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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.


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Expression of Kv4. 3 and Nav1.8 proteins in DRG neurons. Fresh frozen DRG sections (10 μm) were treated with specific antibodies against either Kv4 or Nav1.8 proteins. The expression was then evaluated by immunofluorescence and confocal microscopy (left column, α-channel; see Materials and Methods). The cell nuclei were stained with DAPI or TO-PRO-3 (α-nucleus, middle column). The merging of the images in the left and middle columns is shown on the right column. (A) The Kv4.2 protein does not express in the DRG. (B) The Kv4.3 protein is widely expressed in a heterogeneous population of DRG neurons. (C) The expression of the Nav1.8 protein is more restricted but limited to DRG neurons. The Nav1.8 protein is a marker of nociceptors (see Results). (D) Although several DRG neurons were found to express either Nav1.8 or Kv4.3 proteins exclusively, a sub-population of nociceptive neurons co-expresses Kv4.3 and Nav1.8. Similar results were obtained in experiments from two additional rats. Scale bar = 50 μm.
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Figure 5: Expression of Kv4. 3 and Nav1.8 proteins in DRG neurons. Fresh frozen DRG sections (10 μm) were treated with specific antibodies against either Kv4 or Nav1.8 proteins. The expression was then evaluated by immunofluorescence and confocal microscopy (left column, α-channel; see Materials and Methods). The cell nuclei were stained with DAPI or TO-PRO-3 (α-nucleus, middle column). The merging of the images in the left and middle columns is shown on the right column. (A) The Kv4.2 protein does not express in the DRG. (B) The Kv4.3 protein is widely expressed in a heterogeneous population of DRG neurons. (C) The expression of the Nav1.8 protein is more restricted but limited to DRG neurons. The Nav1.8 protein is a marker of nociceptors (see Results). (D) Although several DRG neurons were found to express either Nav1.8 or Kv4.3 proteins exclusively, a sub-population of nociceptive neurons co-expresses Kv4.3 and Nav1.8. Similar results were obtained in experiments from two additional rats. Scale bar = 50 μm.

Mentions: Ten micrometer fresh frozen tissue sections were prepared on a Shandon Cryotome (Thermo Scientific Inc., Waltham, MA, USA) and directly mounted onto glass slides. DRG tissue sections were fixed in 4% paraformaldehyde and blocked in Phosphate Buffered Solution (PBS) containing 0.1% Triton X-100 and 10% of appropriate serum (goat or donkey). Doubling the concentration of detergent resulted in similar staining patterns (data not shown). Primary antibodies were diluted (1:100) in blocking solution and incubated overnight. Secondary antibodies were diluted (1:400) in blocking solution and incubated for 1 h. Nuclear DNA was stained by either treating tissue sections with TO-PRO-3 (Invitrogen, Carlsbad, CA, USA) for 1 h and mounting slides in ProLong Gold Antifade reagent (Invitrogen, Carlsbad, CA, USA) or directly mounting slides in ProLong Gold Antifade reagent containing DAPI (Invitrogen, Carlsbad, CA, USA). Images were captured using a scanning confocal microscope (Zeiss LSM 510 META-UV) at Kimmel Cancer Center Bioimaging Facility (Thomas Jefferson University, Philadelphia, PA, USA) and images were acquired using the AxioVision v4.6 software (Carl Zeiss, Inc, Thornwood, NY, USA). The co-localization of Kv4.3 in nociceptive neurons (Nav1.8-positive) was examined in double labeling experiments (Figure 5).


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

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

Expression of Kv4. 3 and Nav1.8 proteins in DRG neurons. Fresh frozen DRG sections (10 μm) were treated with specific antibodies against either Kv4 or Nav1.8 proteins. The expression was then evaluated by immunofluorescence and confocal microscopy (left column, α-channel; see Materials and Methods). The cell nuclei were stained with DAPI or TO-PRO-3 (α-nucleus, middle column). The merging of the images in the left and middle columns is shown on the right column. (A) The Kv4.2 protein does not express in the DRG. (B) The Kv4.3 protein is widely expressed in a heterogeneous population of DRG neurons. (C) The expression of the Nav1.8 protein is more restricted but limited to DRG neurons. The Nav1.8 protein is a marker of nociceptors (see Results). (D) Although several DRG neurons were found to express either Nav1.8 or Kv4.3 proteins exclusively, a sub-population of nociceptive neurons co-expresses Kv4.3 and Nav1.8. Similar results were obtained in experiments from two additional rats. Scale bar = 50 μm.
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Figure 5: Expression of Kv4. 3 and Nav1.8 proteins in DRG neurons. Fresh frozen DRG sections (10 μm) were treated with specific antibodies against either Kv4 or Nav1.8 proteins. The expression was then evaluated by immunofluorescence and confocal microscopy (left column, α-channel; see Materials and Methods). The cell nuclei were stained with DAPI or TO-PRO-3 (α-nucleus, middle column). The merging of the images in the left and middle columns is shown on the right column. (A) The Kv4.2 protein does not express in the DRG. (B) The Kv4.3 protein is widely expressed in a heterogeneous population of DRG neurons. (C) The expression of the Nav1.8 protein is more restricted but limited to DRG neurons. The Nav1.8 protein is a marker of nociceptors (see Results). (D) Although several DRG neurons were found to express either Nav1.8 or Kv4.3 proteins exclusively, a sub-population of nociceptive neurons co-expresses Kv4.3 and Nav1.8. Similar results were obtained in experiments from two additional rats. Scale bar = 50 μm.
Mentions: Ten micrometer fresh frozen tissue sections were prepared on a Shandon Cryotome (Thermo Scientific Inc., Waltham, MA, USA) and directly mounted onto glass slides. DRG tissue sections were fixed in 4% paraformaldehyde and blocked in Phosphate Buffered Solution (PBS) containing 0.1% Triton X-100 and 10% of appropriate serum (goat or donkey). Doubling the concentration of detergent resulted in similar staining patterns (data not shown). Primary antibodies were diluted (1:100) in blocking solution and incubated overnight. Secondary antibodies were diluted (1:400) in blocking solution and incubated for 1 h. Nuclear DNA was stained by either treating tissue sections with TO-PRO-3 (Invitrogen, Carlsbad, CA, USA) for 1 h and mounting slides in ProLong Gold Antifade reagent (Invitrogen, Carlsbad, CA, USA) or directly mounting slides in ProLong Gold Antifade reagent containing DAPI (Invitrogen, Carlsbad, CA, USA). Images were captured using a scanning confocal microscope (Zeiss LSM 510 META-UV) at Kimmel Cancer Center Bioimaging Facility (Thomas Jefferson University, Philadelphia, PA, USA) and images were acquired using the AxioVision v4.6 software (Carl Zeiss, Inc, Thornwood, NY, USA). The co-localization of Kv4.3 in nociceptive neurons (Nav1.8-positive) was examined in double labeling experiments (Figure 5).

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