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. 2000 Mar 1;19(5):942-55.
doi: 10.1093/emboj/19.5.942.

Regulation of ATP-sensitive potassium channel function by protein kinase A-mediated phosphorylation in transfected HEK293 cells

Y F Lin  1 , Y N Jan, L Y Jan
Affiliations

Regulation of ATP-sensitive potassium channel function by protein kinase A-mediated phosphorylation in transfected HEK293 cells

Y F Lin et al. EMBO J. .

Abstract

ATP-sensitive potassium (K(ATP)) channels regulate insulin secretion, vascular tone, heart rate and neuronal excitability by responding to transmitters as well as the internal metabolic state. K(ATP) channels are composed of four pore-forming alpha-subunits (Kir6.2) and four regulatory beta-subunits, the sulfonylurea receptor (SUR1, SUR2A or SUR2B). Whereas protein kinase A (PKA) phosphorylation of serine 372 of Kir6.2 has been shown biochemically by others, we found that the phosphorylation of T224 rather than S372 of Kir6.2 underlies the catalytic subunits of PKA (c-PKA)- and the D1 dopamine receptor-mediated stimulation of K(ATP) channels expressed in HEK293 cells. Specific changes in the kinetic properties of channels treated with c-PKA, as revealed by single-channel analysis, were mimicked by aspartate substitution of T224. The T224D mutation also reduced the sensitivity to ATP inhibition. Alteration of channel gating and a decrease in the apparent affinity for ATP inhibition thus underlie the positive regulation of K(ATP) channels by PKA phosphorylation of T224 in Kir6.2, which may represent a general mechanism for K(ATP) channel regulation in different tissues.

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Figures

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Fig. 1. Enhancement of Kir6.2ΔC36 channel activity by PKA phosphorylation of residue T224. The (mouse) Kir6.2ΔC36 channel was expressed in HEK293 cells in the absence of SUR subunit. Recordings were performed in symmetrical potassium solutions at room temperature and voltage was clamped at –60 mV. (A) Single-channel current traces of a Kir6.2ΔC36 channel obtained from an inside-out patch prior to c-PKA treatment (left, control), during c-PKA (50 μg/ml) treatment (center, c-PKA) and during c-PKA plus PKIP (200 μg/ml) application (right). Upward deflections represent openings from closed states. No ATP was included in the bath solution. Segments of raw recordings underlined are shown in successive traces at increasing temporal resolution, revealing singular openings (*) and bursts of openings (**). (B) Normalized open probability (NPo) of Kir6.2ΔC36 channel currents obtained during application of c-PKA (open column), c-PKA plus PKIP (filled column) or PKIP alone (hatched column). ATP (1 mM) was included in the drug solutions. NPo was normalized to the corresponding control in an ATP-free bath (taken as 100%) in individual patches. Dotted lines indicate control levels. Data are presented as the mean ± SEM of 4–12 patches. (C) Single-channel current traces of a Kir6.2ΔC36T224A channel in an inside-out patch prior to and during c-PKA application illustrated at increasing time resolution. T224 is the only putative PKA phosphorylation site in the truncated Kir6.2 channel. Alanine was introduced to disrupt this PKA site. (D) Single-channel current traces of a Kir6.2ΔC36T224D channel in an inside-out patch before and during c-PKA treatment. Aspartate was introduced to mimic the charge effect of protein phosphorylation (Li et al., 1993).
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Fig. 2. Enhancement of Kir6.2/SUR1 channel activity by PKA phosphorylation of the T224 site of Kir6.2. Currents were obtained from HEK293 cells co-expressing mouse Kir6.2 (full-length) and hamster SUR1 subunits. (A) Single-channel current traces of a Kir6.2/SUR1 channel in an inside-out patch prior to (upper trace, control) and during c-PKA treatment (lower trace). (B) NPo of Kir6.2/SUR1 channels obtained during application of c-PKA (open column), c-PKA plus PKIP (filled column) or PKIP alone (stippled column). Owing to the greater sensitivity of Ki6.2/SUR channels to ATP inhibition, 0.3 mM ATP (instead of 1 mM) was included in c-PKA and c-PKA plus PKIP solutions. Concentrations of c-PKA and PKIP were the same as in Figure 1. Data are presented as the mean ± SEM of 3–5 patches. (C–F) Single-channel current traces of Kir6.2(T224A,S372A)/SUR1 (C), Kir6.2T224A/SUR1 (D), Kir6.2S372A/SUR1 (E) and Kir6.2S372D/SUR1 (F) channels in inside-out patches before and during c-PKA application. T224 and S372 are the two putative PKA phosphorylation sites in full-length Kir6.2. The PKA effect was compared with control recordings of the same patch for each channel construct. (G) NPo of wild-type and mutant Kir6.2/SUR1 channels obtained in the presence of c-PKA. The PKA effect obtained from a Kir6.2ΔC36 channel is included for comparison. NPo was normalized in individual patches as described in Figure 1B. One-way ANOVA followed by Bonferroni's multiple comparison tests was performed (Fig. 2G).
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Fig. 3. Enhancement of Kir6.2/SUR2A channel activity by c-PKA. Currents were recorded from HEK293 cells co-expressing mouse Kir6.2 (full-length) and rat SUR2A subunits. (A) Single-channel current traces of a Kir6.2/SUR2A channel in an inside-out patch prior to (upper trace, control) and during c-PKA treatment (50 μg/ml) (lower trace). (B) Single-channel current traces of a Kir6.2(T224A,S372A)/SUR2A channel in an inside-out patch before and during c-PKA treatment. The bath was ATP free, and ATP (0.3 mM) was included in the drug solution.
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Fig. 4. Increase in the activity of Kir6.2ΔC36 and Kir6.2/SUR1 channels in cell-attached patches by D1R stimulation. D1R was co-expressed with the channels in HEK293 cells. (A) Single-channel current traces of a Kir6.2ΔC36 channel obtained from a cell-attached patch before, during and after exposing the cell to dopamine (10 μM). (B) NPo of Kir6.2ΔC36 channel currents obtained during application of dopamine (open column) and during co-application of dopamine and the D1/D2R antagonist tetrahydropalmatine (100 μM, filled column). NPo was normalized to the corresponding control recordings from the same patch (taken as 100%) before drug application in individual patches. Data are presented as the mean ± SEM of six and three patches, respectively. (C) Single-channel current traces of a Kir6.2/SUR1 channel obtained from a cell-attached patch in the control, during dopamine application and during co-application of dopamine and tetrahydropalmatine. (D) NPo of Kir6.2/SUR1 channel currents obtained during application of dopamine (open column) and during co-application of dopamine and tetrahydropalmatine (filled column). Data are presented as the mean ± SEM of three and two patches, respectively. (E) Single-channel current traces of a Kir6.2T224A/SUR1 channel in a cell-attached patch prior to and during dopamine application.
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Fig. 5. Effect of phosphorylation of T224 on the ATP sensitivity of reconstituted KATP channels. The relationship of ATP concentration to normalized open probability was determined in Kir6.2/SUR1 (○, seven patches), Kir6.2T224D/SUR1 (•, seven patches) and Kir6.2T224A/SUR1 (□, three patches) channels in inside-out patches excised from HEK293 cells. NPo was normalized to the control at zero ATP in individual patches and is presented as the mean ± SEM. Solid and dashed curves represent non-linear regression fits to the averaged NPo.
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Fig. 6. The frequency histograms of the open, closed and burst duration distributions of the Kir6.2ΔC36 channel obtained from an inside-out patch. Frequency histograms arrayed from top to bottom are open duration distribution (left column), closed duration distribution (center column) and burst duration distribution (right column), respectively. Frequency histograms of duration distributions fitted from events obtained (A) prior to c-PKA treatment, (B) during c-PKA (50 μg/ml) treatment and (C) during co-application of PKIP (200 μg/ml) plus c-PKA (50 μg/ml). Duration histograms were constructed as described in Materials and methods. See the text for details.
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Scheme 1.
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Scheme 1.
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Scheme 2.
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