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Kv4 Channels Underlie the Subthreshold-Operating A-type K-current in Nociceptive Dorsal Root Ganglion Neurons.

Phuket TR, Covarrubias M - Front Mol Neurosci (2009)

Bottom Line: Recent studies have demonstrated DRG hyperexcitability associated with downregulation of A-type K(+) channels; however, the molecular correlate of the corresponding A-type K(+) current (I(A)) has remained hypothetical.Among Kv4 transcripts, the DRG expresses significant levels of Kv4.1 and Kv4.3 mRNAs.Contrasting the expression patterns of Kv4 channels in the central and peripheral nervous systems, we discuss possible functional roles of these channels in primary sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College of Thomas Jefferson University Philadelphia, PA, USA.

ABSTRACT
The dorsal root ganglion (DRG) contains heterogeneous populations of sensory neurons including primary nociceptive neurons and C-fibers implicated in pain signaling. Recent studies have demonstrated DRG hyperexcitability associated with downregulation of A-type K(+) channels; however, the molecular correlate of the corresponding A-type K(+) current (I(A)) has remained hypothetical. Kv4 channels may underlie the I(A) in DRG neurons. We combined electrophysiology, molecular biology (Whole-Tissue and Single-Cell RT-PCR) and immunohistochemistry to investigate the molecular basis of the I(A) in acutely dissociated DRG neurons from 7- to 8-day-old rats. Whole-cell recordings demonstrate a robust tetraethylammonium-resistant (20 mM) and 4-aminopyridine-sensitive (5 mM) I(A). Matching Kv4 channel properties, activation and inactivation of this I(A) occur in the subthreshold range of membrane potentials and the rate of recovery from inactivation is rapid and voltage-dependent. Among Kv4 transcripts, the DRG expresses significant levels of Kv4.1 and Kv4.3 mRNAs. Also, single small-medium diameter DRG neurons ( approximately 30 mum) exhibit correlated frequent expression of mRNAs encoding Kv4.1 and Nav1.8, a known nociceptor marker. In contrast, the expressions of Kv1.4 and Kv4.2 mRNAs at the whole-tissue and single-cell levels are relatively low and infrequent. Kv4 protein expression in nociceptive DRG neurons was confirmed by immunohistochemistry, which demonstrates colocalization of Kv4.3 and Nav1.8, and negligible expression of Kv4.2. Furthermore, specific dominant-negative suppression and overexpression strategies confirmed the contribution of Kv4 channels to I(A) in DRG neurons. Contrasting the expression patterns of Kv4 channels in the central and peripheral nervous systems, we discuss possible functional roles of these channels in primary sensory neurons.

No MeSH data available.


Related in: MedlinePlus

Isolation of IA in small DRG neurons. (A) Voltage-clamp protocol (top) to elicit the total K+ current (IK, bottom). From a holding voltage of −65 mV, a 1-s conditioning pulse at −100 mV precedes 500-ms step depolarizations delivered in 10-mV increments (for clarity, 20-mV increments are shown only). The start-to-start interval was 5 s. (B) Voltage-clamp protocol (top) to isolate the delayed-rectifier K+ current (IDR, bottom). The conditioning pulse at −30 mV inactivates the A-type K+ current (IA) (Figure 2). (C) The subtraction of IDR from IK yielded IA (bottom). These currents are from a DRG neuron (31 μm, 38 pF) exhibiting a dominant IA (∼90% IA and ∼10% IDR; see Results).
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Figure 1: Isolation of IA in small DRG neurons. (A) Voltage-clamp protocol (top) to elicit the total K+ current (IK, bottom). From a holding voltage of −65 mV, a 1-s conditioning pulse at −100 mV precedes 500-ms step depolarizations delivered in 10-mV increments (for clarity, 20-mV increments are shown only). The start-to-start interval was 5 s. (B) Voltage-clamp protocol (top) to isolate the delayed-rectifier K+ current (IDR, bottom). The conditioning pulse at −30 mV inactivates the A-type K+ current (IA) (Figure 2). (C) The subtraction of IDR from IK yielded IA (bottom). These currents are from a DRG neuron (31 μm, 38 pF) exhibiting a dominant IA (∼90% IA and ∼10% IDR; see Results).

Mentions: We conducted whole-cell patch-clamp measurements to ask whether the electrophysiological properties of the IA in small-medium diameter nociceptive DRG neurons (∼30 μm, ∼45 pF) are consistent with the expression of Kv4 channels. These channels are characteristically resistant to TEA (Amarillo et al., 2008; Birnbaum et al., 2004; Jerng and Covarrubias, 1997, 2004; Pak et al., 1991). Therefore, external TEA (20 mM) was present at all times to eliminate TEA-sensitive Kv channels (see Materials and Methods). In addition, by exploiting the distinct voltage dependence of inactivation of Kv4 channels, we employed voltage-clamp protocols to isolate the IA (Figure 1). To measure the total K+ current (IK), neurons were held at −65 mV, and a 1-s conditioning pulse to −100 mV was delivered prior to 500-ms step depolarizations that typically activate Kv channels. The conditioning pulse permits ∼90% recovery of the total IA that inactivated at −65 mV. Then, the IA was isolated from IK by a depolarizing 1-s conditioning prepulse to −30 mV. This depolarization is sufficient to inactivate the IA and, therefore, the remaining outward current evoked by subsequent step depolarizations is mostly comprised of a TEA-resistant delayed rectifying current (IDR). IA was finally revealed from the off-line subtraction of IDR from IK (Figure 1). About two-thirds the recorded neurons (30/46) exhibited a relatively fast IA component that inactivates ≥80% in 500 ms and accounts for 87 ± 1% of the total peak IK (range = 75–100%). After subtraction, the resulting currents of the remaining neurons displayed mostly slow-inactivating IDR and small non-inactivating IDR (data not shown). For further characterization, we focused on the population of neurons expressing relatively high levels of IA. The absolute IA ranges between 2.2 and 16.5 nA, and the mean IA density is 275 ± 34 pA/pF (at +30 mV, n = 30).


Kv4 Channels Underlie the Subthreshold-Operating A-type K-current in Nociceptive Dorsal Root Ganglion Neurons.

Phuket TR, Covarrubias M - Front Mol Neurosci (2009)

Isolation of IA in small DRG neurons. (A) Voltage-clamp protocol (top) to elicit the total K+ current (IK, bottom). From a holding voltage of −65 mV, a 1-s conditioning pulse at −100 mV precedes 500-ms step depolarizations delivered in 10-mV increments (for clarity, 20-mV increments are shown only). The start-to-start interval was 5 s. (B) Voltage-clamp protocol (top) to isolate the delayed-rectifier K+ current (IDR, bottom). The conditioning pulse at −30 mV inactivates the A-type K+ current (IA) (Figure 2). (C) The subtraction of IDR from IK yielded IA (bottom). These currents are from a DRG neuron (31 μm, 38 pF) exhibiting a dominant IA (∼90% IA and ∼10% IDR; see Results).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724030&req=5

Figure 1: Isolation of IA in small DRG neurons. (A) Voltage-clamp protocol (top) to elicit the total K+ current (IK, bottom). From a holding voltage of −65 mV, a 1-s conditioning pulse at −100 mV precedes 500-ms step depolarizations delivered in 10-mV increments (for clarity, 20-mV increments are shown only). The start-to-start interval was 5 s. (B) Voltage-clamp protocol (top) to isolate the delayed-rectifier K+ current (IDR, bottom). The conditioning pulse at −30 mV inactivates the A-type K+ current (IA) (Figure 2). (C) The subtraction of IDR from IK yielded IA (bottom). These currents are from a DRG neuron (31 μm, 38 pF) exhibiting a dominant IA (∼90% IA and ∼10% IDR; see Results).
Mentions: We conducted whole-cell patch-clamp measurements to ask whether the electrophysiological properties of the IA in small-medium diameter nociceptive DRG neurons (∼30 μm, ∼45 pF) are consistent with the expression of Kv4 channels. These channels are characteristically resistant to TEA (Amarillo et al., 2008; Birnbaum et al., 2004; Jerng and Covarrubias, 1997, 2004; Pak et al., 1991). Therefore, external TEA (20 mM) was present at all times to eliminate TEA-sensitive Kv channels (see Materials and Methods). In addition, by exploiting the distinct voltage dependence of inactivation of Kv4 channels, we employed voltage-clamp protocols to isolate the IA (Figure 1). To measure the total K+ current (IK), neurons were held at −65 mV, and a 1-s conditioning pulse to −100 mV was delivered prior to 500-ms step depolarizations that typically activate Kv channels. The conditioning pulse permits ∼90% recovery of the total IA that inactivated at −65 mV. Then, the IA was isolated from IK by a depolarizing 1-s conditioning prepulse to −30 mV. This depolarization is sufficient to inactivate the IA and, therefore, the remaining outward current evoked by subsequent step depolarizations is mostly comprised of a TEA-resistant delayed rectifying current (IDR). IA was finally revealed from the off-line subtraction of IDR from IK (Figure 1). About two-thirds the recorded neurons (30/46) exhibited a relatively fast IA component that inactivates ≥80% in 500 ms and accounts for 87 ± 1% of the total peak IK (range = 75–100%). After subtraction, the resulting currents of the remaining neurons displayed mostly slow-inactivating IDR and small non-inactivating IDR (data not shown). For further characterization, we focused on the population of neurons expressing relatively high levels of IA. The absolute IA ranges between 2.2 and 16.5 nA, and the mean IA density is 275 ± 34 pA/pF (at +30 mV, n = 30).

Bottom Line: Recent studies have demonstrated DRG hyperexcitability associated with downregulation of A-type K(+) channels; however, the molecular correlate of the corresponding A-type K(+) current (I(A)) has remained hypothetical.Among Kv4 transcripts, the DRG expresses significant levels of Kv4.1 and Kv4.3 mRNAs.Contrasting the expression patterns of Kv4 channels in the central and peripheral nervous systems, we discuss possible functional roles of these channels in primary sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College of Thomas Jefferson University Philadelphia, PA, USA.

ABSTRACT
The dorsal root ganglion (DRG) contains heterogeneous populations of sensory neurons including primary nociceptive neurons and C-fibers implicated in pain signaling. Recent studies have demonstrated DRG hyperexcitability associated with downregulation of A-type K(+) channels; however, the molecular correlate of the corresponding A-type K(+) current (I(A)) has remained hypothetical. Kv4 channels may underlie the I(A) in DRG neurons. We combined electrophysiology, molecular biology (Whole-Tissue and Single-Cell RT-PCR) and immunohistochemistry to investigate the molecular basis of the I(A) in acutely dissociated DRG neurons from 7- to 8-day-old rats. Whole-cell recordings demonstrate a robust tetraethylammonium-resistant (20 mM) and 4-aminopyridine-sensitive (5 mM) I(A). Matching Kv4 channel properties, activation and inactivation of this I(A) occur in the subthreshold range of membrane potentials and the rate of recovery from inactivation is rapid and voltage-dependent. Among Kv4 transcripts, the DRG expresses significant levels of Kv4.1 and Kv4.3 mRNAs. Also, single small-medium diameter DRG neurons ( approximately 30 mum) exhibit correlated frequent expression of mRNAs encoding Kv4.1 and Nav1.8, a known nociceptor marker. In contrast, the expressions of Kv1.4 and Kv4.2 mRNAs at the whole-tissue and single-cell levels are relatively low and infrequent. Kv4 protein expression in nociceptive DRG neurons was confirmed by immunohistochemistry, which demonstrates colocalization of Kv4.3 and Nav1.8, and negligible expression of Kv4.2. Furthermore, specific dominant-negative suppression and overexpression strategies confirmed the contribution of Kv4 channels to I(A) in DRG neurons. Contrasting the expression patterns of Kv4 channels in the central and peripheral nervous systems, we discuss possible functional roles of these channels in primary sensory neurons.

No MeSH data available.


Related in: MedlinePlus