Transcriptional expression of voltage-gated Na⁺ and voltage-independent K⁺ channels in the developing rat superficial dorsal horn

Abstract

Neurons within the superficial dorsal horn (SDH) of the rodent spinal cord exhibit distinct firing properties during early life. While this may reflect a unique combination of voltage-gated Na(+) (Na(v)) and voltage-independent (i.e. "leak'') K(+) channels which strongly influence neuronal excitability across the CNS, surprisingly little is known about which genes encoding for Na(v) and leak K(+) channels are expressed within developing spinal pain circuits. The goal of the present study was therefore to characterize the transcriptional expression of these channels within the rat SDH at postnatal days (P) 3, 10, 21 or adulthood using quantitative real-time polymerase chain reaction. The results demonstrate that Na(v) isoforms are developmentally regulated at the mRNA level in a subtype-specific manner, as Na(v)1.2 and Na(v)1.3 decreased significantly from P3 to adulthood, while Na(v)1.1 was up-regulated during this period. The data also indicate selective, age-dependent changes in the mRNA expression of two-pore domain (K(2P)) K(+) channels, as TWIK-related acid-sensitive K(+) channels TASK-1 (KCNK3) and TASK-3 (KCNK9) were down-regulated during postnatal development in the absence of any changes in the tandem of pore domains in a weak inward rectifying K(+) channel (TWIK) isoforms examined (KCNK1 and KCNK6). In addition, a developmental shift occurred within the TREK subfamily due to decreased TREK-2 (KCNK10) mRNA within the mature SDH. Meanwhile, G-protein-coupled inward rectifying K(+) channels (K(ir)3.1 and K(ir)3.2) were expressed in the SDH at mature levels from birth. Overall, the results suggest that the transcription of ion channel genes occurs in a highly age-dependent manner within the SDH, raising the possibility that manipulating the expression or function of ion channels which are preferentially expressed within immature nociceptive networks could yield novel approaches to relieving pain in infants and children.

Figure 1. Age-dependent changes in the expression of voltage-gated Na⁺ channel (Na_v) genes in the superficial dorsal horn (SDH)

A: Plot of developmental expression ratios (relative to adult; see Methods) for selected voltage-gated Na⁺ channel isoforms at P3, P10 and P21 illustrating significant developmental changes in the mRNA levels of Na_v1.1, Na_v1.2 and Na_v1.3 with no alterations in Na_v1.6. *p<0.05, **p<0.01; Kruskal-Wallis test with Dunn's Multiple Comparison post-test. B: Plot of mRNA amounts relative to a reference gene (rREF) index (see Methods) to allow for the qualitative comparison of relative expression levels between the different Na_v isoforms, demonstrating that Na_v expression at P3 is dominated by Na_v1.2 and Na_v1.3 with minimal expression of Na_v1.1.

Figure 2. Developmental reduction in TASK K⁺ channel mRNA expression in the SDH

A: Both KCNK3 (i.e. TASK-1) and KCNK9 (TASK-3) exhibited a significant decrease in expression over the course of postnatal development. *p<0.05, Kruskal-Wallis test with Dunn's post-test. B: KCNK9 was more highly expressed in lamina I–II compared to KCNK3 throughout life.

Figure 3. Immunohistochemical localization of TASK channels within the developing SDH

Top: TASK-1 expression (green) is co-localized (yellow arrow) with neuronal marker NeuN (red), and also expressed in NeuN-negative profiles (white arrow). Middle: NeuN and TASK-3 are co-expressed (yellow arrow) and TASK-3 is also expressed in the absence of NeuN (white arrow). Bottom: Omitting the primary antibody to the TASK channels or NeuN produces no visible signal. The illustrated borders of lamina I–II (left) were estimated based on prior work showing that lamina I measures ~40 μm thick throughout postnatal development (Lorenzo et al., 2008).

Figure 4. Postnatal expression of TWIK K⁺ channel transcripts within spinal nociceptive circuits

A: mRNA levels of KCNK1 (TWIK-1) and KCNK6 (TWIK-2) were not significantly different between P3 and adulthood, though a transient up-regulation of KCNK1 was observed at P21. *p<0.05, Kruskal-Wallis test with Dunn's post-test. B: KCNK6 was minimally expressed in lamina I–II throughout the postnatal period.

Figure 5. Subtype-specific regulation of TREK K⁺ channel mRNA expression within the developing SDH

A: While KCNK4 (TRAAK) mRNA levels were significantly increased during the first three weeks of life, KCNK10 (TREK-2) expression was reduced during the same time period. *p<0.05, **p<0.01; Kruskal-Wallis test with Dunn's post-test. B: KCNK4 and KCNK10 are the predominant TREK channels expressed within the SDH throughout development.

Figure 6. Stable expression of G-protein-coupled inward rectifying K⁺ channels (GIRKs) within the developing SDH

A: Neither K_ir3.1 (GIRK1) nor K_ir3.2 (GIRK2) exhibited significant age-dependent alterations in mRNA levels within lamina I–II of the spinal cord. B: The two isoforms are expressed at comparative levels throughout the postnatal period.