TREK-1 and TRAAK Are Principal K+ Channels at the Nodes of Ranvier for Rapid Action Potential Conduction on Mammalian Myelinated Afferent Nerves

Abstract

Rapid conduction of nerve impulses is critical in life and relies on action potential (AP) leaps through the nodes of Ranvier (NRs) along myelinated nerves. While NRs are the only sites where APs can be regenerated during nerve conduction on myelinated nerves, ion channel mechanisms underlying the regeneration and conduction of APs at mammalian NRs remain incompletely understood. Here, we show that TREK-1 and TRAAK, the thermosensitive and mechanosensitive two-pore-domain potassium (K2P) channels, are clustered at NRs of rat trigeminal Aβ-afferent nerves with a density over 3,000-fold higher than that on their somas. These K2P channels, but not voltage-gated K⁺ channels as in other parts of nerves, are required for rapid AP repolarization at the NRs. Furthermore, these channels permit high-speed and high-frequency AP conduction along the myelinated afferent nerves, and loss of function of these channels at NRs retards nerve conduction and impairs sensory behavioral responses in animals.

Keywords: action potential; conduction velocity; leak K(+) currents; myelinated nerve; node of Ranvier; saltatory conduction; sensory; temperature; two-pore-domain K(+) channels.

Figure 1.. Nodes of Ranvier of myelinated afferent nerves display unconventional action potentials and high leak K⁺ conductance.

A) Ex-vivo trigeminal afferent nerve preparation. B) Fluorescent image shows patch-clamp recordings at a node of Ranvier (NR) of a trigeminal Aβ-afferent nerve. The electrode contained Alexa-555 to trace the NR (arrow indicated) and other axonal regions. C) Traces illustrate typical action potentials (APs) recorded at NRs in the absence (control, left) and presence of the voltage-gated K⁺ channel blockers of intracellular 135 mM Cs⁺ plus extracellular 20 mM TEA (Cs⁺+TEA, right). Bar graph shows widths of APs at NRs in the absence (control, n = 9) and presence of Cs⁺+TEA (n = 11). D) Traces illustrate APs recorded from a soma of a trigeminal Aβ-afferent nerve in the absence (top, control) and presence of Cs⁺+TEA (bottom). Bar graph shows AP widths in the absence (control, n = 7) and presence of Cs⁺+TEA (n = 6). E) Traces illustrate currents recorded at NRs following voltage steps in the absence (left, control) and presence of Cs⁺+TEA (right). Right panel, I-V curves of non-inactivating currents in control (n = 9) and the presence of Cs⁺+TEA (n = 9). F) Traces illustrate non-inactivating currents at NRs following voltage steps in control with [K⁺]_out/[K⁺]_in = 5 mM/135 mM (left) and symmetric K⁺ with [K⁺]_out/[K⁺]_in = 135 mM/135 mM (right). Right panel, I-V curves of non-inactivating currents recorded at NRs in control (n = 7) and symmetrical K⁺ (n = 7). G) Left, recording setting. Middle, traces illustrate IK_leak single channel currents at different voltages across the nodal membrane patch. Right, I-V curve of IK_leak single channel currents recorded at NRs (n = 15). Data represent Mean ± SEM, ns, not significantly different, ***p < 0.001, Student’s t-Test. See also Fig. S1–4, Table S1

Figure 2.. Leak K⁺ conductance at nodes of Ranvier of myelinated afferent nerves are thermosensitive and mechanosensitive channels.

A-C) Sample traces (A), I-V curves (B), whole-cell conductance (C) of IK_leak recorded at NRs at 35°C (n = 8), 24°C (control, n = 8),15°C (n = 8), and 10°C (n = 6). IK_leak conductance is the slope between − 72 to −82 mV in I-V curves. Recordings were made in whole-cell mode. D-F) Sample traces of IK_leak single channel activity (D), summary (n = 6) of IK_leak single channel events (E), and open probability (F) recorded at 80 mV from NRs at 35, 24, 15 and 10°C. G-I) Sample traces of IK_leak single channel activity (G), summary (n = 6) of IK_leak single channel events (H) and open probability (I) recorded at 80 mV from NRs at 0 mmHg and following intra-electrode applications of −100 mmHg pressures. In D-I, recordings were performed under the cell-attached mode with [K⁺]_in/[K⁺]_out of ~1. Data represent Mean ± SEM, *p < 0.05, ***p < 0.001, one-way ANOVA with the Tukey post hoc test or Student’s t-Test.

Figure 3.. TREK-1 and TRAAK channels are molecular identities of leak K⁺ channels at NRs of myelinated afferent nerves.

A) Left, TREK-1 immunoreactivity (TREK-1-ir) at a NR and MBP immunoreactivity (MBP-ir) on myelin sheath. Right, TREK-1-ir at a NR and CASPR-ir in paranodal regions. B) Left, TRAAK-ir at a NR and MBP-ir on myelin sheath. Right, TRAAK-ir at a NR and CASPR-ir in paranodal regions. C) Similar to A&B except TREK-2-ir was examined and was negative at NRs. In A-C, NRs are indicated by arrows. MBP, myelin basic protein. CASPR, contactin associated protein. D) Summary of immunoreactive nodes for experiments represented in A-C: 112/129 nodes were TREK-1-ir positive, 118/129 nodes were TRAAK-ir positive. 0/129 nodes were TREK-2-ir positive. E) HEK293 cells transfected with TREK-1/eGFP (left), TRAAK/mCherry (middle), and both TREK-1/EGFP and TRAAK/mCherry (right). F) Traces illustrate single channel currents recorded at −80 mV from an HEK293 cell transfected with TREK-1/eGFP (upper, homomeric TREK-1) or an HEK293 cell transfected with TRAAK/mCherry (lower, homomeric TRAAK). Bottom, I-V curves of single channel currents recorded at different transmembrane voltages for homomeric TREK-1 channels (open circles, n = 7) or homomeric TRAAK (solid circles, n = 6). G) Sample traces show two types of single channels recorded at −80 mV from a HEK293 cell co-transfected with TREK-1/eGFP and TRAAK/mCherry plasmids, one type (upper, TREK-1/TRAAK) has unitary currents apparently larger than homomeric channels and another type (lower, TREK-1-like) has unitary currents similar to homomeric TREK-1 channels shown in F. Bottom panel, I-V curves of the currents of TREK-1/TRAAK single channels (n = 12, solid triangles) and TREK-1-like single channels (n = 13, open triangles). H) Summary of single channel conductance at −80 mV (open bars) and 80 mV (closed bars) for homomeric TREK-1 (n = 5 at −80 mV, n = 6 at 80 mV), homomeric TRAAK (n = 6 at both voltages), TREK-1-like (n = 12 at −80 mV, n = 5 at 80 mV), and TREK-1/TRAAK channels (n = 8 at −80 mV, n = 5 at 80 mV) expressed in HEK293 cells. The single channel conductance of nodal K2P channels (n = 13 at both voltages) is also included in the graph for a comparison. The single channel conductance at −80 mV was used for comparison. All recordings were performed under the cell-attached mode. Data represent Mean ± SEM, ns, no significant difference, *p < 0.05, ***p < 0.001, one-way ANOVA with the Tukey post hoc test. See also Fig. S5–9

Figure 4.. Genetic and pharmacological manipulation of TREK-1 and TRAAK activity reveals their roles in the formation of rapid action potentials at nodes of Ranvier of myelinated afferent nerves.

A) Illustration of intra-nerve microinjections of shRNA/AAV preparations. B) Axons of trigeminal Aβ-afferent nerves expressing TREK-1-shRNA/eGFP (shTREK-1, left), TRAAK-shRNA/mCherry (shTRAAK, middle), and TREK-1-shRNA/eGFP plus TRAAK-shRNA/mCherry (shTREK-1+shTRAAK, right) 30 days after in vivo shRNA/AAV microinjections. Arrow in each panel indicates a NR. C) I-V curves of whole-cell IK_leak recorded at NRs of shScramble (n = 7), shTREK-1 (n = 6), shTRAAK (n = 6), and shTREK-1+shTRAAK (n = 6) groups. D) IK_leak conductance at NRs of shScramble (n = 7), shTREK-1 (n = 6), shTRAAK (n = 7), and shTREK-1+shTRAAK (n = 6) groups. E) IK_leak conductance at NRs in control (n = 9), the presence of 5 mM Ba²⁺ (n = 15), 50 μm norfluoxetine (NF, n = 6) and 2 μm ruthenium red (RR, n = 10). F) IK_leak conductance at NRs in control (n = 8), the presence of 10 μm BL 1249 (BL, n = 9), intracellular 20 μm arachidonic acid (AA, n = 9) and intracellular pH of 5 ([pH]_i 5, n = 10). G) Left, traces illustrate APs recorded at NRs of shScramble and shTREK-1 groups. Right, AP widths in shScramble (n = 7), shTREK-1 (n = 6), shTRAAK (n = 6), and shTREK-1+shTRAAK (n = 6) groups. H) AP widths at NRs before (control, n = 9), following the applications of 5 mM Ba²⁺ (n = 15), 50 μm NF (n = 6), or 2 μm RR (n = 10). I) Left, traces on top illustrate currents recorded at NRs under AP-dynamic clamp mode before (control) and following 20 mM TEA application. The trace on the bottom was the AP waveform for AP-dynamic clamp. Right, summary of outward currents at NRs during AP repolarization phase before (n = 6) and following TEA application. J) Left, traces on the top illustrate currents recorded at NRs under AP-dynamic clamp mode before (control) and following 5 mM Ba²⁺ application. The trace on the bottom was the AP waveform for AP-dynamic clamp. Right, summary of outward currents recorded at NRs during AP repolarization before (n = 6) and following Ba²⁺ application. Data represent Mean ± SEM, ns, not significantly different, * p < 0.05, ** p < 0.01, ***p < 0.001, one-way ANOVA with the Tukey post hoc test or Student’s t-Test. See also Fig. S10–12, Table S2.

Figure 5.. TREK-1 and TRAAK channels are required for high-frequency saltatory conduction on myelinated afferent nerves.

A) Experimental setting. B) Traces illustrate APs recorded from the soma (left) and NR (right) of a trigeminal Aβ-afferent nerve following 50 Hz stimulation. Traces were 1^st, 10^th and 20^th APs. Bar graph, AP widths of 20^th APs at somas (n = 7, black) or NRs (n = 7, green) following stimulation at 1 to 200 Hz. Stimulation duration, 20 s. C) Left, two traces illustrate APs recorded at a soma (top) and a NR (bottom) following stimulation at 50 Hz. Tall spikes, successful APs. Right, AP success rates at somas (n = 7) and NRs (n = 7) with stimulation from 1 to 1000 Hz. Dashed lines indicate the frequency at which AP conduction success rate is 50% (FS₅₀). D) AP success rates at NRs of shScramble (n = 6), shTREK-1 (n = 6), shTRAAK (n = 6), and shTREK-1+shTRAAK (n = 6) groups. E) AP success rates at NRs before (control, n = 6), following application of 5 mM Ba²⁺ (n = 7), 50 μm NF (n = 6), or 2 μm RR (n = 6). Recording duration at each frequency was 20 s. Data represent Mean ± SEM, * p < 0.05, **p < 0.01, one-way ANOVA with the Tukey post hoc test. See also Fig. S13&14.

Figure 6.. TREK-1 and TRAAK channels at nodes of Ranvier of myelinated afferent nerves are required for high-speed saltatory conduction and for sensory behavioral responses.

A) Left, experimental setting. Right, traces illustrate APs recorded at NRs of shScramble and shTREK-1 groups. Arrow indicates stimulation marks. B) AP conduction velocity (CV) in shScramble (n = 6), shTREK-1 (n = 6), shTRAAK (n = 6), and shTREK-1+shTRAAK groups (n = 6). C) CV in the absence (control, n = 6), present of 5 mM Ba²⁺ (n = 7), 50 μm NF (n = 6), and 2 μm RR (n = 6). D) CV (n = 7) at NRs at 35°C, 24°C, and 15°C. E) CV at NRs at 15°C without (n = 8), and with 10 μm BL (n = 7), 20 μm AA (n = 6), and intracellular pH of 5 ([pH]_i 5, n = 6). AA and [pH]_i 5 were applied intracellularly via recording electrodes. F) CV recorded at NRs in the absence (control, n = 8) and presence (n = 8) of voltage-gated K⁺ channel blockers TEA (20 mM, extracellular), Cs⁺(135 mM, intracellular), plus 4-AP (1 mM, intracellular). G) Diagram illustrates whisker tactile test. H) Percentage (left) and scores (right) of whisker tactile responses in animals 30 days after microinjection of shScramble (n = 6) and shTREK-1+shTRAAK (n = 6) into D2 whisker hair follicles. I) Percentage (left) and scores (right) of whisker tactile responses in animals 30 days after intra-nerve microinjection of shScramble (n = 6) and shTREK-1 + shTRAAK (n = 6) into D2 whisker nerve branches. Data represent Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with the Tukey post hoc test or Student’s t-Test.