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. 2007 Dec 15;585(Pt 3):867-79.
doi: 10.1113/jphysiol.2007.145649. Epub 2007 Oct 25.

TRESK two-pore-domain K+ channels constitute a significant component of background potassium currents in murine dorsal root ganglion neurones

Tina Dobler  1 , Andreas Springauf, Stefanie Tovornik, Maruschka Weber, Angelika Schmitt, Reinhard Sedlmeier, Erhard Wischmeyer, Frank Döring
Affiliations

TRESK two-pore-domain K+ channels constitute a significant component of background potassium currents in murine dorsal root ganglion neurones

Tina Dobler et al. J Physiol. .

Abstract

TRESK (TWIK-related spinal cord K(+) channel) is the most recently identified member of the two-pore-domain potassium channel (K(2P)) family, the molecular source of background potassium currents. Human TRESK channels are not affected by external acidification. However, the mouse orthologue displays moderate pH dependence isolated to a single histidine residue adjacent to the GYG selectivity filter. In the human protein, sequence substitution of tyrosine by histidine at this critical position generated a mutant that displays almost identical proton sensitivity compared with mouse TRESK. In contrast to human TRESK, which is specifically located in spinal cord, we detected mouse TRESK (mTRESK) mRNA in several epithelial and neuronal tissues including lung, liver, kidney, brain and spinal cord. As revealed by endpoint and quantitative RT-PCR, mTRESK channels are mainly expressed in dorsal root ganglia (DRG) and on the transcript level represent the most important background potassium channel in this tissue. DRG neurones of all sizes were labelled by in situ hybridizations with TRESK-specific probes. In DRG neurones of TRESK[G339R] functional knock-out (KO) mice the standing outward current IK(so) was significantly reduced compared with TRESK wild-type (WT) littermates. Different responses to K(2P) channel regulators such as bupivacaine, extracellular protons and quinidine corroborated the finding that approximately 20% of IK(so) is carried by TRESK channels. Unexpectedly, we found no difference in resting membrane potential between DRG neurones of TRESK[WT] and TRESK[G339R] functional KO mice. However, in current-clamp recordings we observed significant changes in action potential duration and amplitude of after-hyperpolarization. Most strikingly, cellular excitability of DRG neurones from functional KO mice was significantly augmented as revealed by reduced rheobase current to elicit action potentials.

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Figures

Figure 1
Figure 1. Extracellular proton sensitivity of wild-type and mutant TRESK channels from mouse and human
A and C, two-electrode voltage-clamp recordings from Xenopus oocytes injected with cRNA of mouse (A) and human (C) TRESK. Current–voltage relations are shown from oocytes expressing wild-type (left panels) and mutant (right panels) TRESK channels in response to voltage ramps from −150 mV to +60 mV at physiological and acidic extracellular pH as indicated. B and D, pH–current response curves of mouse (B) and human (D) TRESK channels expressed in Xenopus oocytes. Relative amplitudes (I/IpH7.4) of wild-type and mutant TRESK currents are plotted against pH.
Figure 2
Figure 2. Relative expression profile of mouse TRESK channels
A, using endpoint PCR, an amplicon of 288 bp indicates expression of TRESK in different mouse tissues as indicated (upper panel). To monitor sample integrity a 424 bp fragment of GAPDH was PCR-amplified in parallel (lower panel). Bar graph in B shows normalized gene expression in DRGs of diverse K2P channels as indicated. Data are presented in arbitrary units as mean ± s.d. calculated from four individual tissue samples. General presence of various K2P channels in DRGs was monitored by endpoint PCR as depicted in the agarose gel of the inset.
Figure 3
Figure 3. In situ hybridization of TRESK mRNA in mouse dorsal root ganglia
Neuronal expression of TRESK was detected with digoxigenin-labelled cRNA probes. A, bright-field photomicrograph using Nomarski optics shows stained sections of ganglia with transcripts of TRESK located in DRG neurones of all sizes. Different signal intensities do not correlate with the size of the neurones. Scale bar, 50 μm. B and C, high-power magnification of Nissl-stained section with TRESK mRNA detected in most DRG neurones (B) and signal specificity documented by unlabelled neurones hybridized with sense cRNA probes (C). As indicated by the arrow, a single DRG neurone in B does not express TRESK mRNA. Small-diameter satellite glial cells surrounding DRG neurones are visualized by dark staining. Scale bar, 20 μm.
Figure 4
Figure 4. Whole-cell voltage-clamp recordings from cultured DRG neurones
Left panel, depolarizing step from −70 mV holding potential to −25 mV elicits a standing outward current that is inhibited by bupivacaine, acidification and quinidine. Bar graph to right depicts the normalized remaining current after application of inhibitors as indicated.
Figure 5
Figure 5. Comparison of IKso in DRG neurones from TRESK[WT] and TRESK[G339R] mice
A, upper traces show standing outward currents from TRESK[WT] and TRESK[G339R] mice upon an initial depolarizing step to −25 mV. Bupivacaine-derived subtraction currents (lower traces) reversed at −84 ± 9.9 mV (TRESK[WT], black trace) and −82 ± 23 mV (TRESK[G339R], grey trace), respectively. Bar graph in B quantifies IKso amplitude at a depolarizing step to −25 mV for TRESK[WT] and TRESK[G339R] mice. Bar graph in C compares inhibition of IKso in both genotypes as indicated.
Figure 6
Figure 6. Current-clamp recordings in DRG neurones from TRESK[WT] and TRESK[G339R] mice
A, current injections of 50 pA steps depolarized DRG neurones from their normal resting potential to the threshold of action potential (AP) induction. Neurones from TRESK[WT] mice (left recording) were less excitable than neurones from TRESK[G339R] mice (right recording). B, bar graph shows significant difference between genotypes (TRESK[WT], filled columns; TRESK[G339R], open columns) in rheobase current, but not in overall amplitude of APs and resting membrane potential. C, current-clamp whole-cell recordings display differences in AP duration and amplitude of after-hyperpolarization between genotypes (left, TRESK[WT]; right, TRESK[G339R]). Bar graph in D compares AP duration and amplitude of after-hyperpolarization in both genotypes.
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