SEARCH

SEARCH BY CITATION

Keywords:

  • RNA editing;
  • Kv1.1 channel;
  • 4-aminopyridine

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

In rat brain slices, the Kv channel blocker 4-aminopyridine (4-AP) induces seizure-like events. This effect is absent in slices from chronic epileptic rats generated using the kainic acid model. The reason for this phenomenon remained elusive as an altered expression level of Kv channels was ruled out as a mechanism. We recently described that the Ile400Val RNA editing of Kv1.1 generates 4-AP–insensitive Kv1 channels (Kv1.1I400V). We therefore hypothesized that altered RNA editing levels account for the reduced ictogenic potency of 4-AP in chronic epileptic rats. We found fourfold increased RNA editing ratios in the entorhinal cortex of chronic epileptic animals compared to healthy control animals. Electrophysiologic recordings in Xenopus oocytes revealed that the observed increased Kv1.1I400V editing level can in fact lead to significant loss of 4-AP sensitivity. Our data suggest that altered Kv1.1I400V RNA editing contributes to the reduced ictogenic potential of 4-AP in chronic epileptic rats.

4-Aminopyridine (4-AP) is known as a potent convulsant in vivo (Spyker et al., 1980) as well as in vitro (Zahn et al., 2008). When applied to rat brain slices, 4-AP induces seizure-like events (SLEs). In a previous study we reported that 4-AP lacks this effect in brain slices from chronic epileptic rats of the kainic acid model (Zahn et al., 2008). A similar phenomenon was observed in human brain slices of patients with pharmacoresistant temporal lobe epilepsy (Gabriel et al., 2004). At ictogenic concentrations of 4-AP (50–100 μm) the drug blocks potassium channels of the Kv1 and Kv3 family. The generally accepted mechanism for the ictogenic potential of 4-AP action is that the blockade of Kv1 and Kv3 channels results in an increased transmitter release. Quantitative polymerase chain reaction (PCR) and immunocytochemical experiments did not reveal significant changes in the expression level of Kv1 and Kv3 family members between healthy and epileptic animals. Only the expression level of Kv3.4 was altered, but this channel has only a low 4-AP affinity (Zahn et al., 2008). Therefore, it remained unclear why 4-AP loses its ictogenic potential in chronic epileptic rats. The Kv1.1 channel is expressed in all parts of the examined brain slices, including all layers of the entorhinal and perirhinal cortex. Kv1.1 mRNA is a target for enzymatic deamination by adenosine deaminase acting on RNA (ADAR2). Mice with a knock-out of the ADAR2 gene are prone to epileptic seizures and die within a few weeks after birth (Higuchi et al., 2000). A lack of GluR-B editing leads to Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors leading to epilepsy (Sommer et al., 1991; Brusa et al., 1995). Kv1.1 editing by ADAR2 results in channels with an Ile400Val exchange in the S6 segment. We recently reported that Ile400Val–edited Kv1.1 channels (written as Kv1.1I400V in the following) have a reduced drug affinity. In addition, Kv1.1I400V subunits form tetrameric channels with other Kv1.x subunits, resulting in altered pharmacology for the complete Kv1 channel family (Decher et al., 2010).

The objective of the present study was to test whether RNA editing of Kv1.1 accounts for the failure of 4-AP to induce SLEs in chronic epilepsy. We found increased levels of Kv1.1I400V editing in chronic epileptic rats. Our electrophysiologic recordings indicate that the observed increase in Kv1.1I400V editing might cause a significant loss in 4-AP sensitivity of Kv1 channels. Our study suggests a novel mechanism for the understanding of changed electrophysiology and pharmacology in chronic epilepsy.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Animal groups and status epilepticus induction

For the induction of status epilepticus (SE) and later epilepsy, kainic acid (5 mg/kg) was injected intraperitoneally (n = 13; 175–250 g) in Sprague-Dawley rats (Harlan CPB Laboratories, Zeist, The Netherlands). Seizure activity was rated according to Racine’s scale [class I–V seizures; (Racine, 1972)] during the injection period and at least 4 h thereafter (Tolner et al., 2007). The control group consisted of age-matched rats (n = 8) treated with saline. At 2.5 months after SE, the rats were observed by video recording for 36 h to determine their epileptic condition (Zahn et al., 2008). All animal procedures were conducted in accordance with the guidelines of the European Communities Council directive 86/609/EEC and were approved by the Regional Berlin Animal Ethics Committee (LaGeTSi No. G 0328/98).

Slice preparation from control and chronic epileptic rats

Slices were prepared from chronic epileptic rats and control rats at 2.5–10 months (mean = 5.2 months) after injections, as described previously (Zahn et al., 2008). Combined entorhinal cortex–hippocampal horizontal slices (400 μm) were prepared as described previously (Dreier & Heinemann, 1991), using a Vibroslicer (World Precision Instruments, Berlin, Germany). Slices were transferred immediately to a custom-made interface chamber maintained at 34°C and perfused with artificial cerebrospinal fluid CSF saturated with carbogen gas. Field potential recordings and aCSF solution were used as described previously (Zahn et al., 2008).

Quantification of RNA editing in rat neurons

We amplified via PCR the S6 segments using gene-specific primers binding to the S5-pore linker and the C-termini of the Kv1.1 channel. The PCR products were subcloned in the pGEM-T Easy vector and transformed into DH5-α cells. After transformation of the PCR fragments, multiple clones were picked and sequenced to quantify the fraction of edited transcripts. For more information see Data S1.

Two electrode voltage clamp

Isolation of Xenopus oocytes, cRNA synthesis, and two-electrode voltage-clamp recordings of Kv1.1 were performed as described previously (Decher et al., 2010). As voltage protocol one depolarizing step to 0 mV was repeated every 10 s from the holding potential of −80 mV. Data are expressed as mean ± standard error of the mean (SEM).

Results

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Kv1.1I400V editing might modulate the ictogenic potential of 4-AP in epileptic rats

We have recently observed that in slices of the perirhinal and the entorhinal cortex of kainic acid–treated chronic epileptic rats, 4-AP has a reduced ictogenic potential as compared to slices from control rats (Zahn et al., 2008). In these experiments, 50–100 μm 4-AP induced SLEs in slices from control rats but not from chronic epileptic rats. Similar experiments serve as a control in the present study to prove the lack of 4-AP sensitivity in our set of animals (Fig. 1A–B). In our RNA editing experiments, we focused on the entorhinal cortex, as it is known to be a common site of seizure onset in human patients with temporal lobe epilepsy (Spencer & Spencer, 1994). When we analyzed the extent of I400V editing in the entorhinal cortex of chronic epileptic and control rats, we found that only 5.1% of the Kv1.1 mRNA transcripts were edited in wild-type entorhinal cortex, whereas in chronic epileptic rats 21.5% of the transcripts were edited (Fig. 1C).

image

Figure 1.   Reduced ictogenic potential of 4-AP and increased Kv1.1I400V RNA editing in the entorhinal cortex (EC) of rats with chronic epilepsy. (A) Sample records showing seizure-like events (SLEs) triggered by 100 μm 4-AP in control animals. (B) Sample records from our animals with chronic epilepsy where 100 μm 4-AP failed to induce SLEs. Inset shows that even over a long period of time no SLEs were observed. (C) Quantification of the fraction of edited Kv1.1 RNA in samples of EC from healthy and epileptic rats (n = 50).

Download figure to PowerPoint

Next we constructed a Kv1.1I400V mutant for pharmacologic experiments after heterologous expression in Xenopus oocytes. This mutant mimicking edited Kv1.1 channels was coexpressed with wild-type Kv1.1 subunits. Utilizing this approach, we analyzed if an increased editing ratio can account for the observed changes in 4-AP sensitivity. Therefore, we injected either wild-type Kv1.1 cRNA or a mixture of 75% Kv1.1 and 25%“edited” Kv1.1I400V cRNA in Xenopus oocytes to reproduce the observed RNA editing of wild-type and epileptic animals. We then tested the 4-AP sensitivity of the resulting currents (Fig. 2). 500 μm 4-AP blocked unedited Kv1.1 currents by 80.9 ± 0.7% (n = 5). In contrast, after expressing a Kv1.1 cRNA mix containing 25% Kv1.1I400V transcripts, the currents were blocked only by 49.2 ± 2.9% (n = 5) (Fig. 2A–B). At 100 μm of 4-AP, a concentration previously used to induce ictaform events (Zahn et al., 2008), the block was reduced from 44.4 ± 4.2% (n = 8) to 20.6 ± 3.7% (n = 9) by the Kv1.1I400V editing (Fig. 2B). In Xenopus oocytes the half maximal inhibitory concentration (IC50) for 4-AP was increased from from 98 to 619 μm, reflecting a 6.3-fold change. The Kv1.1I400V editing reduced the affinity for 4-AP, whereas the kinetics of block—that is, the voltage dependence of block, voltage dependence of activation, and the time course of onset of block—were not changed compared to wild-type Kv1.1 channels (Fig. S1).

image

Figure 2.   Partial Kv1.1I400V editing as found in the EC of epileptic rats changes the sensitivity of Kv1.1 channels to 4-AP. (A) Inhibition of Kv1.1 channels by 500 μm 4-AP in Xenopus oocytes after injection of 100% Kv1.1 or 75% Kv1.1 plus 25% Kv1.1I400V cRNA. (B) Statistics of block by 500 or 100 μm 4-AP measured at 0 mV after injection of 100% Kv1.1 or 75% Kv1.1 plus 25% Kv1.1I400V cRNA. **Mark a p-value below 0.01.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Kortenbruck et al. (2001) found that the ADAR2–mediated editing of GluR5 and GluR6 subunits is upregulated in patients with epilepsy. The authors propose that increased editing levels reduce the Ca2+ permeability providing a compensatory mechanism for chronic epilepsy. We observed in our study that increased RNA editing of Kv1.1 channels in chronic epilepsy alters the IC50 of Kv channels for 4-AP. This can at least partially explain the previously observed reduction of ictogenic potential of 4-AP in chronic epileptic rats. Therefore, RNA editing of Kv1.1 in chronic epilepsy might contribute to the reorganization of the EC and may have anticonvulsive effects. The editing of Kv1.1 mRNA could therefore constitute a compensating mechanism in brain areas that are origins of epileptic seizures. The edited site in the pore-forming S6 segment of the channels is part of the binding site for many channel blockers (Decher et al., 2010). In a recent publication, we described the dominant negative effect of a single edited Kv1.1 subunit on the block of tetrameric Kv1 channels by different blockers (Decher et al., 2010). Interestingly, also the effect of the endogenous Kv channel blocker arachidonic acid is drastically reduced by Kv1.1I400V editing. Therefore, the regulation of Kv1.1 by arachidonic acid might also be altered in the epileptic brain. We assume that the altered Kv1.1 RNA editing might also influence the action of other convulsive agents affecting Kv channels. The physiologic relevance of this remains to be elucidated. In addition, it has to be considered that the increased editing ratios may not be restricted to the mRNA of Kv1.1. Other nervous system targets were described to be edited by ADAR2 (Hoopengardner et al., 2003), including several GluR subunits and the 5-HT2C receptor. However, those targets are unlikely to account for the reduced potency of 4-AP, as neither GluRs nor 5-HT2C receptors are sensitive to 4-AP. Hoopengardner et al. (2003) have described five different RNA editing sites in the Drosophila Shaker channel, the ortholog of the Kv1.x channels. However, it appears that editing of human Kv1.x channels is restricted to Kv1.1I400V (Bhalla et al., 2004; Li et al., 2009; Decher et al., 2010).

In future experiments, a precise analysis of changes in Kv1.1I400V editing in different brain regions might provide more insight to the electrical remodeling in chronic epilepsy. We conclude that an increased Kv1.1I400V editing ratio is an important factor for the altered electrophysiology and pharmacology in chronic epilepsy.

Acknowledgments

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

This work was supported by the Deutsche Forschungsgemeinschaft DE-1482/3-1 and DE-1482/2-1 to ND, by Research Grants of the University Medical Center Giessen and Marburg to ND, by the P.E. Kempkes Stiftung 12/07 to ND, and by the SFB TR3 as well as by the EU via EpiCure. We would like to thank Andrea Schubert for technical support.

Disclosure

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

References

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
  • Bhalla T, Rosenthal JJ, Holmgren M, Reenan R. (2004) Control of human potassium channel inactivation by editing of a small mRNA hairpin. Nat Struct Mol Biol 11:950956.
  • Brusa R, Zimmermann F, Koh DS, Feldmeyer D, Gass P, Seeburg PH, Sprengel R. (1995) Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice. Science 270:16771680.
  • Decher N, Streit AK, Rapedius M, Netter MF, Marzian S, Ehling P, Schlichthorl G, Craan T, Renigunta V, Kohler A, Dodel RC, Navarro-Polanco RA, Preisig-Muller R, Klebe G, Budde T, Baukrowitz T, Daut J. (2010) RNA editing modulates the binding of drugs and highly unsaturated fatty acids to the open pore of Kv potassium channels. EMBO J 29:21012113.
  • Dreier JP, Heinemann U. (1991) Regional and time dependent variations of low Mg2+ induced epileptiform activity in rat temporal cortex slices. Exp Brain Res 87:581596.
  • Gabriel S, Njunting M, Pomper JK, Merschhemke M, Sanabria ER, Eilers A, Kivi A, Zeller M, Meencke HJ, Cavalheiro EA, Heinemann U, Lehmann TN. (2004) Stimulus and potassium-induced epileptiform activity in the human dentate gyrus from patients with and without hippocampal sclerosis. J Neurosci 24:1041610430.
  • Higuchi M, Maas S, Single FN, Hartner J, Rozov A, Burnashev N, Feldmeyer D, Sprengel R, Seeburg PH. (2000) Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 406:7881.
  • Hoopengardner B, Bhalla T, Staber C, Reenan R. (2003) Nervous system targets of RNA editing identified by comparative genomics. Science 301:832836.
  • Kortenbruck G, Berger E, Speckmann EJ, Musshoff U. (2001) RNA editing at the Q/R site for the glutamate receptor subunits GLUR2, GLUR5, and GLUR6 in hippocampus and temporal cortex from epileptic patients. Neurobiol Dis 8:459468.
  • Li JB, Levanon EY, Yoon JK, Aach J, Xie B, Leproust E, Zhang K, Gao Y, Church GM. (2009) Genome-wide identification of human RNA editing sites by parallel DNA capturing and sequencing. Science 324:12101213.
  • Racine RJ. (1972) Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32:281294.
  • Sommer B, Kohler M, Sprengel R, Seeburg PH. (1991) RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67:1119.
  • Spencer SS, Spencer DD. (1994) Entorhinal-hippocampal interactions in medial temporal lobe epilepsy. Epilepsia 35:721727.
  • Spyker DA, Lynch C, Shabanowitz J, Sinn JA. (1980) Poisoning with 4-aminopyridine: report of three cases. Clin Toxicol 16:487497.
  • Tolner EA, Frahm C, Metzger R, Gorter JA, Witte OW, Lopes da Silva FH, Heinemann U. (2007) Synaptic responses in superficial layers of medial entorhinal cortex from rats with kainate-induced epilepsy. Neurobiol Dis 26:419438.
  • Zahn RK, Tolner EA, Derst C, Gruber C, Veh RW, Heinemann U. (2008) Reduced ictogenic potential of 4-aminopyridine in the perirhinal and entorhinal cortex of kainate-treated chronic epileptic rats. Neurobiol Dis 29:186200.

Supporting Information

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Data S1. Methods.

Figure S1. Kinetics of Kv1.1 channel block by 100 μm 4-AP, studied in Xenopusoocytes.

FilenameFormatSizeDescription
EPI_2986_sm_figS1.pdf171KSupporting info item
EPI_2986_sm_SupportingMethods.pdf58KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.