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

2000; Springer Nature; Volume: 19; Issue: 5 Linguagem: Inglês

10.1093/emboj/19.5.942

1460-2075

Yu‐Fung Lin, Yuh Nung Jan, Lily Yeh Jan,

Cardiac electrophysiology and arrhythmias

Article1 March 2000free access Regulation of ATP-sensitive potassium channel function by protein kinase A-mediated phosphorylation in transfected HEK293 cells Yu-Fung Lin Yu-Fung Lin Howard Hughes Medical Institute, Departments of Physiology and Biochemistry, University of California, San Francisco, CA, 94143-0725 USA Search for more papers by this author Yuh Nung Jan Yuh Nung Jan Howard Hughes Medical Institute, Departments of Physiology and Biochemistry, University of California, San Francisco, CA, 94143-0725 USA Search for more papers by this author Lily Yeh Jan Corresponding Author Lily Yeh Jan Howard Hughes Medical Institute, Departments of Physiology and Biochemistry, University of California, San Francisco, CA, 94143-0725 USA Search for more papers by this author Yu-Fung Lin Yu-Fung Lin Howard Hughes Medical Institute, Departments of Physiology and Biochemistry, University of California, San Francisco, CA, 94143-0725 USA Search for more papers by this author Yuh Nung Jan Yuh Nung Jan Howard Hughes Medical Institute, Departments of Physiology and Biochemistry, University of California, San Francisco, CA, 94143-0725 USA Search for more papers by this author Lily Yeh Jan Corresponding Author Lily Yeh Jan Howard Hughes Medical Institute, Departments of Physiology and Biochemistry, University of California, San Francisco, CA, 94143-0725 USA Search for more papers by this author Author Information Yu-Fung Lin1, Yuh Nung Jan1 and Lily Yeh Jan 1 1Howard Hughes Medical Institute, Departments of Physiology and Biochemistry, University of California, San Francisco, CA, 94143-0725 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:942-955https://doi.org/10.1093/emboj/19.5.942 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info ATP-sensitive potassium (KATP) channels regulate insulin secretion, vascular tone, heart rate and neuronal excitability by responding to transmitters as well as the internal metabolic state. KATP channels are composed of four pore-forming α-subunits (Kir6.2) and four regulatory β-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 KATP 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 KATP channels by PKA phosphorylation of T224 in Kir6.2, which may represent a general mechanism for KATP channel regulation in different tissues. Introduction ATP-sensitive potassium (KATP) channels, widely distributed in the central and peripheral nervous system, pituitary, cardiac muscle, smooth muscle, skeletal muscle and pancreatic β-cells, serve a variety of cellular functions (Babenko et al., 1998). Not only are they modulated by transmitters, but KATP channels are inhibited by intracellular ATP and stimulated by Mg-ADP and thus couple cellular metabolic status to changes in transmembrane potassium fluxes and cellular excitability. In the brain, KATP channels contribute to the glucose-sensing mechanism involved in appetite control (Ashford et al., 1990). They also protect central neurons and cardiac myocytes under metabolic stress (Mercuri et al., 1994; Roeper and Ashcroft, 1995; Stanford and Lacey, 1995; Watts et al., 1995; Seutin et al., 1996; Isomoto et al., 1997; Nichols and Lopatin, 1997; Yokoshiki et al., 1998). In pancreatic β-cells, KATP channels regulate insulin secretion in response to glucose metabolism (Ashcroft and Rorsman, 1989). In smooth muscles, transmitter-mediated modulation of KATP channels controls vascular tone: vasodilators stimulate KATP channels whereas vasoconstrictors inhibit them (Ashcroft, 1996; Isomoto et al., 1997; Nichols and Lopatin, 1997; Quayle and Nelson, 1997; Seino et al., 1997; Babenko et al., 1998; Yokoshiki et al., 1998). Indeed, KATP channels have become the therapeutic targets for a variety of diseases including angina, hypertension and diabetes (Nichols and Lopatin, 1997). The KATP channel is an octamer (Clement et al., 1997; Inagaki et al., 1997; Shyng and Nichols, 1997; Babenko et al., 1998). It is composed of four pore-forming α-subunits (Kir6.2) (Inagaki et al., 1995; Sakura et al., 1995) and four regulatory β-subunits (SUR1 or SUR2) (Aguilar-Bryan et al., 1995; Inagaki et al., 1996; Isomoto et al., 1996). Partial complexes with fewer than eight subunits do not reach the cell membrane because of the exposure of an endoplasmic reticulum (ER) retention/retrieval signal that is present in each subunit (Zerangue et al., 1999). Deleting the last 36 amino acids containing this ER retention/retrieval signal therefore permits functional expression of Kir6.2ΔC36 channels in the absence of SUR (Tucker et al., 1997). Native KATP channels in different tissues comprise the same Kir6.2 (α) subunits, which form a weakly inwardly rectifying potassium channel, but different SUR (β) subunits. KATP channels in pancreatic β-cells and some central neurons contain Kir6.2 and SUR1 (Inagaki et al., 1995; Aguilar-Bryan et al., 1998). Mutations of either Kir6.2 or SUR1 cause persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a disease associated with unregulated insulin secretion (Thomas et al., 1995). Cardiac and skeletal muscle KATP channels are composed of Kir6.2 and SUR2A (Inagaki et al., 1996; Okuyama et al., 1998). KATP channels in smooth muscle and certain central neurons in the substantia nigra (SN) comprise Kir6.2 and SUR2B instead (Isomoto et al., 1996; Yamada et al., 1997; Liss et al., 1999a). The sites mediating ATP inhibition are located in the Kir6.2 subunits, but SUR may modulate the ATP sensitivity further (Nichols et al., 1996; Gribble et al., 1997; Shyng et al., 1997b; Trapp et al., 1997; Tucker et al., 1997; John et al., 1998). Moreover, the SUR subunits, members of the ATP-binding cassette (ABC) superfamily, mediate channel inhibition by sulfonylurea drugs and stimulation by Mg-ADP and potassium channel openers (KCOs) (Ashcroft, 1996; Isomoto et al., 1997; Nichols and Lopatin, 1997; Seino et al., 1997; Tucker et al., 1997; Babenko et al., 1998). Compared with SUR2A- or SUR2B-containing KATP channels, SUR1-containing KATP channels exhibit higher affinity for sulfonylurea drugs such as tolbutamide (Isomoto and Kurachi, 1997; Babenbo et al., 1998; Gribble et al., 1998) and higher sensitivity to metabolic stress (Ashcroft and Gribble, 1998; Liss et al., 1999a). Protein kinases play important roles in the physiological regulation of KATP channels, typically by mediating the effects of transmitters. The induction of ischemic pre-conditioning in the heart may involve activation of KATP channels via protein kinase C (PKC) (Lawson and Downey, 1993; Light et al., 1995; Light, 1996). Vasodilators and vasoconstrictors modulate smooth muscle KATP via protein kinase A (PKA) (Quayle et al., 1994) and PKC (Hatakeyama et al., 1995), respectively. Vasodilatation of the renal artery may be mediated by activation of the D1 dopamine receptor (D1R) (Goldberg et al., 1978) that is coupled to G-αs and stimulates adenylate cyclase and hence PKA (Missale et al., 1988). Whether the D1R action is due to KATP channel activation has not been determined. In the mammalian brain, D1R is widely distributed (Civelli et al., 1993) and participates in a number of dopamine-mediated functions, including locomotor activity, cognition, emotion, positive reinforcement, food intake and endocrine regulation (Missale et al., 1998). Dopaminergic neurons as well as γ-aminobutyric acid (GABA)ergic neurons in the SN express not only D1-like receptors but also KATP channels (Gerfen et al., 1990; Le Moine et al., 1991; Nicola et al., 1996; Liss et al., 1999a). In the weaver mouse, a model for neurodegeneration, the surviving subpopulation of SN dopaminergic neurons contain Kir6.2 and SUR1 exclusively, in contrast to wild-type dopaminergic neurons, which express Kir6.2, SUR1 and/or SUR2B (Liss et al., 1999a,b). It is hence suggested that KATP channels may confer resistance to neurodegeneration in the SN, origin of the nigrostriatal dopaminergic system. Whether D1R can activate KATP channels in these and other central neurons is therefore an interesting open question. To determine whether D1R stimulates KATP channels and whether such stimulation involves PKA phosphorylation of KATP channels, and to identify the channel subunit(s) and the specific residue(s) responsible for such PKA modulation of KATP channels, it is necessary to use heterologous expression systems. By examining HEK293 cells transiently transfected with various combinations of mutant or wild-type KATP channel subunits and D1R, we found that D1R activation by dopamine caused an increase in KATP channel activities. Moreover, application of c-PKA, the catalytic subunit of PKA, to the cytoplasmic side of inside-out membrane patches also stimulated KATP channels. To test whether the positive c-PKA effect is mediated by phosphorylation of Kir6.2, we expressed Kir6.2ΔC36 alone without SUR1. Indeed, these tetrameric channels were stimulated by c-PKA. The involvement of SUR1 or SUR2A in channel stimulation by PKA appears to be negligible, since c-PKA failed to stimulate KATP channels containing mutant Kir6.2 devoid of PKA consensus sites. The Kir6.2 sequence contains two putative phosphorylation sites: T224 and S372. Only the former site is present in Kir6.2ΔC36 channels. Mutation of a single residue in Kir6.2, T224, abolished channel stimulation by either c-PKA or D1R, indicating that D1R activates KATP channels via PKA. A recent biochemical study has shown persistent PKA phosphorylation of S372 but not T224 of Kir6.2 (Béguin et al., 1999). We found, however, that T224 rather than S372 was necessary for PKA stimulation of channel activities: mutations of T224 but not S372 abolished the ability of c-PKA and D1R to enhance channel activities. Moreover, the T224D mutation mimicked c-PKA-mediated channel activation by increasing the opening frequency and decreasing the mean closed duration of the Kir6.2ΔC36 channel. The elevated activity of the T224D mutant was persistent, in contrast to the transient stimulation of wild-type channels following a brief application of c-PKA, making it feasible to determine whether the presence of a negatively charged group at position 224 of Kir6.2 affects the ATP inhibition of the channel. The physiological significance of PKA phosphorylation affecting KATP channel gating and ATP sensitivity, as well as the intriguingly different effects of PKA phosphorylation of T224 and S372 of Kir6.2, will be discussed. Results c-PKA enhanced the single-channel currents of Kir6.2ΔC36 channels in transiently transfected HEK293 cells The KATP channels in different tissues differ in their SUR subunits (e.g. SUR1 in pancreas, SUR2A in myocardium, SUR2B in smooth muscle and SUR1 or SUR2 isoforms in brain), but share the same inwardly rectifying channel subunit, Kir6.2 (Karschin et al., 1997). Thus, we first examined the PKA effects on Kir6.2 channels. The expression of full-length Kir6.2 does not result in functional channels in the absence of SUR due to the ER retention/retrieval signal in the C-terminus of Kir6.2 (Zerangue et al., 1999). Truncation of the last 36 amino acids removes the retention signal, thereby allowing functional expression of Kir6.2ΔC36 channels in the absence of SUR (Tucker et al., 1997). We therefore expressed the Kir6.2ΔC36 channels in HEK293 cells by transient transfection. Channel activities were recorded from excised, inside-out patches that were exposed to 140 mM potassium solutions on both sides and voltage clamped at −60 mV. Single-channel openings, most frequently at a conductance level of ∼70 pS (chord conductance between −30 and −100 mV), appear as upward deflections at −60 mV (Figure 1). At increasing temporal resolution (Figure 1A, control, top to bottom), single-channel openings of Kir6.2ΔC36 channels exposed to ATP-free solutions could be resolved into singular openings (*) and bursts of openings (**) that were separated by closures shorter than a critical time (burst terminator) determined in individual patches (see Materials and methods). After recording the basal activity (Figure 1A, control), 30 μl or less of c-PKA (50 μg/ml) and Mg-ATP (1 mM) were applied to the patch through a glass capillary by pressure ejection (see Materials and methods). The c-PKA application lasted for 30–60 s; subsequent mixing with the 2 ml solution in the bath would reduce the c-PKA concentration to <25 U/ml. The apparent opening frequency was increased during c-PKA application (Figure 1A, c-PKA), and gradually reverted to control levels in a few minutes. This c-PKA effect was reproducible; a second application of c-PKA caused a similar stimulation (data not shown). The increase in the apparent opening frequency resulted in simultaneous openings of 2–3 channels (Figure 1A, c-PKA), which were only observed infrequently prior to c-PKA application (Figure 1A, control). The conductance level was not affected by c-PKA (Figure 1A). When the inhibitory peptide specific for PKA, PKIP (200 μg/ml), was applied together with c-PKA (50 μg/ml) and Mg-ATP (1 mM), there was no effect on single-channel conductance or opening frequency (Figure 1A, PKIP + c-PKA). As shown in Figure 1B, the normalized open probability (NPo) during c-PKA application was increased to 222.3 ± 35.2% (mean ± SEM, 12 patches; Table I, p <0.05, paired t-test), whereas the application of PKIP plus c-PKA and Mg-ATP (75.0 ± 14.1%, six patches) or PKIP alone (71.9 ± 17.6%, four patches) failed to increase channel activity (not significantly different, paired t-test). c-PKA also exerted significant effects on other single-channel properties of the channel, such as opening frequency, mean closed duration and bursting frequency (Table I, details described later in the text). Because these effects were abolished when PKIP was applied with c-PKA, the channel stimulation is most probably due to PKA phosphorylation rather than to a non-specific effect of the kinase. Figure 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). Download figure Download PowerPoint Table 1. Effects of c-PKA on normalized open, closed and burst properties of mouse Kir6.2ΔC36 channels expressed in acutely transfected HEK293 cells Properties Control PKA PKIP + PKA Open probability (%) 100 222.3 ± 35.2* 75.0 ± 14.1 Opening frequency (%) 100 205.1 ± 30.8** 81.7 ± 6.1 Mean open duration (%) 100 108.1 ± 2.5 86.7 ± 13.2 Mean closed duration (%) 100 58.3 ± 8.9** 133.3 ± 17.0 Mean burst duration (%) 100 124.4 ± 13.8 82.2 ± 16.8 Bursting frequency (%) 100 176.9 ± 18.5* 91.3 ± 5.7 Opening per burst (%) 100 113.7 ± 11.5 90.5 ± 8.4 Single-channel recordings of Kir6.2ΔC36 channels in inside-out patches were obtained at −60 mV in symmetrical 140 mM K+ solutions. Control recordings were obtained in an ATP-free bath prior to drug application. c-PKA (50 μg/ml, 12 patches) or c-PKA plus PKIP (200 μg/ml, six patches) was applied to the cytoplasmic surface of patches via pressure ejection. ATP (1 mM) was included in both drug solutions. Data were normalized to the corresponding controls obtained in individual patches (taken as 100%), averaged and are presented as the mean ± SEM (as a percentage). Significance levels are: * p <0.05; ** p <0.01 (paired t-tests). c-PKA failed to enhance single-channel currents when the T224 residue of Kir6.2ΔC36 channel was substituted with alanine To test whether c-PKA increased Kir6.2ΔC36 channel function directly by phosphorylation of the channel protein, we substituted the only PKA consensus site of the Kir6.2ΔC36 channel, threonine (T) 224, with alanine (A). The single-channel conductance level of Kir6.2ΔC36T224A was similar to that of Kir6.2ΔC36. However, unlike with Kir6.2ΔC36, treatment with c-PKA (50 μg/ml) in the presence of Mg-ATP (1 mM) did not stimulate the single-channel currents of Kir6.2ΔC36T224A (Figure 1C, control versus c-PKA). The averaged absolute NPo of Kir6.2ΔC36T224A was 0.8 ± 0.3% in ATP-free control and 0.3 ± 0.2% during application of c-PKA and Mg-ATP (two patches, Table II). In the absence of SUR1, Kir6.2ΔC36 channels are inhibited by millimolar ATP (Tucker et al., 1997). The modest reduction in the apparent opening frequency and increase in the mean closed duration of Kir6.2ΔC36T224A during c-PKA application (Table II) were probably due to the ability of ATP, which was applied together with c-PKA, to inhibit the channel. The abolition of a positive c-PKA effect by T224A mutation indicates that T224 is the phosphorylation site through which PKA modulates Kir6.2ΔC36 channel function. Table 2. Abolition of c-PKA effects on the single-channel kinetic properties of Kir6.2ΔC36T224A and Kir6.2ΔC36T224D channels Properties Control c-PKA Kir6.2ΔC36T224A Open probability (%) 0.8 ± 0.3 0.3 ± 0.2 Opening frequency (s−1) 8.4 ± 0.7 4.3 ± 2.6 Mean open duration (ms) 0.9 ± 0.3 0.4 ± 0.1 Mean closed duration (ms) 112.1 ± 13.7 404.6 ± 249.8 Kir6.2ΔC36T224D Open probability (%) 37.5 ± 18.8 23.9 ± 15.6 Opening frequency (s−1) 125.8 ± 58.2 74.4 ± 48.9 Mean open duration (ms) 2.3 ± 0.5 2.3 ± 0.6 Mean closed duration (ms) 29.3 ± 23.2 71.2 ± 45.0 Recordings were performed at −60 mV in symmetrical K+ solutions. Single-channel open and closed properties were determined from records obtained before (control) and during c-PKA (50 μg/ml) application in patches expressing Kir6.2ΔC36T224A (two patches) or Kir6.2ΔC36T224D (four patches). ATP (1 mM) was included in the drug solution. Data were averaged and are presented as the mean ± SEM. Paired t-tests of data pairs (control and c-PKA-treated) obtained in the same patch revealed no significant differences. Aspartate substitution of the T224 residue in Kir6.2ΔC36 increased basal single-channel activity, but eliminated the c-PKA effects To mimic the effect of phosphorylation, we used aspartate (D) to replace T224. Compared with wild-type channels, Kir6.2ΔC36T224D exhibited similar single-channel conductance, but increased channel activity (Figure 1D). Significant differences were detected for open probability, opening frequency and mean closed duration between Kir6.2ΔC36 and Kir6.2ΔC36T224D channels (Table III). In addition to these parameters, the mean open duration was also significantly different between Kir6.2ΔC36T224A and Kir6.2ΔC36T224D channels (Table III). Treatment with c-PKA and Mg-ATP did not increase further the single-channel currents of Kir6.2ΔC36T224D channels (Figure 1D, control versus c-PKA; Table II, paired t-test); the averaged NPo of Kir6.2ΔC36T224D during application of c-PKA and Mg-ATP was 53.9 ± 19.2% of control (four patches). The inability of c-PKA treatment to stimulate the T224D mutant channels indicates again that the T224 residue is responsible for the c-PKA-mediated modulation of Kir6.2ΔC36 channel function. Furthermore, the T224D mutation mimicked c-PKA stimulation of Kir6.2ΔC36 by increasing the opening frequency as well as reducing the mean closed duration (Tables I and III), indicating that the PKA stimulatory effect arises from the attachment of a negatively charged group to the residue at position 224. Table 3. Changes in basal single-channel properties by aspartate or alanine substitution at T224 of the Kir6.2 subunit Properties Kir6.2ΔC36 Kir6.2ΔC36T224A Kir6.2ΔC36T224D Open probability (%) 3.83 ± 0.91a 0.59 ± 0.13b 31.59 ± 15.73 Opening frequency (s−1) 22.63 ± 3.53a 4.73 ± 1.07c 108.20 ± 48.36 Mean open duration (ms) 1.47 ± 0.11 1.29 ± 0.30d 2.26 ± 0.41 Mean closed duration (ms) 78.39 ± 20.39e 339.10 ± 114.40b 28.36 ± 17.98 No. of openings 70 938 2137 33 620 No. of patches 21 7 5 Recordings were made on inside-out patches in an ATP-free bath at −60 mV. Comparisons were made by performing one-way analysis of variance (ANOVA) on data obtained from Kir6.2ΔC36, Kir6.2ΔC36T224A and Kir6.2ΔC36T224D channels, followed by Bonferroni's multiple comparison tests. Data are presented as the mean ± SEM. Significance levels are: a Kir6.2ΔC36 versus Kir6.2ΔC36T224D (p <0.001); b Kir6.2ΔC36T224A versus Kir6.2ΔC36T224D (p <0.01); c Kir6.2ΔC36T224A versus Kir6.2ΔC36T224D (p <0.001); d Kir6.2ΔC36T224A versus Kir6.2ΔC36T224D (p <0.05); and e Kir6.2ΔC36 versus Kir6.2ΔC36T224D (p <0.01). PKA enhanced currents of recombinant Kir6.2/SUR1 channel, a neuronal/pancreatic isoform of KATP Owing to the higher sensitivity of Kir6.2/SUR1 channels to ATP inhibition than that of Kir6.2ΔC36 channels, a lower concentration of Mg-ATP (0.3 mM instead of 1 mM) was co-applied with c-PKA. Application of c-PKA (50 μg/ml) and Mg-ATP (0.3 mM) to the cytoplasmic surface of inside-out patches of HEK293 cells transfected with Kir6.2 and SUR1 did not alter the single-channel conductance, but caused an increase in the apparent opening frequency (Figure 2A, control versus c-PKA). c-PKA significantly increased the averaged NPo of the Kir6.2/SUR1 channel to 226.6 ± 17.2% of control (four patches; p <0.01, one-sample t-test). No significant stimulation was observed for co-application of c-PKA with PKIP (200 μg/ml) and Mg-ATP (0.3 mM) (43.1 ± 20.5%, three patches, one-sample t-test; Figure 2B), or application of PKIP alone (91.5 ± 5.2%, five patches, one-sample t-test; Figure 2B). KATP channels containing SUR1 and Kir6.2 are highly sensitive to ATP inhibition (Nichols et al., 1996; Gribble et al., 1997; Shyng et al., 1997b; Trapp et al., 1997; Tucker et al., 1997). Exposure to 0.3 mM Mg-free ATP would reduce NPo to 1.9 ± 1.0% (seven patches, Figure 5). KATP channel activity is also reactivated or refreshed by a brief application of a high concentration (mM) of Mg-ATP (Ohno-Shosaku et al., 1987). This 'refreshment' effect does not require the nucleotide-binding domains (NBDs) of SUR1 (Gribble et al., 1997) and becomes apparent when the inhibitory effects of ATP subside following removal of Mg-ATP (for a review, see Babenko et al., 1998). In our hands, the refreshment effect on channels became evident after terminating the application of 30 μl of 0.3 mM Mg-ATP to the patch and was long-lasting. It was therefore essential to examine the PKA effect during the 30–60 s period of c-PKA application. As evident from the controls, within this period of pressure ejection the inhibitory effects of ATP outweighed the refreshment effect of Mg-ATP. Moreover, the stimulatory effects of Kir6.2/SUR1 channel activity obtained during c-PKA application were abolished by PKIP, indicating a specific PKA phosphorylation effect. Figure 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). Download figure Download PowerPoint Figure 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. Download figure Download PowerPoint Alanine substitution of Kir6.2T224 of the Kir6.2/SUR1 channel resulted in insensitivity of the channels to c-PKA While Kir6.2ΔC36 contains only one putative PKA phosphorylation site, T224, another PKA phosphorylation site, S372, is located in the C-terminus of Kir6.2 (Inagaki et al., 1995) and there are at least three consensus phosphorylation sites for PKA on SUR1 (Aguilar-Bryan et al., 1995). Biochemical studies have revealed PKA phosphorylation of S372 and one site on human SUR1 (S1571), but not on SUR1 from other species or on SUR2 (Béguin et al., 1999). We thus examined Kir6.2T224A/SUR1, Kir6.2(T224A,S372A)/SUR1, Kir6.2S372A/SUR1 and Kir6.2S372D/SUR1 channels in inside-out patches to look for possible effects of PKA mediated through Kir6.2 or SUR1. Application of c-PKA (50 μg/ml) and Mg-ATP (0.3 mM) failed to increase the single-channel currents of Kir6.2(T224A,S372A)/SUR1 (Figure 2C) and Kir6.2T224A/SUR1 (Figure 2D) channels; the channel activities appeared to be reduced, probably due to the ATP inhibition of the channel. By contrast, the apparent opening frequency of Kir6.2S372A/SUR1 (Figure 2E) and Kir6.2S372D/SUR1 (Figure 2F) was enhanced by the same treatment. Treatment with c-PKA reduced the NPo to 54.0 ± 13.8% for Kir6.2T224A/SUR1 (five patches) and to 46.0 ± 14.2% for Kir6.2(T224A,S372A)/SUR1 (five patches), but increased the NPo to 304.3 ± 34.1% for Kir6.2S372A/SUR1 (two patches) and to 253.5 ± 30.1% for Kir6.2S372D/SUR1 (two patches), respectively. In comparison, c-PKA increased the NPo to 226.6 ± 17.3% of control for Kir6.2/SUR1 (four patches, Figure 2B) and to 323.8 ± 20.8% for Kir6.2ΔC36 channels (four patches, Figure 2G). Bonferroni's multiple comparison tests following one-way ANOVA revealed significant differences when Kir6.2(T224A,S372A)/SUR1 was compared with Kir6.2/SUR1, Kir6.2S372A/SUR1 or Kir6.2S372D (p <0.001 in each pairwise comparison). Significant differences were also found when Kir6.2T224A/SUR1 was compared with Kir6.2/SUR1, Kir6.2S372A/SUR1 or Kir6.2S372D (p <0.001 in each case). These data demonstrate that T224 is primarily responsible for the PKA stimulation of the KATP channel. PKA enhanced currents of recombinant Kir6.2/SUR2A channel, a cardiac/skeletal muscle isoform of KATP Application of c-PKA (50 μg/ml) and Mg-ATP (0.3 mM) to the cytoplasmic surface of inside-out patches excised from HEK293 cells transiently transfected with SUR2A and Kir6.2 increased the apparent o