Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase

Abstract

ATP-sensitive potassium channels (K(ATP) channels) regulate cell excitability in response to metabolic changes. K(ATP) channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K(+) channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), activate K(ATP) channels and antagonize ATP inhibition of K(ATP) channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP(2) levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed K(ATP) channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K(1/2), the half maximal inhibitory concentration, approximately 60 microM) than the sensitivities from control cells (K(1/2) approximately 10 microM). An inactive form of the PIP5K had little effect on the K(1/2) of wild-type channels but increased the ATP-sensitivity of a mutant K(ATP) channel that has an intrinsically lower ATP sensitivity (from K(1/2) approximately 450 microM to K(1/2) approximately 100 microM), suggesting a decrease in membrane PIP(2) levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP(2) and PI-3,4,5-P(3) levels, is a significant determinant of the physiological nucleotide sensitivity of K(ATP) channels.

Figure 1

Expression and distribution of the PIP5K:WT and PIP5K:Δ1–238 mutant. (A) Cells were transfected with Sindbis virus expressing GFP-PIP5K:WT or GFP-PIP5K:Δ1–238 for different times as indicated, and PIP5K proteins were analyzed by Western blotting with anti-GFP antibodies. (B) Distribution of PIP5K:WT and PIP5K:Δ1–238 mutant in membrane and cytosol. After infection, cytosolic (cy) and membrane (mb) fractions were prepared as described in Materials and Methods, and the distributions of PIP5K protein were analyzed by Western blotting with anti-GFP antibodies.

Figure 2

Overexpression of PIP5K:WT decreases the sensitivity of K_ATP channels to inhibition by ATP. (A) Representative wild-type K_ATP channel currents recorded in inside-out membrane patches from control cells, cells overexpressing PIP5K:WT (WT Kinase), or cells overexpressing PIP5K:Δ1–238 (ΔN Kinase). Currents were recorded at −50 mV and are shown as upward deflections. The patch was exposed to differing [ATP] as indicated by the bars above the record. (B) K_1/2 estimated for wild-type K_ATP channels in cells overexpressing PIP5K:WT or cells overexpressing PIP5K:Δ1–238, from fits of the Hill equation {I_rel = 1/[1 + ([ATP]/K_1/2)^H]}, with I_rel being the current in ATP, relative to the current in the absence of ATP. The Hill coefficient H was fixed at 1.3. Each data point represents the average of 15–26 patches. The error bar is the SEM. (Inset) PIP₂ levels measured in control cells, cells overexpressing PIP5K:WT, or cells overexpressing PIP5K:Δ1–238. Results represent the means ± SD of triplicate determinations.

Figure 3

Overexpression of PIP5K:Δ1–238 increases the sensitivity of a mutant K_ATP channel (Kir6.2[I154C, C166S] + SUR1) to inhibition by ATP. (A) Representative (Kir6.2[I154C, C166S] + SUR1) K_ATP channel currents recorded in inside-out membrane patches from control cells or cells overexpressing PIP5K:Δ1–238 (ΔN Kinase). Currents were recorded at −50 mV and are shown as upward deflections. The patch was isolated and exposed to differing [ATP] as indicated by the bars above the record. The dashed line indicates zero current. (B) To estimate the K_1/2 in control cells or cells overexpressing PIP5K:Δ1–238, data points were fitted to the Hill equation {I_rel = 1/[1 + ([ATP]/K_1/2)^H]}, with I_rel being the current relative to the current in the absence of ATP and with H being fixed at 1.3. Each data point represents the average of 4–6 patches, with the error bar being the SEM. (Inset) Response of (Kir6.2[I154C, C166S] + SUR1) K_ATP channels in an isolated inside-out membrane patch from cells overexpressing PIP5K:Δ1–238 to exogenous PIP₂. Currents were recorded at −50 mV and are shown as upward deflections. The patch was exposed to differing [ATP] and 5 μg/ml PIP₂ as indicated by the bars above the record. The K_1/2 increased from 219 μM to >5 mM with continued PIP₂ application.