Limits...
A unique voltage sensor sensitizes the potassium channel AKT2 to phosphoregulation.

Michard E, Lacombe B, Porée F, Mueller-Roeber B, Sentenac H, Thibaud JB, Dreyer I - J. Gen. Physiol. (2005)

Bottom Line: We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation.The phosphorylation-induced reduction of the activation energy in AKT2 is approximately 6 kT larger than in the K197S mutant.It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it.

View Article: PubMed Central - PubMed

Affiliation: Universität Potsdam, Institut für Biochemie und Biologie, Abteilung Molekularbiologie, D-14476 Potsdam-Golm, Germany.

ABSTRACT
Among all voltage-gated K+ channels from the model plant Arabidopsis thaliana, the weakly rectifying K+ channel (K(weak) channel) AKT2 displays unique gating properties. AKT2 is exceptionally regulated by phosphorylation: when nonphosphorylated AKT2 behaves as an inward-rectifying potassium channel; phosphorylation of AKT2 abolishes inward rectification by shifting its activation threshold far positive (>200 mV) so that it closes only at voltages positive of +100 mV. In its phosphorylated form, AKT2 is thus locked in the open state in the entire physiological voltage range. To understand the molecular grounds of this unique gating behavior, we generated chimeras between AKT2 and the conventional inward-rectifying channel KAT1. The transfer of the pore from KAT1 to AKT2 altered the permeation properties of the channel. However, the gating properties were unaffected, suggesting that the pore region of AKT2 is not responsible for the unique K(weak) gating. Instead, a lysine residue in S4, highly conserved among all K(weak) channels but absent from other plant K+ channels, was pinpointed in a site-directed mutagenesis approach. Substitution of the lysine by serine or aspartate abolished the "open-lock" characteristic and converted AKT2 into an inward-rectifying channel. Interestingly, phosphoregulation of the mutant AKT2-K197S appeared to be similar to that of the K(in) channel KAT1: as suggested by mimicking the phosphorylated and dephosphorylated states, phosphorylation induced a shift of the activation threshold of AKT2-K197S by about +50 mV. We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation. The phosphorylation-induced reduction of the activation energy in AKT2 is approximately 6 kT larger than in the K197S mutant. It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it.

Show MeSH

Related in: MedlinePlus

Exchange of the AKT2 pore by the KAT1 pore does not affect gating but alters the permeation properties of the chimeric channel. Characteristics of AKT2 (left) and the chimera AKT2-S5-P-S6-KAT1 (right) expressed in COS cells. In the chimera the fragment AKT2-E204_N314 was replaced by the fragment KAT1-E186_N297. (A) Currents elicited by voltage steps (AKT2: 3 s; chimera: 1.6 s) from a holding potential of +40 mV to voltages from +40 mV to −180 mV (20-mV decrements). The dashed lines indicate the zero current level. (B) Steady-state current–voltage characteristics. Currents were measured in standard solution (black circles) and after the addition of 10 mM Cs+ to external standard solution (white circles). Data displayed in A and B are representative for at least three repeats. (C) Normalized current–voltage characteristics measured at pH 7.4 (black) and pH 5.6 (white). Currents were normalized to the current values measured at −160 mV at pH 7.4 (ISS[−160 mV; pH 7.4] = −1). Data are displayed as mean ± SD (n = 3–8). (D) Normalized current–voltage characteristics measured in the presence (standard solution; black) and absence of external potassium (white; KCl in standard solution was replaced by NaCl). Currents were normalized to the current values measured at +60 mV in standard solution (ISS[+60 mV; 150 mM K+] = 1). Data are displayed as mean ± SD (n = 3). (E) Normalized current–voltage characteristics measured in bath solutions containing 1 mM Ca2+ (standard solution; black) and 20 mM Ca2+ (standard solution + 19 mM CaCl2; white). Currents were normalized to the current values measured at −160 mV in 1 mM Ca2+ (ISS[−160 mV; 1 mM Ca2+] = −1). Data are displayed as mean ± SD (n = 3). It should be noted that, with respect to the permeation properties, the chimera behaved essentially like KAT1 (Hedrich et al., 1995; Véry et al., 1995; Becker et al., 1996; Hoth et al., 1997; Dreyer et al., 1998), the donor of the pore.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2266593&req=5

fig2: Exchange of the AKT2 pore by the KAT1 pore does not affect gating but alters the permeation properties of the chimeric channel. Characteristics of AKT2 (left) and the chimera AKT2-S5-P-S6-KAT1 (right) expressed in COS cells. In the chimera the fragment AKT2-E204_N314 was replaced by the fragment KAT1-E186_N297. (A) Currents elicited by voltage steps (AKT2: 3 s; chimera: 1.6 s) from a holding potential of +40 mV to voltages from +40 mV to −180 mV (20-mV decrements). The dashed lines indicate the zero current level. (B) Steady-state current–voltage characteristics. Currents were measured in standard solution (black circles) and after the addition of 10 mM Cs+ to external standard solution (white circles). Data displayed in A and B are representative for at least three repeats. (C) Normalized current–voltage characteristics measured at pH 7.4 (black) and pH 5.6 (white). Currents were normalized to the current values measured at −160 mV at pH 7.4 (ISS[−160 mV; pH 7.4] = −1). Data are displayed as mean ± SD (n = 3–8). (D) Normalized current–voltage characteristics measured in the presence (standard solution; black) and absence of external potassium (white; KCl in standard solution was replaced by NaCl). Currents were normalized to the current values measured at +60 mV in standard solution (ISS[+60 mV; 150 mM K+] = 1). Data are displayed as mean ± SD (n = 3). (E) Normalized current–voltage characteristics measured in bath solutions containing 1 mM Ca2+ (standard solution; black) and 20 mM Ca2+ (standard solution + 19 mM CaCl2; white). Currents were normalized to the current values measured at −160 mV in 1 mM Ca2+ (ISS[−160 mV; 1 mM Ca2+] = −1). Data are displayed as mean ± SD (n = 3). It should be noted that, with respect to the permeation properties, the chimera behaved essentially like KAT1 (Hedrich et al., 1995; Véry et al., 1995; Becker et al., 1996; Hoth et al., 1997; Dreyer et al., 1998), the donor of the pore.

Mentions: To understand the apparent differences in phosphoregulation between the Kin channel KAT1 and the Kweak channel AKT2, we constructed recombinant chimeric channels between both. We swapped (a) the region between S4 and the COOH terminus comprising the P domain, (b) the COOH termini, and (c) the entire regions downstream the S4 segment (Figs. 1–4). The electrophysiological phenotypes of the chimeras were analyzed after expression in COS cells and Xenopus oocytes (Figs. 2–4). When the fragment between S4 and the COOH terminus of AKT2 was replaced by the corresponding region of KAT1, the chimera exhibited a voltage-dependent gating identical to the AKT2 wild-type characterized by the two K+ current components: an instantaneous “leak-like” component and a hyperpolarization-activated time-dependent component (Fig. 2 A). The two current components of both channels were blocked by extracellular Cs+ ions (Fig. 2 B). A more refined pharmacological fingerprint, however, unmasked the chimera. In contrast to wild-type AKT2, the chimera was neither blocked by protons (Fig. 2 C) nor sensitive to a reduction in the external K+ concentration (Fig. 2 D). Additionally, the chimera was less Ca2+ sensitive when compared with AKT2 (Fig. 2 E). Consequently, with respect to its susceptibility toward H+, K+, and Ca2+, the chimera had the features of the KAT1 pore (Hedrich et al., 1995; Véry et al., 1995; Becker et al., 1996; Hoth et al., 1997; Dreyer et al., 1998) rather than the features of the AKT2 pore (Fig. 2, left; Hoth et al., 2001; Geiger et al., 2002). Thus, the replacement of the pore altered the properties of the permeation pathway. However, this exchange left the gating properties unaffected.


A unique voltage sensor sensitizes the potassium channel AKT2 to phosphoregulation.

Michard E, Lacombe B, Porée F, Mueller-Roeber B, Sentenac H, Thibaud JB, Dreyer I - J. Gen. Physiol. (2005)

Exchange of the AKT2 pore by the KAT1 pore does not affect gating but alters the permeation properties of the chimeric channel. Characteristics of AKT2 (left) and the chimera AKT2-S5-P-S6-KAT1 (right) expressed in COS cells. In the chimera the fragment AKT2-E204_N314 was replaced by the fragment KAT1-E186_N297. (A) Currents elicited by voltage steps (AKT2: 3 s; chimera: 1.6 s) from a holding potential of +40 mV to voltages from +40 mV to −180 mV (20-mV decrements). The dashed lines indicate the zero current level. (B) Steady-state current–voltage characteristics. Currents were measured in standard solution (black circles) and after the addition of 10 mM Cs+ to external standard solution (white circles). Data displayed in A and B are representative for at least three repeats. (C) Normalized current–voltage characteristics measured at pH 7.4 (black) and pH 5.6 (white). Currents were normalized to the current values measured at −160 mV at pH 7.4 (ISS[−160 mV; pH 7.4] = −1). Data are displayed as mean ± SD (n = 3–8). (D) Normalized current–voltage characteristics measured in the presence (standard solution; black) and absence of external potassium (white; KCl in standard solution was replaced by NaCl). Currents were normalized to the current values measured at +60 mV in standard solution (ISS[+60 mV; 150 mM K+] = 1). Data are displayed as mean ± SD (n = 3). (E) Normalized current–voltage characteristics measured in bath solutions containing 1 mM Ca2+ (standard solution; black) and 20 mM Ca2+ (standard solution + 19 mM CaCl2; white). Currents were normalized to the current values measured at −160 mV in 1 mM Ca2+ (ISS[−160 mV; 1 mM Ca2+] = −1). Data are displayed as mean ± SD (n = 3). It should be noted that, with respect to the permeation properties, the chimera behaved essentially like KAT1 (Hedrich et al., 1995; Véry et al., 1995; Becker et al., 1996; Hoth et al., 1997; Dreyer et al., 1998), the donor of the pore.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266593&req=5

fig2: Exchange of the AKT2 pore by the KAT1 pore does not affect gating but alters the permeation properties of the chimeric channel. Characteristics of AKT2 (left) and the chimera AKT2-S5-P-S6-KAT1 (right) expressed in COS cells. In the chimera the fragment AKT2-E204_N314 was replaced by the fragment KAT1-E186_N297. (A) Currents elicited by voltage steps (AKT2: 3 s; chimera: 1.6 s) from a holding potential of +40 mV to voltages from +40 mV to −180 mV (20-mV decrements). The dashed lines indicate the zero current level. (B) Steady-state current–voltage characteristics. Currents were measured in standard solution (black circles) and after the addition of 10 mM Cs+ to external standard solution (white circles). Data displayed in A and B are representative for at least three repeats. (C) Normalized current–voltage characteristics measured at pH 7.4 (black) and pH 5.6 (white). Currents were normalized to the current values measured at −160 mV at pH 7.4 (ISS[−160 mV; pH 7.4] = −1). Data are displayed as mean ± SD (n = 3–8). (D) Normalized current–voltage characteristics measured in the presence (standard solution; black) and absence of external potassium (white; KCl in standard solution was replaced by NaCl). Currents were normalized to the current values measured at +60 mV in standard solution (ISS[+60 mV; 150 mM K+] = 1). Data are displayed as mean ± SD (n = 3). (E) Normalized current–voltage characteristics measured in bath solutions containing 1 mM Ca2+ (standard solution; black) and 20 mM Ca2+ (standard solution + 19 mM CaCl2; white). Currents were normalized to the current values measured at −160 mV in 1 mM Ca2+ (ISS[−160 mV; 1 mM Ca2+] = −1). Data are displayed as mean ± SD (n = 3). It should be noted that, with respect to the permeation properties, the chimera behaved essentially like KAT1 (Hedrich et al., 1995; Véry et al., 1995; Becker et al., 1996; Hoth et al., 1997; Dreyer et al., 1998), the donor of the pore.
Mentions: To understand the apparent differences in phosphoregulation between the Kin channel KAT1 and the Kweak channel AKT2, we constructed recombinant chimeric channels between both. We swapped (a) the region between S4 and the COOH terminus comprising the P domain, (b) the COOH termini, and (c) the entire regions downstream the S4 segment (Figs. 1–4). The electrophysiological phenotypes of the chimeras were analyzed after expression in COS cells and Xenopus oocytes (Figs. 2–4). When the fragment between S4 and the COOH terminus of AKT2 was replaced by the corresponding region of KAT1, the chimera exhibited a voltage-dependent gating identical to the AKT2 wild-type characterized by the two K+ current components: an instantaneous “leak-like” component and a hyperpolarization-activated time-dependent component (Fig. 2 A). The two current components of both channels were blocked by extracellular Cs+ ions (Fig. 2 B). A more refined pharmacological fingerprint, however, unmasked the chimera. In contrast to wild-type AKT2, the chimera was neither blocked by protons (Fig. 2 C) nor sensitive to a reduction in the external K+ concentration (Fig. 2 D). Additionally, the chimera was less Ca2+ sensitive when compared with AKT2 (Fig. 2 E). Consequently, with respect to its susceptibility toward H+, K+, and Ca2+, the chimera had the features of the KAT1 pore (Hedrich et al., 1995; Véry et al., 1995; Becker et al., 1996; Hoth et al., 1997; Dreyer et al., 1998) rather than the features of the AKT2 pore (Fig. 2, left; Hoth et al., 2001; Geiger et al., 2002). Thus, the replacement of the pore altered the properties of the permeation pathway. However, this exchange left the gating properties unaffected.

Bottom Line: We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation.The phosphorylation-induced reduction of the activation energy in AKT2 is approximately 6 kT larger than in the K197S mutant.It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it.

View Article: PubMed Central - PubMed

Affiliation: Universität Potsdam, Institut für Biochemie und Biologie, Abteilung Molekularbiologie, D-14476 Potsdam-Golm, Germany.

ABSTRACT
Among all voltage-gated K+ channels from the model plant Arabidopsis thaliana, the weakly rectifying K+ channel (K(weak) channel) AKT2 displays unique gating properties. AKT2 is exceptionally regulated by phosphorylation: when nonphosphorylated AKT2 behaves as an inward-rectifying potassium channel; phosphorylation of AKT2 abolishes inward rectification by shifting its activation threshold far positive (>200 mV) so that it closes only at voltages positive of +100 mV. In its phosphorylated form, AKT2 is thus locked in the open state in the entire physiological voltage range. To understand the molecular grounds of this unique gating behavior, we generated chimeras between AKT2 and the conventional inward-rectifying channel KAT1. The transfer of the pore from KAT1 to AKT2 altered the permeation properties of the channel. However, the gating properties were unaffected, suggesting that the pore region of AKT2 is not responsible for the unique K(weak) gating. Instead, a lysine residue in S4, highly conserved among all K(weak) channels but absent from other plant K+ channels, was pinpointed in a site-directed mutagenesis approach. Substitution of the lysine by serine or aspartate abolished the "open-lock" characteristic and converted AKT2 into an inward-rectifying channel. Interestingly, phosphoregulation of the mutant AKT2-K197S appeared to be similar to that of the K(in) channel KAT1: as suggested by mimicking the phosphorylated and dephosphorylated states, phosphorylation induced a shift of the activation threshold of AKT2-K197S by about +50 mV. We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation. The phosphorylation-induced reduction of the activation energy in AKT2 is approximately 6 kT larger than in the K197S mutant. It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it.

Show MeSH
Related in: MedlinePlus