KV4 channels in isolectin B4 muscle dorsal root ganglion neurons of rats with experimental peripheral artery disease: effects of bradykinin B1 and B2 receptors

Abstract

Muscle afferent nerve-activated reflex sympathetic nervous and blood pressure responses are exaggerated during exercise in peripheral artery diseases (PAD). However, the precise signaling pathways and molecular mediators responsible for these abnormal autonomic responses in PAD are poorly understood. Our previous study suggests that A-type voltage-gated K⁺ (K_V4) channels regulate the excitability in muscle dorsal root ganglion (DRG) neurons of PAD rats; however, it is still lacking regarding the effects of PAD on characteristics of K_V4 currents and engagement of bradykinin (BK) subtype receptors. Thus, we examined K_V4 currents in two distinct muscle DRG neurons, namely isolectin B₄-positive and B₄-negative (IB4+ and IB4-) DRG neurons. IB4+ neurons express receptors for glial cell line-derived neurotrophic factor (GDNF), whereas IB4- DRG neurons are depending on nerve growth factors for survival. Our data showed that current density in muscle DRG neurons of PAD rats was decreased and this particularly appeared in IB4+ DRG neurons as compared with IB4- DRG neurons. We also showed that stimulation of BK B1 and B2 receptors led to a greater inhibitory effect on K_V4 currents in IB4+ muscle DRG neurons and siRNA knockdown of K_V4 subunit K_V4.3 decreased the activity of K_V4 currents in IB4+ DRG neurons. In conclusion, our data suggest that limb ischemia and/or ischemia-induced BK inhibit activity of K_V4 channels in a subpopulation of the thin fiber muscle afferent neurons depending on GDNF, which is likely a part of signaling pathways involved in the exaggerated blood pressure response during activation of muscle afferent nerves in PAD.

Keywords: A-type voltage-gated K+ channels; bradykinin receptors; dorsal root ganglion; limb ischemia; peripheral artery disease.

Figure 1.

A-type voltage-gated K⁺ (K_V4) currents in different phenotypes of muscle dorsal root ganglion (DRG) neurons. A: representative of isolectin B₄-positive (IB4+) and isolectin B₄-negative (IB4−) muscle DRG neurons. Scale bar: 50 µm. B: representative traces of K_V4 currents in muscle neurons of the control limbs. Protocol 1 was used to record the total tetraethylammonium (TEA)-resistant K⁺ currents (I_Ktotal), and protocol 2 to record the delayed rectifying K+ current (I_{K DR}), Kv4 currents = I_Ktotal − I_{K DR}, calculated by pClampfit 10.1. Representative traces for I_Ktotal, I_{K DR}, and K_V4 currents are indicated by red, black, and blue color, respectively. C: histogram of averaged K_V4 current density at 60 mV (Density_{60 mV}) in IB4+ and IB4− muscle DRG neurons. Averaged data showing that density of K_V4 currents is greater in IB4+ DRG neurons than that in IB4− DRG neurons. *P = 0.002 between two groups of neurons. Open circles, individual data. “n” in all the figures of this study indicates the number of the recorded DRG neurons.

Figure 2.

A-type voltage-gated K⁺ (K_V4) currents in isolectin B₄-positive (IB4+) and isolectin B₄-negative (IB4−) dorsal root ganglion (DRG) neurons of control limbs and occluded limbs. A: representative traces of K_V4 currents in muscle DRG neurons of both control limbs and occluded limbs. Two individual step depolarization protocols with an increment of 10 mV were used to record Kv4 currents. Protocol 1 was for tetraethylammonium (TEA)-resistant I_Ktotal, and protocol 2 for I_Ktotal. B: current densities of muscle DRG neurons with different voltages applied. *P < 0.05 between control and occlusion groups. C: histogram of averaged data showing that Density_{60 mV} of K_V4 currents is smaller in IB4+ muscle DRG neurons of occluded limbs (n = 32) than that in those neurons of control limbs (n = 56). *P = 0.004 between control and occlusion groups. No significant difference in Density_{60 mV} of K_V4 currents was observed in IB4− DRG neurons between the control (n = 35) and occluded groups (n = 22; P = 0.176 between two groups). Open circles, individual data.

Figure 3.

Percentage inhibition of bradykinin (BK), and BK B1 and B2 receptor agonists on A-type voltage-gated K⁺ (K_V4) currents in isolectin B₄-positive (IB4+) and isolectin B₄-negative (IB4−) muscle dorsal root ganglion (DRG) neurons of control limbs and occluded limbs. A: representative traces of K_V4 currents following application control vehicle, BK, Lys-BK, and Phe-BK onto muscle DRG neurons of control limbs and occluded limbs. B: BK led to a greater inhibitory effect on IB4+ DRG neurons than that on IB4− muscle DRG neurons in both control limbs and occluded limbs. *P = 0.005 between two groups of neurons (IB4+ and IB4−) of the control limbs; and P = 0.002 between two groups of DRG neurons of the occluded limbs. There was no significant difference observed in the inhibition of K_V4 currents by BK in IB4+ and IB4− DRG neurons between control limbs and occluded limbs. Open circles, individual data. C: *P = 0.002 between two groups of neurons (IB4+ and IB4−). No significant difference was observed in inhibition of Lys-BK (B1 receptor agonist) on K_V4 currents in muscle IB4+ and IB4− DRG neurons between control limbs and occluded limbs. D: Phe-BK (B2 receptor agonist) inhibited K_V4 currents to a greater degree in muscle IB4+ and IB4− DRG neurons of occluded limbs than in control limbs. *P = 0.032 between IB4+ and IB4− in the control limbs; and *P = 0.049 between IB4+ and IB4− in the occluded limbs. *P = 0.045 between control limbs and occluded limbs for IB4+ neurons; and *P = 0.012 between control limbs and occluded limbs for IB4− neurons. “n”, number of recorded DRG neurons is indicated in B–D.

Figure 4.

Distribution of A-type voltage-gated K⁺ (K_V4.1) and K_V4.3 subunits in dorsal root ganglion (DRG) neurons in situ. Immunofluorescence was used to examine the presence of K_V4.1/K_V4.3 channels within isolectin B₄-positive (IB4+) dorsal root ganglion (DRG) neurons of healthy control rats. Representative photomicrographs show that K_V4.1 (top) and K_V4.3 (bottom) exist within IB4+ DRG neurons. After the images were merged, neuronal cells positive for both K_V4.1/K_V4.3 and IB4+ appear to be yellow color. Scale bar = 50 µm.

Figure 5.

Contribution of A-type voltage-gated K⁺ (K_V)4.1 and K_V4.3 to K_V4 currents in muscle dorsal root ganglion (DRG) neurons. A: representatives of K_V4.1 and K_V4.3 subunits’ distribution in rat dissociated DRG neurons. The isolectin B₄-positive (IB4+) DRG neurons were labeled green and the neurons containing K_V4.1 (top) and K_V4.3 (bottom) labeled red. The DRG neurons appear yellow as colocalization of K_V4 subunits and IB4+. Scale bar = 50 µm. B and C: approach of siRNA-K_V4.1/siRNA-K_V4.3 knockdown was used to decrease the expression levels of K_V4.1/K_V4.3 in DRG neurons and then the specific contribution of K_V4.1 and/or K_V4.3 subunit to K_V4 currents was determined. The effectiveness of siRNA-K_V4.1 and siRNA-K_V4.3 knockdown was verified (B). Histogram of averaged Density_{60 mV} of K_V4 currents in both IB4+ and isolectin B₄-negative (IB4−) muscle DRG neurons (C) showing that after knockdown of K_V4.3 the density of K_V4 currents was significantly decreased in IB4+ muscle DRG neurons compared with scramble control group, but this was not observed after the knockdown of K_V4.1 (P = 0.5002 between scramble and knockdown). *P = 0.0019 between K_V4.3 knockdown and scramble in IB4+ muscle DRG neurons. Open circles, individual data. “n,” number of recorded DRG neurons.