interface. (A high-quality digital representation of this figure is available in the online issue.)
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F3
Lysates of COSm6 cells expressing recombinant channels were assayed by immunoblotting (Fig. 3A). The activity of WT and mutant KATP channels was further examined in inside-out membrane patch-clamp experiments (Figs. 3-5). Following patch excision, V290M channels inactivated with time constant of 2.0 +- 0.5 s (n = 5), while WT channels showed essentially no inactivation following excision (Fig. 3B).
Mutant V290M channels have unaltered ATP-sensitivity and intact MgADP activation. A: Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT or mutant V290M KATP channels at -50 mV membrane potential (+50 mV pipette). Patches were exposed to different concentrations of ATP as indicated. B: Mean steady-state patch current following isolation (+- SEM). C: Steady-state dependence of membrane current on ATP relative to current in the absence of ATP. Solid line represents mutant V290M channels while dashed line represents WT channels fitted with the Hill equation by least-squares method. D: Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT or mutant V290M KATP channels at -50 mV membrane potential (+50 mV pipette). Patches were exposed to different concentrations of ATP, and ADP (in the presence of 0.5. mM free Mg2+) as indicated.
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F4
The activity of WT and mutant KATP channels was further examined in inside-out membrane patch-clamp experiments (Figs. 3-5). Steady-state ATP sensitivity of V290M channels was similar to WT (Fig. 4A and C), but current density was considerably lower than WT (Fig. 4B), reflecting the dramatic inactivation that occurs. Physiologically, the major determinant of channel activation is stimulation by Mg-nucleotides and, as shown in Fig. 4D, MgADP activation is intact in V290M channels, although inactivation follows the MgADP activation, reducing steady-state currents in MgADP.
Inactivation is PIP2-sensitive. Current recordings of COSm6 patches expressing WT (A) and mutant V290M (B) channels. In WT channels, the current approximately doubles following addition of PIP2. However, in V290M channels, steady-state channel activity increases several-fold, accompanied by a marked reduction in inactivation following exposures to PIP2, a channel open-state stabilizer.
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F5
The activity of WT and mutant KATP channels was further examined in inside-out membrane patch-clamp experiments (Figs. 3-5). Following patch excision, exogenously applied phosphatidylinositol bisphosphate (PIP2) incorporates into the membrane inner leaflet and typically increases activity of WT channels by two- to threefold as a result of increased open-state stability (Fig. 5). During PIP2 exposure, inactivation became progressively slower and less complete in response to application and removal of ATP (Fig. 5B).
Kinetic mechanism of inactivation. A: Fraction of inactivating current (following ATP removal) vs. steady-state current (relative to maximal attained following repeat PIP2 exposure-relative open probability) at selected time points following exposures to PIP2 in V290M channels. Individual patches are indicated by different symbols; large symbols indicate starting values prior to first exposure to PIP2. B: A 3-state model of channel activity (with an unliganded open and closed state, and with ATP binding to the closed state) is adequate to explain multiple features of KATP gating. A fourth, inactivated, state (CIN), which is coupled to the unliganded closed state, can explain both the phenomenon of inactivation and the finding that PIP2 (which acts to shift the KCO equilibrium toward the open state) both reduces and slows inactivation.
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F6
During this process, the rate and extent of inactivation both decreased in correlation with the estimated open probability (Fig. 6A) as expected for an inactivation process occurring from the closed state (discussion).