A Critical Gating Switch at a Modulatory Site in Neuronal Kir3 Channels

Abstract

Inwardly rectifying potassium channels enforce tight control of resting membrane potential in excitable cells. The Kir3.2 channel, a member of the Kir3 subfamily of G-protein-activated potassium channels (GIRKs), plays several roles in the nervous system, including key responsibility in the GABAB pathway of inhibition, in pain perception pathways via opioid receptors, and is also involved in alcoholism. PKC phosphorylation acts on the channel to reduce activity, yet the mechanism is incompletely understood. Using the heterologous Xenopus oocyte system combined with molecular dynamics simulations, we show that PKC modulation of channel activity is dependent on Ser-196 in Kir3.2 such that, when this site is phosphorylated, the channel is less sensitive to PKC inhibition. This reduced inhibition is dependent on an interaction between phospho-Ser (SEP)-196 and Arg-201, reducing Arg-201 interaction with the sodium-binding site Asp-228. Neutralization of either SEP-196 or Arg-201 leads to a channel with reduced activity and increased sensitivity to PKC inhibition. This study clarifies the role of Ser-196 as an allosteric modulator of PKC inhibition and suggests that the SEP-196/Arg-201 interaction is critical for maintaining maximal channel activity.

Significance statement: The inwardly rectifying potassium 3.2 (Kir3.2) channel is found principally in neurons that regulate diverse brain functions, including pain perception, alcoholism, and substance addiction. Activation or inhibition of this channel leads to changes in neuronal firing and chemical message transmission. The Kir3.2 channel is subject to regulation by intracellular signals including sodium, G-proteins, ethanol, the phospholipid phosphatidylinositol bis-phosphate, and phosphorylation by protein kinases. Here, we take advantage of the recently published structure of Kir3.2 to provide an in-depth molecular view of how phosphorylation of a specific residue previously thought to be the target of PKC promotes channel gating and acts as an allosteric modulator of PKC-mediated inhibition.

Keywords: GIRK; Kir3; PIP2; PKC.

Figure 1.

Kir3.2*(Kir3.2_E152D) has slower Ci-VSP-mediated inhibition and faster recovery versus wild-type Kir3.2. A, Left, Voltage protocols (in mV) used for inhibition (top) and recovery (bottom). Right, Sample inhibition traces for Kir3.2* (top) and recovery (bottom). B, Kir3.2* displays slower current inhibition compared with wild-type Kir3.2. C, Tau of inhibition for Kir3.2* is significantly slower than wild-type. *p < 0.05 compared with control. D, Kir3.2* has faster recovery than wild-type Kir3.2 at −80 mV.

Figure 2.

Kir3.2* has lower PMA inhibition relative to Kir3.4* and both PMA effects are blocked by PKC inhibition. A, Time course of PMA inhibition in control Kir3.2_E152D (Kir3.2*). Single arrows indicate I_max, and double arrows indicate I_post-PMA. B, Time course of PMA inhibition in Kir3.2* after Bis pretreatment. C, Time course of PMA inhibition in control Kir3.4_S143T (Kir3.4*). D, Time course of PMA inhibition in Kir3.4* after Bis pretreatment. E, Summary of PMA inhibition with and without Bis pretreatment.

Figure 3.

PMA inhibition and normalized currents for Kir3.2* and Kir3.4* phosphorylation site mutants. A, PMA inhibition for Kir3.4* Ser-191 mutants. **p < 0.05 between indicated groups; *p < 0.05 compared with control. B, Normalized currents of Kir3.4* Ser-191 mutants. C, PMA inhibition for Kir3.2* Ser-196 mutants. D, Normalized currents of Kir3.2* Ser-196 mutants. E, Purified Kir3.2 from P. pastoris treated with alkaline phosphatase reveals constitutive phosphorylation of the channel protein.

Figure 4.

Kir3.2* mutants show similar sensitivity to PIP₂ as the Kir3.2* control and PMA sensitivity of S196Q is independent of PIP₂ manipulation. A, Normalized Ci-VSP inhibition of Kir3.2* and Ser-196 mutants. B, Normalized recovery of Kir3.2* and Ser-196 mutants. Protocols for inhibition and recovery were identical to experiments shown in Figure 1. C, PMA inhibition for Kir3.2* Ser-196 mutants in control and with wortmannin pretreatment. *p < 0.05 compared with control condition. D, PMA inhibition of Kir3.2_I234L Ser-196 mutants. *p < 0.05 compared with Kir3.2_I234L control. N.S., Not significant.

Figure 5.

Intersubunit helix bundle crossing distance at F192 increases in S196E, but not S196Q. A, Time course for S196Q for A–B and C–D intersubunit minimal distances. B, Time course for S196E showing an increase in the A–B distance. C, An 80 ns snapshot showing the F192 A–B distance in S196Q. Chain A is colored in red and chain B is colored in blue. D, An 80 ns snapshot showing the F192 A–B distance in S196E.

Figure 6.

Comparison of simulation results for S196Q and S196E in Kir3.2* background. A, Simulation time course for average distance per subunit for Arg-201(CZ)/Gln-196(CD) and Arg-201(CZ)/Asp-228(CG). B, Time course for Arg-201(CZ)/Glu-196(CD) and Arg-201(CZ)/Asp-228(CG). C, An 80 ns snapshot of MD simulation depicting Gln-196, Arg-201, and Asp-228. D, An 80 ns snapshot depicting Glu-196, Arg-201, and Asp-228.

Figure 7.

Comparison of simulation results for Kir3.2* and Kir3.2*_SEP196. A, Simulation time course for average distance per subunit for Ser-196(CB)/Arg-201(CZ) and Arg-201(CZ)/Asp-228(CG). B, Time course for SEP-196(P)/Arg-201(CZ) and Arg-201(CZ)/Asp-228(CG). C, 80 ns snapshot depicting Arg-201, Ser-196, and Asp-228. D, An 80 ns snapshot depicting Arg-201, SEP196, and Asp-228.

Figure 8.

Neutralization of R201 results in low currents with increased sensitivity to PMA in the Kir3.2*_D228N background. A, Kir3.2*_D228N has higher currents than Kir3.2* control. B, Kir3.2*_R201A_D228N has reduced currents compared with Kir3.2*_D228N. **, Kir3.2*_R201A_D228N has significantly higher currents than Kir3.2*_R201A at p < 0.05. C, PMA inhibition is increased in Kir3.2*_D228N_R201A compared with Kir3.2*_D228N control. *p < 0.05 compared with control.

Figure 9.

Schematic of PKC inhibition depending on phosphorylation state of Ser-196 and salt–bridge formation with Arg-201. Top, In PKC-sensitive channels, Q196 or dephosphorylated S196 cannot interact with R201 and are stabilized by D228. Bottom, In PKC-insensitive channels, R201 interacts with E196 or phosphorylated S196, stabilizing the open state of the helix-bundle-crossing gate.