Inwardly rectifying potassium channel Kir4.1 is responsible for the native inward potassium conductance of satellite glial cells in sensory ganglia

Abstract

Satellite glial cells (SGCs) surround primary afferent neurons in sensory ganglia, and increasing evidence has implicated the K(+) channels of SGCs in affecting or regulating sensory ganglion excitability. The inwardly rectifying K(+) (Kir) channel Kir4.1 is highly expressed in several types of glial cells in the central nervous system (CNS) where it has been implicated in extracellular K(+) concentration buffering. Upon neuronal activity, the extracellular K(+) concentration increases, and if not corrected, causes neuronal depolarization and uncontrolled changes in neuronal excitability. Recently, it has been demonstrated that knockdown of Kir4.1 expression in trigeminal ganglia leads to neuronal hyperexcitability in this ganglia and heightened nociception. Thus, we investigated the contribution of Kir4.1 to the membrane K(+) conductance of SGCs in neonatal and adult mouse trigeminal and dorsal root ganglia. Whole cell patch clamp recordings were performed in conjunction with immunocytochemistry and quantitative transcript analysis in various mouse lines. We found that in wild-type mice, the inward K(+) conductance of SGCs is blocked almost completely with extracellular barium, cesium and desipramine, consistent with a conductance mediated by Kir channels. We then utilized mouse lines in which genetic ablation led to partial or complete loss of Kir4.1 expression to assess the role of this channel subunit in SGCs. The inward K(+) currents of SGCs in Kir4.1+/- mice were decreased by about half while these currents were almost completely absent in Kir4.1-/- mice. These findings in combination with previous reports support the notion that Kir4.1 is the principal Kir channel type in SGCs. Therefore Kir4.1 emerges as a key regulator of SGC function and possibly neuronal excitability in sensory ganglia.

Figure 1. EGFP is colocalizes with glutamine synthetase (GS) in trigeminal and dorsal root ganglia SGCs

(A-C) Immunostaining for GS and intrinsic fluorescence of EGFP in Kir4.1-EGFP trigeminal ganglion. EGFP and GS co-localized in small fusiform cells (arrows) surrounding larger neurons (stars). The expression of GS and cell morphology indicate that EGFP+ cells constitute SGCs. (D-F) Immunostaining for GS and intrinsic fluorescence of EGFP in the Kir4.1-EGFP dorsal root ganglion. There is again co-localization of Kir4.1 and EGFP signals in SGCs (arrows) enveloping larger neurons (stars). Scale bars: 50 μm.

Figure 2. SGCs from wild-type trigeminal ganglion express a barium-sensitive inward conductance

(A) Electrophysiological recordings were performed on SGCs in partially intact ganglia from Kir4.1-EGFP mice. DIC image in the left panel shows the recording pipette on the fluorescent cell shown in the right panel (arrow). Right panel shows epifluorescence image of fluorescent SGCs. (B) Inward and outward currents were evoked by voltage steps from -160 to +30 mV for 300 ms in the in the absence (○) and presence (●) of 100μM barium in the bath. Dashed lines indicate zero current level. (C) Current magnitudes at the end of the voltage step are plotted against membrane potential for a representative cell the absence (○) and presence (●) of 100μM barium in the bath. (D) Average barium-sensitive currents plotted against membrane potential show an inward rectification profile (n=9). Scale bar: (A) 50μm.

Figure 3. SGCs from wild-type trigeminal ganglion express cesium-sensitive and desipramine-sensitive inward conductances

(A) Inward and outward currents evoked upon voltage steps from -160 to +30 mV in the absence (○, left panel) and presence (●, middle panel) of 1 mM extracellular cesium. Right panel shows the current-voltage relationship for a representative cell in the absence (○) and presence (●) of 1 mM extracellular cesium, demonstrating the strong blockade of inward currents at membrane potentials more hyperpolarized than -130mV. Dashed lines represent zero current level. (B) Inward and outward currents evoked upon voltage steps from -160 to +30 mV in the absence (○, left panel) and presence (●, middle panel) of 100 μM desipramine in the bath. Right panel shows the current-voltage relationship for a representative cell in the absence (○) and presence (●) of 100 μM desipramine in the bath. Current-voltage relationships for evoked currents show some relief of blockade at membrane potentials more hyperpolarized than -140 mV. Dashed lines represent zero current level. (C) Summary of the relative blockade at -140 mV for cesium (Cs), barium (Ba) or desipramine (Des) shows a relative blockade between 80 to 90% of the inward currents at -140 mV.

Figure 4. Kir4.1 transcript levels and inward currents are diminished in SGCs from trigeminal ganglia from Kir4+/- mice

(A-B) Kir4.1 transcript levels were analyzed in trigeminal ganglia from Kir4.1 +/- and wild-type littermates using real time RT-PCR. Summary of Kir4.1 mRNA expression in P30-P50 (left panel) and P8-P9 (right panel) shows the significant reduction of transcript levels in the Kir4.1 +/- mice (P<0.05). Dashed lines represent 50% of wild-type transcript levels. (C) Summary of the current density for SGCs from adult wild-type (circle) and heterozygous Kir4.1 +/- (dot) trigeminal ganglions (measured at -140 mV). Significantly smaller currents were recorded from SGCs in Kir4.1 +/- mice compared to wild-type (P<0.05). (D) Currents in the SGCs of Kir4.1 +/- mice were normalized against the currents obtained in wild-type littermates. Normalized current density for SGCs from (C) shows a significant reduction of 46% in current density recorded from heterozygous (dot) mice compared to wild-type (P<0.05). Dashed line indicates the half of the normalized mean value of wild-type current density.

Figure 5. Neonatal (P8-P9) wild-type mouse trigeminal ganglia SGCs express large inward currents sensitive to extracellular barium

(A) Left panel shows inward and outward currents evoked upon voltage steps of -160 to +30 mV in a representative trigeminal ganglion SGC from a wild-type neonatal (P8-P9) mouse. In the right panel the cell was exposed to 100 μM extracellular barium with blockade of inward current with little effect on outward currents. Dashed lines indicate zero current level. (B) I-V relationships in the absence (○) and presence (●) of 100μM barium in the bath for a representative cell from the SGC shown in (A). (C) Mean I-V relationships of barium-sensitive currents (n=6) in neonatal wild-type mouse trigeminal ganglia SGCs. (D) Summary of the relative blockade at -140 mV for cesium (Cs), barium (Ba) or desipramine (Des).

Figure 6. Lack of inward currents and barium-sensitive currents in the neonatal (P8-P9) Kir4.1 -/- mice SGCs

(A) Left panel shows inward and outward currents evoked upon voltage steps of -160 to +30 mV in a representative trigeminal ganglion SGC from Kir4.1 -/- mouse. Notice the almost complete absence of inward currents. In the right panel the cell was exposed to 100 μM extracellular barium with no apparent effect. Dashed lines indicate zero current level. (B) I-V relationships from the cell recorded in (A). (C) Mean I-V relationships of barium-sensitive currents recorded from Kir4.1 -/- mouse SGCs (n=9). (D) Summary of the relative blockade of currents at -140 mV for cesium (Cs), barium (Ba) or desipramine (Des) from Kir4.1 -/- mice shows the lack of expression of Kir channels. (E) Current density for SGCs from neonatal wild-type (circle), Kir4.1 +/- (dot) and Kir4.1 -/- (triangle) trigeminal ganglions (measured at -140 mV, 300ms) shows a graded reduction of inward currents with decreased expression of Kir4.1.

Figure 7. Evidence for functional Kir4.1-Kir5.1 heteromultimers in of adult (P30-P50) wild-type trigeminal ganglion SGCs

(A) Current traces from a representative cell before (left panel) and after (middle panel) application of 0.1 μM of the PKC activator PMA show the partial inhibition of inward currents upon PMA application. Right panel shows I-V relationship for the same cell in the absence (○) and presence (●) of 0.1μmM of the PKC activator PMA. Dashed lines represent zero current level. (B) Current traces from a representative cell before (left panel) and after (middle panel) application of the inactive analog of PMA 4α-PDD at 0.1μmM, which failed to exhibit any changes. Right panel shows I-V relationship for the same cell in the absence (○) and presence (●) of 4α-PDD at 0.1μmM. The inactive analog of PMA 4α-PDD at 0.1μmM failed to elicit any changes in either inward or outward currents Dashed lines represent zero current level. (C) Summary of the relative blockade of currents at -140 mV for PMA and 4a-PDD shows significant inhibition for PMA (P<0.05).