Metabolic and thermal stimuli control K(2P)2.1 (TREK-1) through modular sensory and gating domains

Abstract

K(2P)2.1 (TREK-1) is a polymodal two-pore domain leak potassium channel that responds to external pH, GPCR-mediated phosphorylation signals, and temperature through the action of distinct sensors within the channel. How the various intracellular and extracellular sensory elements control channel function remains unresolved. Here, we show that the K(2P)2.1 (TREK-1) intracellular C-terminal tail (Ct), a major sensory element of the channel, perceives metabolic and thermal commands and relays them to the extracellular C-type gate through transmembrane helix M4 and pore helix 1. By decoupling Ct from the pore-forming core, we further demonstrate that Ct is the primary heat-sensing element of the channel, whereas, in contrast, the pore domain lacks robust temperature sensitivity. Together, our findings outline a mechanism for signal transduction within K(2P)2.1 (TREK-1) in which there is a clear crosstalk between the C-type gate and intracellular Ct domain. In addition, our findings support the general notion of the existence of modular temperature-sensing domains in temperature-sensitive ion channels. This marked distinction between gating and sensory elements suggests a general design principle that may underlie the function of a variety of temperature-sensitive channels.

Figure 1

Extracellular potassium antagonizes regulation of K_2P2.1 (TREK-1) by membrane potential. (A) Effect of membrane potential (E_M) on K_2P2.1 (TREK-1) activity in Xenopus laevis oocytes measured by two-electrode voltage clamp in 2 mM [K⁺]_o pH 7.4. Current–voltage (I-V) curves show exemplar voltage-clamp recordings of K_2P2.1 (TREK-1) activity after 4 min of holding at −100 and 0 mV, consecutively. (B) Cartoon diagram of a single K_2P2.1 (TREK-1) subunit, transmembrane segments 1–4 (M1–M4), pore helix 1 and pore helix 2 (PH1, PH2), and the positions of key residues are indicated. (C) Exemplar I–V curves showing the effect of E_M on K_2P2.1 (TREK-1) S300A. (D) Channel activity from a representative cell in response to changes in E_M. Channel activity was assayed every 15 s by a ramp from −150 to 50 mV. Following current stabilization at −100 mV, E_M was changed to 0 mV, and channel activity was assayed by the ramp protocol until current reached minimal values (usually within 4–5 min). Channel activity was reversed by returning E_M to −100 mV. Each point represents channel activity from the ramp curves at 0 or +50 mV for 2 mM and 90 [K⁺]_o solutions, respectively. Data presented as fraction relative to activity after initial stabilization at −100 mV. (E) Quantification of maximal inhibition of WT or mutant K_2P2.1 (TREK-1) by prolonged incubation at 0 mV (mean±s.e., n⩾6, N⩾2, where ‘n’ is the number of oocytes and ‘N’ in the number of independent oocyte batches). (F) I–V curves showing exemplar voltage-clamp recordings of K_2P2.1 (TREK-1) in 90 mM [K⁺]_o pH 7.4 after stabilization at, consecutively, −100 and 0 mV.

Figure 2

PH1 is critical for K_2P channels C-type gating. (A) Amino-acid alignment of the PH1 region of the indicated K_2P channels. The GXG selectivity filter sequence is highlighted in grey. (B, C, E, F, H, I) Exemplar recordings of the response of the indicated K_2P channels to the external pH (pH_o) changes in 2 mM [K⁺]_o solution. Currents were elicited by a voltage ramp from −150 to +50 mV, from a holding potential of −80 mV. (D, G, J) Quantitation of the response of the K_2P channels to changes in pH_o. Data (mean±s.e., n⩾6, N⩾2) was taken at 0 mV, normalized to activity at pH 9.0, and fitted to the Hill equation.

Figure 3

Mutations that stabilize the C-type gate antagonize regulation of K_2P2.1 (TREK-1) by membrane potential. (A, B) Exemplar recordings from oocytes expressing the indicated K_2P2.1 (TREK-1) mutants after prolonged incubation at, consecutively, −100 and 0 mV in 2 mM [K⁺]_o pH 7.4. After current stabilization at −100 mV, the membrane potential was changed to 0 mV, and the current was recorded every 15 s using a voltage ramp from −150 to +50 mV. (C) Exemplar time resolution of channel activity from a representative cell in response to fluctuating E_M in 2 mM [K⁺]_o pH 7.4. Each point represents channel activity from the ramp curves at 0 mV. Data presented as fraction relative to activity after initial stabilization at −100 mV. (D) Quantification of maximal K_2P2.1 (TREK-1) inhibition by prolonged incubation at 0 mV (mean±s.e., n⩾6, N⩾2). (E) Ribbon diagram showing the location of Trp275 and Gly137 of K_2P2.1 (TREK-1) on the crystal structure of K_2P4.1 (TRAAK) (Brohawn et al, 2012)(PDB ID 3UM7). M4, transmembrane helix 4. PH1, pore helix 1. Blue spheres depict potassium ions.

Figure 4

M4–Ct junction is critical for cross-talk between Ct and the C-type gate. (A) Amino-acid sequence of the M4–Ct junction region of K_2P2.1 (TREK-1) showing the location of the 3G and 3A mutations. Dashed line indicates a predicted boundary between M4 and Ct. (B) Exemplar time resolution of channel activity from a representative cell in response to fluctuating E_M in 2 mM [K⁺]_o pH_o 7.4. Each point represents channel activity from the ramp curves at 0 mV. Data presented as fraction relative to activity after initial stabilization at −100 mV. (C) Quantification of maximal K_2P2.1 (TREK-1) inhibition by prolonged incubation at 0 mV (mean±s.e., n⩾6, N⩾2). (D–F) Normalized responses of the indicated channels to pH_o changes in 2 mM [K⁺]_o. Currents were elicited by a voltage ramp from −150 to +50 mV, from a holding potential of −80 mV. Data (mean±s.e., n⩾6, N⩾2) was taken at 0 mV, normalized to activity at pH 9.0 and fitted to the Hill equation.

Figure 5

Ct domains act cooperatively to affect K_2P2.1 (TREK-1) function. (A) Immunoblot analysis of lysates from oocytes expressing HA-tagged WT, HA-WT or tandem HA-WT-WT K_2P2.1 (TREK-1) channels. Lysates were pre-incubated with or without EndoH for 1 h at 4^oC or at room temperature (RT). Before electrophoresis, all samples were treated with 2% SDS and 2% β-mercaptoethanol for 15 min at 50^oC to dissociate K_2P2.1 (TREK-1) subunits. Asterisks denote deglycolyslated forms of WT and tandem channels. (B–D) Normalized responses of the indicated K_2P2.1 (TREK-1) channels to pH_o changes 2 mM [K⁺]_o. Currents were elicited by a voltage ramp from −150 to +50 mV, from a holding potential of −80 mV. Data (mean±s.e., n⩾6, N⩾2) was taken at 0 mV, normalized to activity at pH 9.0 and fitted to the Hill equation.

Figure 6

Both C-terminal domains are required for the K_2P2.1 (TREK-1) temperature response. (A–C) Exemplar two-electrode voltage clamp recordings of K_2P2.1 (TREK-1) (A), K_2P2.1-3G (B), and K_2P2.1-3A (C) responses to temperature in 2 mM [K⁺]_o pH 7.4. Currents were elicited by a ramp from −150 to +50 mV, from a −80 mV holding potential. (D–F) Quantification of the temperature responses. Data (mean±s.e., n⩾6, N⩾2) was taken at 0 mV and normalized to channel activity at 14°C. (G) Cartoon model of how Ct couples to the C-type gate of K_2P2.1 (TREK-1). M4, transmembrane segment 4. Channel elements come from a single subunit. Transmembrane segments M1–M3 and pore helix 2 are not depicted. Dashed regions indicate connections to parts of the subunit that are not shown. Green circles represent potassium ions in the selectivity filter.