PMC

Chemical structures of the agents used in this study. The anionic forms of the compounds are shown. The sulphonylurea moiety and benzamide group of glibenclamide are indicated.

Mitiglinide inhibits CFTR Cl⁻ currents. (a) Time-course of CFTR Cl⁻ current in an excised inside-out membrane patch. ATP (0.3 mM), PKA (75 nM), and mitiglinide (50–500 μM) were present in the intracellular solution during the times indicated by the bars; other details as in . (b) Hill plot of the effect of mitiglinide concentration on CFTR Cl⁻ currents. Data are means±s.e.mean; n=4–5 observations at each concentration. The continuous line is the fit of a first-order regression to the data. Other details as in (a).

Effect of mitiglinide on the single-channel activity of CFTR. (a) Representative recordings show the effect of mitiglinide (250 μM) on the activity of a single CFTR Cl⁻ channel. ATP (0.3 mM) and PKA (75 nM) were continuously present in the intracellular solution; voltage was −50 mV. Scale bar, 2 s and 200 ms for prolonged and expanded recordings, respectively. (b) and (c) Effect of mitiglinide (250 μM) on i and P_o, respectively. Columns and error bars indicate means±s.e.mean (n=6) for each concentration. Mitiglinide (250 μM) was without effect on i (n=6; P>0.05), but decreased P_o to 41±5% (n=6; P<0.05). Other details as in (a).

Effect of repaglinide on the single-channel activity of CFTR. (a) Representative recordings show the effect of repaglinide (50 μM) on the activity of a single CFTR Cl⁻ channel. ATP (0.3 mM) and PKA (75 nM) were continuously present in the intracellular solution; voltage was −50 mV. Scale bar, 2 s and 200 ms for prolonged and expanded recordings, respectively. (b) and (c) Effect of repaglinide (50 μM) on i and P_o, respectively. Columns and error bars indicate means±s.e.mean (n=6) for each concentration. Repaglinide (50 μM) decreased i to 91±2% of the control value (n=6; P=0.005), but was without effect on P_o (n=6; P>0.05). Other details as in (a).

Voltage-dependent inhibition of CFTR Cl⁻ channels by meglitinide and mitiglinide. (a) and (b) Relationships between P_o and voltage in the absence (filled symbols) and presence (open symbols) of meglitinide (250 μM) and mitiglinide (250 μM) when the membrane patch was bathed in symmetrical 147 mM Cl⁻ solutions. Data points are means±s.e.mean (meglitinide: n=3–5; mitiglinide: n=3–4) at each voltage. (c) Relationship between K_D and voltage for the data shown in (a) and (b). K_D values were calculated as described in Results and the continuous lines are the fit of first-order regressions to the data. For comparison, the dotted line shows the relationship between K_D and voltage for glibenclamide under similar experimental conditions. Data for glibenclamide are from .

When compared to glibenclamide, repaglinide only weakly inhibits CFTR Cl⁻ currents. (a) Time-course of CFTR Cl⁻ current in an excised inside-out membrane patch. ATP (1.0 mM), PKA (75 nM), repaglinide (50 μM), and glibenclamide (50 μM) were present in the intracellular solution during the times indicated by the bars; other details as in . (b) Comparison of the effects of repaglinide (50 μM) and glibenclamide (50 μM) on CFTR Cl⁻ currents. Columns and error bars indicate means±s.e.mean (n=5) for each concentration. Values represent the average current recorded during an intervention normalised to that measured under control conditions at the start of the experiment. Repaglinide (50 μM) reduced CFTR Cl⁻ current to 87±3% of control (n=5; P<0.05), whereas glibenclamide (50 μM) reduced CFTR Cl⁻ current to 37±4% of control (n=5; P<0.001). Other details as in (a). In the presence of both glibenclamide (50 μM) and repaglinide (50 μM) CFTR Cl⁻ currents were reduced to 37±3% of control (n=5), a value not significantly different from that observed in the presence of glibenclamide (50 μM; n=5; P>0.05).

Meglitinide inhibits CFTR Cl⁻ currents. (a) Time-course of CFTR Cl⁻ current in an excised inside-out membrane patch. ATP (0.3 mM), PKA (75 nM), and meglitinide (50–500 μM) were present in the intracellular solution during the times indicated by the bars. Voltage was −50 mV, and there was a large Cl⁻ concentration gradient across the membrane patch (internal [Cl⁻]=147 mM; external [Cl⁻]=10 mM). Each point is the average current for a 4 s period and no data were collected while solutions were changed. For the purpose of illustration, the time-course has been inverted so that an upward deflection represents an inward current. (b) Hill plot of the effect of meglitinide concentration on CFTR Cl⁻ currents. Data are means±s.e.mean; n=3–6 observations at each concentration. The continuous line is the fit of a first-order regression to the data. Other details as in (a).

Open and closed time histograms of single CFTR Cl⁻ channels inhibited by meglitinide and mitiglinide. (a) and (b) Effect of meglitinide and mitiglinide on the open (top) and closed (bottom) time histograms of CFTR, respectively. Data are from experiments in which the membrane patch contained only one active channel, studied in the absence and presence of either meglitinide (250 μM) or mitiglinide (250 μM); ATP (0.3 mM) and PKA (75 nM) were present throughout and voltage was −50 mV. For open time histograms, the continuous line is the fit of either a one- (control, meglitinide) or a two- (mitiglinide) component exponential function. For closed time histograms, the continuous line is the fit of either a two- (control, meglitinide) or a three- (mitiglinide) component exponential function. Dashed lines show the individual components of the exponential functions. Logarithmic x-axes with 10 bins decade⁻¹ were used for both open and closed time histograms. Other details as in .

Effect of meglitinide on the single-channel activity of CFTR. (a) Representative recordings show the effect of meglitinide (250 μM) on the activity of a single CFTR Cl⁻ channel. ATP (0.3 mM) and PKA (75 nM) were continuously present in the intracellular solution. Voltage was −50 mV, and there was a large Cl⁻ concentration gradient across the membrane patch (internal [Cl⁻]=147 mM; external [Cl⁻]=10 mM). Scale bar, 2 s and 200 ms for prolonged and expanded recordings, respectively. Dashed lines indicate the closed channel state and downward deflections correspond to channel openings. For the purpose of illustration, records were filtered at 500 Hz and digitised at 1 kHz. (b) and (c) Effect of meglitinide (250 μM) on i and P_o, respectively. Columns and error bars indicate means±s.e.mean (n=7) for each concentration. Meglitinide (250 μM) decreased i to 92±1% of the control value (n=7; P<0.001) and P_o to 42±3% of the control value (n=7; P<0.001). Other details as in (a).