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The KCNQ5 Potassium Channel Mediates a Component of the Afterhyperpolarization Current in Mouse Hippocampus

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The KCNQ5 Potassium Channel Mediates a Component of the Afterhyperpolarization Current in Mouse Hippocampus The KCNQ5 potassium channel mediates a component of the afterhyperpolarization current in mouse hippocampus Anastassios V. Tzingounisa,1,2, Matthias Heidenreichb,1, Tatjana Kharkovetsc, Guillermo Spitzmaulb, Henrik S. Jensend,3, Roger A. Nicolla,4, and Thomas J. Jentschb,c,4 aDepartment of Cellular and Molecular Pharmacology and Department of Physiology, University of California, San Francisco, CA 94143; bLeibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, D-13125, Germany; cZentrum für Molekulare Neurobiologie (ZMNH), Universität Hamburg, Hamburg, D-20251, Germany; and dDepartment of Medical Physiology, The Panum Institute, University of Copenhagen, Copenhagen, DK-2200, Denmark Contributed by Roger A. Nicoll, April 8, 2010 (sent for review February 18, 2010) Mutations in KCNQ2 and KCNQ3 voltage-gated potassium channels brainstem (18). Unlike KCNQ2 and KCNQ3, the function of lead to neonatal epilepsy as a consequence of their key role in KCNQ5 in the brain remains unknown and no neurological dis- regulating neuronal excitability. Previous studies in the brain have orders have been attributed to it. Given KCNQ5’s similar bio- focused primarily on these KCNQ family members, which contribute physical properties to other members of the KCNQ family, to M-currents and afterhyperpolarization conductances in multiple KCNQ5 may also contribute to M-currents (15, 16). To elucidate brain areas. In contrast, the function of KCNQ5 (Kv7.5), which also the physiological functions of KCNQ5 and test whether KCNQ5 displays widespread expression in the brain, is entirely unknown. also has a role in AHP currents, we generated knock-in (KI) mice Here, we developed mice that carry a dominant negative mutation carrying a dominant negative (dn) mutation in KCNQ5. In in the KCNQ5 pore to probe whether it has a similar function as other Kcnq5dn/dn mice, channels containing at least one KCNQ5 subunit KCNQ channels. This mutation renders KCNQ5dn-containing homo- are expected to be nonfunctional, including KCNQ3/KCNQ5 meric and heteromeric channels nonfunctional. We find that heterooligomers that normally yield higher currents than KCNQ5 Kcnq5dn/dn mice are viable and have normal brain morphology. Fur- homooligomers (15, 16). By contrast, the KO of Kcnq5 may par- thermore, expression and neuronal localization of KCNQ2 and adoxically increase M-currents in cells also expressing KCNQ2 KCNQ3 subunits are unchanged. However, in the CA3 area of hip- and KCNQ3 because KCNQ3 subunits that normally associate pocampus, a region that highly expresses KCNQ5 channels, the me- with KCNQ5 may now be free to assemble into more efficient dium and slow afterhyperpolarization currents are significantly KCNQ2/KCNQ3 heteromers. reduced. In contrast, neither current is affected in the CA1 area of Using these mice, we show here that KCNQ5 contributes to the hippocampus, a region with low KCNQ5 expression. Our results the mAHP and sAHP currents (ImAHP and IsAHP) in CA3 demonstrate that KCNQ5 channels contribute to the afterhyperpo- pyramidal neurons of the hippocampus. Our data present larization currents in hippocampus in a cell type-specific manner. a demonstration of a KCNQ5 function in the brain and further support the role of molecularly diverse KCNQ channels in me- M-current | sAHP | calcium | epilepsy | KCNQ diating AHP current components. train of action potentials can be terminated through the Results Aopening of multiple potassium channels activated by depo- Dominant Negative KCNQ5 KI Mice. To investigate the physiological larized voltages, incoming calcium, or both (1, 2). A slow calcium- roles of the KCNQ5 channel, we created a KI mouse carrying activated potassium conductance plays a prominent role in the a dominant negative mutation in KCNQ5. We mutated the first cessation of neuronal firing in most pyramidal neurons (3). This glycine of the GYG signature pore sequence of KCNQ5 to serine potassium conductance is highly regulated by various neuro- (G278S). Equivalent human mutations have been found in KCNQ1 transmitters and neuromodulators and has been implicated in with the dominant form of the long QT syndrome (19) and in KCNQ4 sleep-wake cycles (4), synchronized burst activity in neuronal in a family with dominant deafness (20). These mutations, as well as populations (5), long-term potentiation (6), and learning and equivalent mutations introduced into KCNQ2 and KCNQ3, were memory (7, 8). Recently, we have proposed that the calcium ac- shown to have dominant negative effects in heterologous expression tivation of this potassium slow afterhyperpolarization (sAHP) (15, 20, 21). KCNQ3 can form functional heteromers with KCNQ5 current is through hippocalcin (9), a diffusible neuronal calcium (15, 16). On heterologous expression in Xenopus oocytes, KCNQ3 sensor (NCS) that translocates to the membrane after binding induces currents that are barely above background. Coexpression cytosolic calcium (10). As a result, the sAHP current reports of KCNQ3 with KCNQ5 boosts currents in oocytes more than 2- global cytosolic calcium changes instead of only calcium fluctua- tions close to the membrane. Despite sAHP’s significant contri- bution to regulating neuronal excitability and unique gating Author contributions: A.V.T., R.A.N., and T.J.J. designed research; A.V.T., M.H., T.K., G.S., and H.S.J. performed research; A.V.T., M.H., and G.S. analyzed data; and A.V.T., M.H., G.S., mechanism, the potassium channels mediating this current re- R.A.N., and T.J.J. wrote the paper. main elusive (11). A possible breakthrough has come with the The authors declare no conflict of interest. observation that genetically induced loss of KCNQ2 or KCNQ3 1 function impairs the sAHP current in a cell type-specific manner These authors contributed equally to this work. 2 (12). This has raised the unexpected possibility that KCNQ Present address: Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT 06269. channels, known mediators of the M-current (13) and principal 3Present address: H. Lundbeck A/S, Department of Molecular Neurobiology, Copenhagen, contributors to medium afterhyperpolarization (mAHP) (2, 14), DK 2500, Denmark. might have a dual role in hippocampus. 4To whom correspondence may be addressed. E-mail: [email protected] or jentsch@ In addition to KCNQ2 and KCNQ3, KCNQ5 is widely distrib- fmp-berlin.de. – uted in the brain, including the hippocampus (15 17). In contrast, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. KCNQ4 is found only in a few nuclei and tracts mainly in the 1073/pnas.1004644107/-/DCSupplemental. 10232–10237 | PNAS | June 1, 2010 | vol. 107 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1004644107 Downloaded by guest on September 27, 2021 fold with respect to KCNQ5 homooligomers (15). Dominant nega- (G278S) in a 1:1.5:1.5 ratio exhibited a small current of about 15% of tive effects of KCNQ3(G318S) on KCNQ5 currents have been KCNQ3/KCNQ5 (1:3) current (Fig. 1 A and B), indicating that demonstrated before (15). We now tested whether KCNQ5(G278S), KCNQ5(G278S) also exerts a dominant negative effect on KCNQ3/ which is nonfunctional by itself (15), exerts dominant negative effects KCNQ5 heteromers. Currents were indistinguishable from back- ground when KCNQ3 was coexpressed with KCNQ5(G278S) (Fig. 1 on KCNQ5 and on KCNQ3/KCNQ5 heteromers (an effect on A B KCNQ3 homomers cannot be tested because of low currents). As and ), a situation that may arise in homozygous KI mice. Having verified the dominant negative effects of the KCNQ5 described before (15), currents increased more than 2-fold with re- (G278S) mutant, we introduced this mutation into the murine spect to KCNQ5 homomers when KCNQ3 and KCNQ5 were coin- Kcnq5 gene by homologous recombination in ES cells. For geno- A B jected at a 1:3 ratio (Fig. 1 and ). Coinjection of KCNQ5 WT with typing purposes, we also inserted a BsiWI restriction site (Fig. 1 C G278S mutant (1:1) decreased KCNQ5 current more than 90%, and D). These alterations required the mutation of a total of 4 base demonstrating the strong dominant negative effect of the mutant pairs. A neomycin (NEO) cassette flanked by loxP sites was inserted subunit. Oocytes injected with KCNQ3, KCNQ5, and KCNQ5 into intron 5 for selection. It was later removed by treating ES cells with Cre-recombinase (Fig. 1D). This allele is called Kcnq5dn. Kcnq5dn/dn mice were derived from targeted ES cells. Sequencing of genomic DNA from these mice confirmed the changes in the pore domain (Fig. 1D) and the presence of one remaining loxP site. We next compared KCNQ5 mRNA and protein levels between Kcnq5dn/dn and WT mice. Using quantitative RT-PCR of RNA extracted from total brain, we found no significant differences be- tween WT and Kcnq5dn/dn mice (Fig. 1E). Similarly, Western blot analysis did not reveal any differences in the expression of the KCNQ5 protein levels in whole brain between WT and Kcnq5dn/dn mice (Fig. 1F). Importantly, the expression of KCNQ2 and KCNQ3 was unchanged in Kcnq5dn/dn mice (Fig. 1F and Fig. S1), suggesting that there is no compensatory up-regulation of those other major KCNQ isoforms in Kcnq5dn/dn brain. Moreover, the subcellular lo- calization of KCNQ2 and KCNQ3 subunits to axon initial segments of hippocampal pyramidal cells was not changed (Fig. 2A). Mice carrying the dominant negative allele were born at a less than Mendelian ratio. Among 147 pups resulting from crossing Kcnq5+/dn mice, we observed 41.4% Kcnq5+/+,46.2%Kcnq5+/dn, and 12.4% Kcnq5dn/dn animals. However, Kcnq5dn/dn mice displayed normal postnatal development and lifespan and lacked visible phenotypes. The morphology of the brains of Kcnq5dn/dn mice is grossly normal, as revealed by histological analysis at two different ages, postnatal day (PD) 65 (Fig. 2B) and PD 18 (Fig. S2), in regions of high Kcnq5 expression (15), such as the hippocampus and cortex, or in regions of low expression, such as the cerebellum (Fig.
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