Intracellular nucleotide-mediated gating of SUR/Kir6.0 complex potassium channels expressed in a mammalian cell line and its modification by pinacidil

Abstract

1. We have examined the properties of intracellular nucleotide-mediated gating of K+ channel constructs composed of the sulphonylurea receptor 2B and the inwardly rectifying K+ channel subunits Kir6.1 and Kir6.2 (SUR2B/Kir6.1 and SUR2B/Kir6.2 complex K+ channels) heterologously expressed in human embryonic kidney (HEK) 293T cells. In the cell-attached form, both types of K+ channel were activated by pinacidil. 2. In inside-out (IO) patches, the SUR2B/Kir6.2 channels opened spontaneously and were inhibited by intracellular ATP (ATPi). Pinacidil attenuated the ATPi-mediated channel inhibition in a concentration-dependent manner. In contrast, the SUR2B/Kir6.1 channels required intracellular nucleoside di- or tri-, but not mono-, phosphates for opening. The potency of adenine, guanine or uracil nucleotides to activate SUR2B/Kir6.1 channels was enhanced by pinacidil. 3. In the presence of pinacidil, adenine and guanine, but not uracil, nucleotides exhibited bell-shaped concentration-dependent activating effects on SUR2B/Kir6.1 channels. This was due to channel inhibition caused by adenine and guanine nucleotides, which was unaffected by pinacidil. 4. From power density spectrum analysis of SUR2B/Kir6.1 currents, channel activation could be described by the product of two gates, a nucleotide-independent fast channel gate and a nucleotide-dependent slow gate, which controlled the number of functional channels. Pinacidil specifically increased the potency of nucleotide action on the slow gate. 5. We conclude that Kir6.0 subunits play a crucial role in the nucleotide-mediated gating of SUR/Kir6.0 complex K+ channels and may determine the molecular mode of pinacidil action.

Figure 1. Comparison of nucleotide and pinacidil regulation of SUR2B/Kir6.2 and SUR2B/Kir6.1 channels

Aa, representative single-channel recording of the SUR2B/Kir6.2 channel illustrating the effects of pinacidil (Pin) and glibenclamide (Gli) in the cell-attached and excised (arrow and IO) inside-out membrane patch. Ab, apparent antagonism of ATP-evoked inhibition of SUR2B/Kir6.2 channels induced by pinacidil. Each symbol shows the ATP-induced channel inhibition in the absence (○) or presence of pinacidil (□, at 100 μm; and ▵, 300 μm). The continuous lines were fitted with eqn (1). B, representative single-channel recordings of SUR2B/Kir6.1 channels illustrating the effects of ATP (a), AMP (b) and ADP (b) in the absence or presence of pinacidil. At the points indicated by IO, the patches were excised from their cells. In each membrane current recording the zero current level is indicated by a thin horizontal line.

Figure 2. The effects of pinacidil on NTP-induced SUR2B/Kir6.1 channel openings in inside-out patches

Top panels, representative single-channel current recordings of SUR2B/Kir6.1 channel activity induced by internal ATP (Aa), GTP (Ba) and UTP (Ca) in the absence or presence of pinacidil at the indicated concentrations (10 or 300 μm). In each single-channel current recording, IO indicates the moment when the membrane patch was excised from the cell and the zero current level is indicated by thin horizontal lines. Lower panels, NTP concentration-response relationships for channel activation in the absence (○) or presence of pinacidil (□, at 10 μm; ▵, 100 μm and •, 300 μm). The number of observations for each point are 3–11 for ATP (Ab), 3-8 for GTP (Bb) and 3–8 for UTP (Cb). The thick continuous lines in Ab and Bb were fitted by the multiplication of eqn (2) and eqn (3). Thin continuous lines and dashed lines show the estimated ATP- or GTP-induced activation and inhibition curves, respectively. The continuous lines in Cb were fitted with eqn (2).

Figure 3. Effects of pinacidil on NDP-induced SUR2B/Kir6.1 channel openings in inside-out patches

Aa and b, representative single-channel recordings of ADP-induced channel openings in the absence (a), or presence of 300 μm pinacidil (Pin) (b). Ac, concentration-response relationships for ADP-induced activation of the channels in the absence (○) or presence of 300 μm pinacidil (□). The number of observations for each point was 3-5. Ad, concentration-response relationships for GDP-induced activation of the channels in the absence (○) or presence of 300 μm pinacidil (□). The number of observations for each point was 3-7. Ba, representative single-channel recording of UDP-induced channel openings in the absence or presence of 10 μm pinacidil. Bb, concentration-response relationships for UDP-induced activation of the channels in the absence (○) or presence of pinacidil (□, at 10 μm; and ▵, 300 μm). The number of observations for each point was 3-7. In each single-channel recording, IO indicates the moment when the patch was excised from the cell and the zero current level is indicated by a thin horizontal line. The thick continuous lines in Ac and d were fitted by multiplication of eqn (2) and eqn (3). Thin continuous lines and dashed lines show the estimated ADP- or GDP-induced activation and inhibition curves, respectively. The continuous lines in Bb were fitted with eqn (2).

Figure 4. Effects of UTP and UDP on the power density spectra of current fluctuations of the SUR2B/Kir6.1 channel

A, power density spectra calculated from recordings shown in Fig. 2C a in the presence of 3 mM UTP (a) and 3 mM UTP plus 300 μm pinacidil (b). B, power density spectra calculated from recordings obtained with 10 μm UTP (a) and 100 μm UTP in the presence of 100 μm pinacidil (b). C, power density spectra calculated from the recording shown in Fig. 3B a with 1 mM UDP (a) and 10 mM UDP (b). Each spectrum could be fitted with the sum of two Lorentzian functions. F₁ and F₂ indicate the corner frequencies of the slow and fast Lorentzian components, respectively. S₁/S₂ represents the ratio of the power at 0 Hz of the two components.

Figure 5. Spectral parameters of SUR2B/Kir6.1 channel activation induced by UTP and UDP

A, concentration-dependent activation of SUR2B/Kir6.1 channels by UTP in the absence (○) or presence of pinacidil (□, at 10 μm; ▵, 100 μm; and •, 300 μm). a, F₁ (continuous line) and F₂ (dashed line). b, relative S₁ (rS₁) and relative S₂ (rS₂) and S₁/S₂ ratio. c, the rate constants for the fast gating transitions, α₁ (continuous line) and β₁ (dashed line) in the upper panel, and α₂ (continuous line) and β₂ (dashed line) in the lower panel. B, the rate constants for the fast gating transitions, α₁ (continuous line) and β₁ (dashed line) in the upper panel, and α₂ (continuous line) and β₂ (dashed line) in the lower panel calculated and plotted against [UDP] in the absence (○) or presence of pinacidil (□, at 10 μm; and ▵, 300 μm).

Figure 6. Activation of SUR2B/Kir6.1 channels by UTP, UDP and pinacidil

Data are reproduced from Fig. 2C b for UTP (A) and from Fig. 3B b for UDP (B). The dashed lines represent the fits of these data to eqn (6) which represents the increase in channel activity resulting from the recruitment of ion channels (see text for details).