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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.

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AKT2-KAT1 chimeras show “open-locked” behavior. Chimeras' structure (see also Fig. 1) is indicated (left). Parts originating from AKT2 are illustrated in light gray and parts from KAT1 in dark gray. Current traces (middle) and steady-state current–voltage characteristics (right) of the chimeras. Currents were measured in standard solution (black circles) and after the addition of 10 mM Cs+ to external standard solution (white circles). The dashed lines indicate the zero current level. (A) Expression in COS cells. Currents elicited by 1.6-s voltage steps from a holding potential of +40 mV to voltages from +60 mV to −140 mV (20-mV decrements). (B) Expression in oocytes (100 mM KCl in the bath); 0.5-s voltage steps from 0 mV to voltages from +50 mV to −160 mV (15-mV decrements) followed by a step to −60 mV. Data displayed in A and B are representative for at least three repeats.
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fig3: AKT2-KAT1 chimeras show “open-locked” behavior. Chimeras' structure (see also Fig. 1) is indicated (left). Parts originating from AKT2 are illustrated in light gray and parts from KAT1 in dark gray. Current traces (middle) and steady-state current–voltage characteristics (right) of the chimeras. Currents were measured in standard solution (black circles) and after the addition of 10 mM Cs+ to external standard solution (white circles). The dashed lines indicate the zero current level. (A) Expression in COS cells. Currents elicited by 1.6-s voltage steps from a holding potential of +40 mV to voltages from +60 mV to −140 mV (20-mV decrements). (B) Expression in oocytes (100 mM KCl in the bath); 0.5-s voltage steps from 0 mV to voltages from +50 mV to −160 mV (15-mV decrements) followed by a step to −60 mV. Data displayed in A and B are representative for at least three repeats.

Mentions: When the entire region downstream the S4 segment of AKT2 was replaced by that of KAT1, the resulting chimera mediated only instantaneously activating currents. A time-dependent component was not observable (Fig. 3 A). Similar results were obtained when only the COOH terminus was exchanged (Fig. 3 B), indicating that the COOH terminus of KAT1 is influencing the gating process differently compared with the AKT2 COOH terminus.


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)

AKT2-KAT1 chimeras show “open-locked” behavior. Chimeras' structure (see also Fig. 1) is indicated (left). Parts originating from AKT2 are illustrated in light gray and parts from KAT1 in dark gray. Current traces (middle) and steady-state current–voltage characteristics (right) of the chimeras. Currents were measured in standard solution (black circles) and after the addition of 10 mM Cs+ to external standard solution (white circles). The dashed lines indicate the zero current level. (A) Expression in COS cells. Currents elicited by 1.6-s voltage steps from a holding potential of +40 mV to voltages from +60 mV to −140 mV (20-mV decrements). (B) Expression in oocytes (100 mM KCl in the bath); 0.5-s voltage steps from 0 mV to voltages from +50 mV to −160 mV (15-mV decrements) followed by a step to −60 mV. Data displayed in A and B are representative for at least three repeats.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: AKT2-KAT1 chimeras show “open-locked” behavior. Chimeras' structure (see also Fig. 1) is indicated (left). Parts originating from AKT2 are illustrated in light gray and parts from KAT1 in dark gray. Current traces (middle) and steady-state current–voltage characteristics (right) of the chimeras. Currents were measured in standard solution (black circles) and after the addition of 10 mM Cs+ to external standard solution (white circles). The dashed lines indicate the zero current level. (A) Expression in COS cells. Currents elicited by 1.6-s voltage steps from a holding potential of +40 mV to voltages from +60 mV to −140 mV (20-mV decrements). (B) Expression in oocytes (100 mM KCl in the bath); 0.5-s voltage steps from 0 mV to voltages from +50 mV to −160 mV (15-mV decrements) followed by a step to −60 mV. Data displayed in A and B are representative for at least three repeats.
Mentions: When the entire region downstream the S4 segment of AKT2 was replaced by that of KAT1, the resulting chimera mediated only instantaneously activating currents. A time-dependent component was not observable (Fig. 3 A). Similar results were obtained when only the COOH terminus was exchanged (Fig. 3 B), indicating that the COOH terminus of KAT1 is influencing the gating process differently compared with the AKT2 COOH terminus.

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