The modulation of voltage-gated potassium channels by anisotonicity in trigeminal ganglion neurons

Abstract

Voltage-gated potassium channels (VGPCs) play an important role in many physiological functions by controlling the electrical properties and excitability of cells. Changes in tonicity in the peripheral nervous system can activate nociceptors and produce pain. Here, using whole cell patch clamp techniques, we explore how hypo- and hypertonicity modulate VGPCs in cultured rat and mouse trigeminal ganglion (TG) neurons. We found that hypo- and hypertonicity had different effects on slow-inactivating K+ current (IK) and fast-inactivating K+ current (IA): hypotonicity increased IK but had no effect on IA while hypertonicity depressed both IK and IA. The increase of IK by hypotonicity was mimicked by transient receptor potential vanilloid 4 (TRPV4) receptor activator 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) but hypotonicity did not exhibit increase in TRPV4-/- mice TG neurons, suggesting that TRPV4 receptor was involved in hypotonicity-induced response. We also found that inactivation of PKC selectively reversed the increase of IK by hypotonicity, whereas antagonism of G-protein selectively rescued the inhibitions of IK and IA by hypertonicity, indicating that different intracellular signaling pathways were required for the modulation by hypo- and hypertonicity. In summary, changes in osmolality have various effects on IK and IA and different receptors and second messenger systems are selective for the modulation of VGPCs induced by hypo- versus hypertonicity.

Figure 1. Effect of hypotonicity on I_K

A. The typical recordings show that I_K, evoked by voltage step to +50 mV, was increased from 4.57 nA to 6.38 nA when the external solution was changed from 300mOsm to 220mOsm for 3min and the current recovered to 4.77 nA after washout. B. The peak current-voltage relationship (I–V) was shown before and during hypotonicity treatment. C. In the presence of hypotonicity, G–V curve did not shift significantly. V_0.5 was −0.93±1.30 mV and −0.90±0.71 mV (n=8, paired t test, P>0.05) for 300mOsm and 220mOsm, respectively; k was 16.46±1.13 and 16.07±0.41 (n=8, paired t test, P>0.05), respectively. Data were transformed from the I–V data shown in B. D. The typical recordings show that hypotonicity increased I_K and inactivation–voltage curve significantly shifted to depolarizing direction. V_0.5 was −72.67±1.54 mV and −40.55±5.25 mV (n=8, paired t test, P<0.05) for 300mOsm and 220mOsm, respectively; k was −9.67±1.32 and −10.98±4.61 (n=8, paired t test, P>0.05), respectively.

Figure 2. Effect of hypotonicity on I_A

A. After exposure to hypotonicity (220mOsm), I_A was not affected and the peak amplitude was 5.87 nA, 5.91 nA and 5.93 nA before, during and after hypotonicity application, respectively. B. I–V curve did not change before and during hypotonicity treatment. C. G–V curve did not shift in the presence of hypotonicity. V_0.5 was −3.57±0.18 mV and −3.33±0.72 mV (n=11, paired t test, P>0.05) for 300mOsm and 220mOsm, respectively; k was 19.47±1.20 and 21.08±0.75 (n=11, paired t test, P>0.05), respectively. Data were transformed from the I–V data shown in B. D. Hypotonicity had no effect on inactivation–voltage curve. V_0.5 was −68.47±0.23 mV and −69.42±0.21 mV (n=11, paired t test, P>0.05) for 300mOsm and 220mOsm, respectively; k was −9.37±0.20 and −9.67±0.15 (n=11, paired t test, P>0.05), respectively.

Figure 3. Effect of hypertonicity on I_K

A. The typical recordings show that I_K was reduced from 8.16 nA to 5.32 nA when the external solution was changed from 300mOsm to 350mOsm for 3 min but the current recovered only slightly (5.84 nA) after washout. B. I–V curve was shown before and during hypertonicity treatment. C. In the hypertonic solution G–V curve shifted to the hyperpolarization direction. V_0.5 was −0.92±1.23 mV and −8.16±1.02 mV (n=11, paired t test, P<0.05) for 300mOsm and 350mOsm, respectively; k was 18.13±1.08 and 17.63±0.95 (n=11, paired t test, P>0.05), respectively. Data were transformed from the I–V data shown in B. D. The typical recordings show that the amplitude of I_K was reduced in the presence of hypertonic solution and inactivation–voltage curve shifted to hyperpolarizing direction with V_0.5 being −69.99±1.48 mV and −83.00±1.22 mV (n=12, paired t test, P<0.05) for 300mOsm and 350mOsm, respectively; k being −11.68±1.28 and −15.19±0.87 (n=12, paired t test, P<0.05), respectively.

Figure 4. Effect of hypertonicity on I_A

A. The typical recordings show that I_A was reduced from 8.34 nA to 6.01 nA when the external solution was changed from 300mOsm to 350mOsm but the current almost did not recover (6.75 nA) after washout. B. I–V curve was shown before and during hypertonicity treatment. C. In the hypertonic solution G–V curve shifted to the hyperpolarization direction. V_0.5 was −3.98±0.25 mV and −9.43±0.99 mV (n=10, paired t test, P<0.05) for 300mOsm and 350mOsm, respectively; k was 19.16±0.81 and 20.38±0.94 (n=11, paired t test, P>0.05), respectively. Data were transformed from the I–V data shown in B. D. The typical recordings show that the amplitude of I_A was inhibited upon exposure to hypertonic stimulus and inactivation–voltage curve shifted to the hyperpolarizing direction. V_0.5 was −69.45±0.27 mV and −86.32±0.47 mV (n=12, paired t test, P<0.05) for 300mOsm and 350mOsm, respectively; k was −9.93±0.24 and −16.37±0.28 (n=10, paired t test, P<0.05), respectively.

Figure 5. Effect of anisotonicity on I_K and I_A

A. I_K was enhanced by hypotonic stimuli and inhibited by hypertonic stimuli. B. I_A was not significantly affected when exposed to hypotonicity but inhibited by hypertonic stimuli.

Figure 6. Effect of 4α-PDD on I_K and I_A

A. The plot shows the percentage in increase of I_K by 4α-PDD at concentrations of 0.03, 0.1, 0.3, 1.0, 3.0, 10 and 30 μM. The dose–response curve fits to Hill equation with IC₅₀ being 4.01 μM and n being 0.80. B. G–V curve of I_K did not shift upon exposed to 3.0 μM4α-PDD. V_0.5 was −0.58±0.60 mV and −0.11±0.48 mV (n=9, paired t test, P>0.05) before and during 4α-PDD treatment, respectively; k was 17.83±0.53 and 18.38±0.42 (n=9, paired t test, p>0.05), respectively. C. After application of 3.0 μM 4α-PDD, inactivation–voltage curve of I_K significantly shifted to the depolarizing direction. V_0.5 was −70.44±0.10 mV and −50.40±0.42 mV (n=9, paired t test, P<0.05) before and during 4α-PDD treatment, respectively; k was −10.08±0.08 and −7.60±0.36 (n=9, paired t test, P<0.05), respectively. D. Application of 3.0 μM 4α-PDD had no effect on G–V curve of I_A (300mOsm: V_0.5=−3.46±0.17 mV, k=19.62±0.15; 4α-PDD: V_0.5=−4.42±0.25 mV, k=20.80±0.23, n=8, paired t test, P>0.05). E. Application of 4α-PDD had no effect on inactivation–voltage curve of I_A either (300mOsm: V_0.5=−65.93±0.29 mV, k=−11.49±0.25; 4α-PDD: V_0.5=−66.38±0.74 mV, k=−11.58±0.65, n=8, paired t test, P>0.05).

Figure 7. Effects of anisotonicity on I_K and I_A in TG neurons from TRPV4^+/+ and TRPV4^−/− mice

A. Upon exposure to hypotonicity (220mOsm), I_K was increased by 29.21±9.81% (n=15) in TRPV4^+/+ mice TG neurons, which was significantly different from that in TRPV4^−/− mice (1.41±2.72%, n=17) (unpaired t test, P<0.01). B. After exposure to hypotonicity (220mOsm), I_A was not significantly affected. On the average, in TRPV4^+/+ mice TG neurons, the amplitude was 253.75±31.43 pA/pF and 243.19±33.99 pA/pF (n=14, paired t test, P>0.05) before and during hypotonicity treatment, respectively, and in TRPV4^−/− mice TG neurons I_A was 246.42±35.52 pA/pF and 232.13±32.58 pA/pF (n=13, paired t test, P>0.05), respectively. C. After exposure to hypertonicity (350mOsm), I_K was reduced by 38.82±7.25% (n=14) and 38.52±6.38% (n=16) in TRPV4^+/+ and TRPV4^−/− TG neurons respectively (unpaired t test, P>0.05). D. After exposure to hypertonicity (350mOsm), the inhibition of I_A by hypertonicity was 30.68±7.16% (n=14) and 33.31±6.61% (n=14) in TRPV4^+/+ and TRPV4^−/− mice TG neurons respectively (unpaired t test, P>0.05).

Figure 8. Modulation of PKC system on the effects induced by hypo and hypertonicity

A. Pre-incubation with PKC antagonists, BIM and staurosporine, significantly reversed the increase of I_K by hypotonicity from 34.52±8.58% to 17.48±5.59% (n=12) (unpaired t test, P<0.05) and 15.11±6.17% (n=10) (unpaired t test, P<0.05) respectively. B. Neither BIM nor staurosporine statistically affected the inhibitions of VGPCs by hypertonicity (unpaired t test, P>0.05). In the presence of BIM and staurosporine, I_K was inhibited by 37.88±7.38% (n=11) and 35.43±6.01% (n=8), respectively and I_A by 25.03±6.12% (n=12) and 27.13±5.05% (n=8), respectively.

Figure 9. Modulation of G-protein system on the effects induced by hypo and hypertonicity

A. Neither GTP-γs nor GDP-βs had significant effect on the increase of I_K by hypotonicity (unpaired t test, P>0.05). In hypotonic solution (220mOsm) I_K was enhanced by 36.00±7.91% (n=12) and 38.23±8.01% (n=13) with GTP-γs and GDP-βs in the pipette solution, respectively. B. Both GTP-γs and GDP-βs markedly reversed the inhibitions of I_K (unpaired t test, P<0.05) and I_A (unpaired t test, P<0.05) by hypertonicity. In hypertonic solution (330mOsm) I_K was reduced by 18.73±5.90% (n=12) and 19.29±8.02% (n=12) in the presence of GTP-γs and GDP-βs, respectively and I_A by 11.53±4.19% (n=11) and 13.17±5.26% (n=12), respectively.

Figure 10. Modulation of PKA, lipid cascade and PKG systems on the effects induced by hypo and hypertonicity

A. For PKA system, pre-incubation with PKA inhibitor, H-89, had no significant effect on the modulations of VGPCs by hypo or hypertonicity (unpaired t test, P>0.05). In the presence of 10 μM H-89, I_K was increased 38.04±8.64% (n=10) by hypotonicity, while I_K and I_A were decreased 29.46±7.68% (n=10) and 30.00±6.64% (n=14) by hypertonicity respectively. B. For lipids cascade, in hypotonic solution, I_K was increased by 38.32±7.62% (n=11), 36.61±6.83% (n=14) and 37.86±8.59% (n=12) with Wortmannin, LY294002 and U73122 in the pipette solution, respectively. Compared with the inhibition of hypotonicity with normal pipette solution (34.52±8.58%, n=37), none of them was significantly different (unpaired t test, P>0.05). Similarly, pre-incubation with Wortmannin, LY294002 and U73122 did not affect the inhibition of I_K or IA by hypertonicity (unpaired t test, P<0.05). In the presence of Wortmannin, LY294002 and U73122, the inhibition of I_K by hypertonicity was 34.71±6.72% (n=11), 30.73±7.29% (n=10) and 33.23±7.55% (n=12) respectively and the inhibition of I_A was 27.25±8.02% (n=11), 29.50±6.61% (n=11) and 30.16±8.79% (n=11) respectively. C. For PKG system, KT5823 did not statistically alter the modulation of VGPCs by hypo or hypertonicity (unpaired t test, P>0.05). In the presence of KT5823, I_K was increased 38.33±7.15% (n=13) by hypotonicity, while I_K and I_A were inhibited 31.12±6.12% (n=15) and 25.17±5.67% (n=14) by hypertonicity, respectively.