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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2010 May 13;107(22):10232–10237. doi: 10.1073/pnas.1004644107

The KCNQ5 potassium channel mediates a component of the afterhyperpolarization current in mouse hippocampus

Anastassios V Tzingounis a,1,2, Matthias Heidenreich b,1, Tatjana Kharkovets c, Guillermo Spitzmaul b, Henrik S Jensen d,3, Roger A Nicoll a,4, Thomas J Jentsch b,c,4
PMCID: PMC2890451  PMID: 20534576

Abstract

Mutations in KCNQ2 and KCNQ3 voltage-gated potassium channels lead to neonatal epilepsy as a consequence of their key role in regulating neuronal excitability. Previous studies in the brain have focused primarily on these KCNQ family members, which contribute to M-currents and afterhyperpolarization conductances in multiple brain areas. In contrast, the function of KCNQ5 (Kv7.5), which also displays widespread expression in the brain, is entirely unknown. Here, we developed mice that carry a dominant negative mutation in the KCNQ5 pore to probe whether it has a similar function as other KCNQ channels. This mutation renders KCNQ5dn-containing homomeric and heteromeric channels nonfunctional. We find that Kcnq5dn/dn mice are viable and have normal brain morphology. Furthermore, expression and neuronal localization of KCNQ2 and KCNQ3 subunits are unchanged. However, in the CA3 area of hippocampus, a region that highly expresses KCNQ5 channels, the medium and slow afterhyperpolarization currents are significantly reduced. In contrast, neither current is affected in the CA1 area of the hippocampus, a region with low KCNQ5 expression. Our results demonstrate that KCNQ5 channels contribute to the afterhyperpolarization currents in hippocampus in a cell type-specific manner.

Keywords: M-current, sAHP, calcium, epilepsy, KCNQ

A train of action potentials can be terminated through the opening of multiple potassium channels activated by depolarized voltages, incoming calcium, or both (1, 2). A slow calcium-activated potassium conductance plays a prominent role in the cessation of neuronal firing in most pyramidal neurons (3). This potassium conductance is highly regulated by various neurotransmitters and neuromodulators and has been implicated in sleep-wake cycles (4), synchronized burst activity in neuronal populations (5), long-term potentiation (6), and learning and memory (7, 8). Recently, we have proposed that the calcium activation of this potassium slow afterhyperpolarization (sAHP) current is through hippocalcin (9), a diffusible neuronal calcium sensor (NCS) that translocates to the membrane after binding cytosolic calcium (10). As a result, the sAHP current reports global cytosolic calcium changes instead of only calcium fluctuations close to the membrane. Despite sAHP's significant contribution to regulating neuronal excitability and unique gating mechanism, the potassium channels mediating this current remain elusive (11). A possible breakthrough has come with the observation that genetically induced loss of KCNQ2 or KCNQ3 function impairs the sAHP current in a cell type-specific manner (12). This has raised the unexpected possibility that KCNQ channels, known mediators of the M-current (13) and principal contributors to medium afterhyperpolarization (mAHP) (2, 14), might have a dual role in hippocampus.

In addition to KCNQ2 and KCNQ3, KCNQ5 is widely distributed in the brain, including the hippocampus (15–17). In contrast, KCNQ4 is found only in a few nuclei and tracts mainly in the brainstem (18). Unlike KCNQ2 and KCNQ3, the function of KCNQ5 in the brain remains unknown and no neurological disorders have been attributed to it. Given KCNQ5’s similar biophysical properties to other members of the KCNQ family, KCNQ5 may also contribute to M-currents (15, 16). To elucidate the physiological functions of KCNQ5 and test whether KCNQ5 also has a role in AHP currents, we generated knock-in (KI) mice carrying a dominant negative (dn) mutation in KCNQ5. In Kcnq5dn/dn mice, channels containing at least one KCNQ5 subunit are expected to be nonfunctional, including KCNQ3/KCNQ5 heterooligomers that normally yield higher currents than KCNQ5 homooligomers (15, 16). By contrast, the KO of Kcnq5 may paradoxically increase M-currents in cells also expressing KCNQ2 and KCNQ3 because KCNQ3 subunits that normally associate with KCNQ5 may now be free to assemble into more efficient KCNQ2/KCNQ3 heteromers.

Using these mice, we show here that KCNQ5 contributes to the mAHP and sAHP currents (ImAHP and IsAHP) in CA3 pyramidal neurons of the hippocampus. Our data present a demonstration of a KCNQ5 function in the brain and further support the role of molecularly diverse KCNQ channels in mediating AHP current components.

Results

Dominant Negative KCNQ5 KI Mice.

To investigate the physiological roles of the KCNQ5 channel, we created a KI mouse carrying a dominant negative mutation in KCNQ5. We mutated the first glycine of the GYG signature pore sequence of KCNQ5 to serine (G278S). Equivalent human mutations have been found in KCNQ1 with the dominant form of the long QT syndrome (19) and in KCNQ4 in a family with dominant deafness (20). These mutations, as well as equivalent mutations introduced into KCNQ2 and KCNQ3, were shown to have dominant negative effects in heterologous expression (15, 20, 21). KCNQ3 can form functional heteromers with KCNQ5 (15, 16). On heterologous expression in Xenopus oocytes, KCNQ3 induces currents that are barely above background. Coexpression of KCNQ3 with KCNQ5 boosts currents in oocytes more than 2-fold with respect to KCNQ5 homooligomers (15). Dominant negative effects of KCNQ3(G318S) on KCNQ5 currents have been demonstrated before (15). We now tested whether KCNQ5(G278S), which is nonfunctional by itself (15), exerts dominant negative effects on KCNQ5 and on KCNQ3/KCNQ5 heteromers (an effect on KCNQ3 homomers cannot be tested because of low currents). As described before (15), currents increased more than 2-fold with respect to KCNQ5 homomers when KCNQ3 and KCNQ5 were coinjected at a 1:3 ratio (Fig. 1 A and B). Coinjection of KCNQ5 WT with G278S mutant (1:1) decreased KCNQ5 current more than 90%, demonstrating the strong dominant negative effect of the mutant subunit. Oocytes injected with KCNQ3, KCNQ5, and KCNQ5(G278S) in a 1:1.5:1.5 ratio exhibited a small current of about 15% of KCNQ3/KCNQ5 (1:3) current (Fig. 1 A and B), indicating that KCNQ5(G278S) also exerts a dominant negative effect on KCNQ3/KCNQ5 heteromers. Currents were indistinguishable from background when KCNQ3 was coexpressed with KCNQ5(G278S) (Fig. 1 A and B), a situation that may arise in homozygous KI mice.

Fig. 1.

Fig. 1.

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Generation of dominant negative Kcnq5 KI mice. (A) Effect of KCNQ5(G278S) on currents elicited by KCNQ5 homomers and KCNQ3/KCNQ5 heteromers. Oocytes were injected with the same total amount of RNA with the indicated concentration ratios. t, time. (Inset) Oocytes were clamped for 5 s to +40 mV. (B) Averaged currents (at +40 mV) from oocytes injected with the indicated combinations of KCNQ3, KCNQ5, and KCNQ5(G278S) RNAs. Error bars, SEM. Number of oocytes (from three or more different batches) is indicated above columns. Scheme of mouse genomic WT Kcnq5 sequence (C) and mouse genomic dominant-negative Kcnq5 sequence (D). A NEO selection cassette flanked by loxP sites was introduced into intron 5, together with three point mutations in exon 6 that replace glycine 278 by serine and additionally generate a BsiWI restriction site. The NEO cassette was removed by Cre-recombinase in vitro (exons shown in red). (E) Quantitative real-time RT-PCR analysis of Kcnq5(G278S) mRNA expression in brain, normalized to Kcnq5 mRNA found in WT brain. (F) Western analysis of whole-brain membrane preparations (P65) of KCNQ2, KCNQ3, and KCNQ5 from Kcnq5+/+ and Kcnq5dn/dn mice. Lysates of transfected tsA cells were used as controls for antibody specificity. Kcnq5dn/dn mice have normal expression levels of KCNQ2, KCNQ3, and KCNQ5.

Having verified the dominant negative effects of the KCNQ5(G278S) mutant, we introduced this mutation into the murine Kcnq5 gene by homologous recombination in ES cells. For genotyping purposes, we also inserted a BsiWI restriction site (Fig. 1 C and D). These alterations required the mutation of a total of 4 base pairs. A neomycin (NEO) cassette flanked by loxP sites was inserted 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 between 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 localization of KCNQ2 and KCNQ3 subunits to axon initial segments of hippocampal pyramidal cells was not changed (Fig. 2A).

Fig. 2.

Fig. 2.

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Normal subcellular localization of KCNQ2 and KCNQ3 and unchanged brain histomorphology in Kcnq5dn/dn mice. (A) KCNQ2 and KCNQ3 are localized to ankyrinG-positive axon initial segments in hippocampus pyramidal cells in both Kcnq5+/+ and Kcnq5dn/dn mice. (B) Nissl staining of sagittal sections of the hippocampus, cortex, and cerebellum shows normal histomorphology in Kcnq5dn/dn mice at PD65. Higher magnification of the CA3 area is shown. (Scale bars: A, 10 μm; B, 500 μm; higher magnification of CA3, 100 μm.)

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. 3A).

Fig. 3.

Fig. 3.

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Expression of KCNQ3 and KCNQ5 proteins in adult brain. (A) Immunohistochemistry revealed strong expression of KCNQ5 in the CA3 region of the hippocampus (arrow) and in the cortex, caudoputamen, and substantia nigra but weak expression in the thalamus and cerebellum. (B) Higher levels of KCNQ5 in hippocampal CA3 pyramidal cells in contrast to lower levels in CA1 and CA2 pyramidal cells and mossy fibers are shown. (C) Strong expression of KCNQ3 was found in the subiculum (arrow) and mossy fibers (arrowhead) of the hippocampus and in the cortex, caudoputamen, substantia nigra, and thalamus. (D) Higher levels of KCNQ3 in hippocampal CA1 and CA2 pyramidal cells and mossy fibers in contrast to lower levels in CA3 pyramidal cells are shown. cb, cerebellum; cp, caudoputamen; ctx, cortex; hp, hippocampus; mf, mossy fibers; sn, substantia nigra; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; th, thalamus. The CA2/CA3 border is indicated by white bars in B and D. (Scale bars: A and C, 0.25 cm; B and D, 500 μm.)

Effect of KCNQ5 Dominant Negative Mutation on the ImAHP and IsAHP in the Hippocampus.

We first examined the distribution of KCNQ5 and KCNQ3 within the brain, particularly within the hippocampus, using immunohistochemistry. KCNQ5 was found to be prominently expressed in the hippocampus, where the highest levels were visible in the CA3 region (Fig. 3 A and B). CA1 and CA2 pyramidal cells as well as dentate gyrus granule cells showed weaker KCNQ5 expression than those in the CA3 region (Fig. 3B). Similar to KCNQ5, KCNQ3 protein showed robust expression in the hippocampus, most prominently in the subiculum, mossy fibers, and CA1 and CA2 pyramidal cells (Fig. 3 C and D). This expression pattern of KCNQ3 and KCNQ5 protein agrees with previous published mRNA data (15, 21) and with data from the Allen brain atlas (22).

To determine the relative contribution of KCNQ5 to the mAHP and sAHP currents, we compared the ImAHP and IsAHP recorded from WT and Kcnq5dn/dn mice. We decided to focus on CA1 and CA3 pyramidal neurons in the hippocampus because they both express KCNQ5 and KCNQ3 but at different expression levels, as shown above. In slices from WT mice and in the presence of 100 nM apamin to block SK channels, a 100-ms depolarizing pulse from −55 mV to +25 mV in CA1 or CA3 pyramidal neurons robustly elicits the ImAHP and IsAHP. We found that in CA1 neurons, the apamin-insensitive mAHP current is unaffected in Kcnq5dn/dn mice, but it is decreased by almost 50% in CA3 pyramidal neurons (Fig. 4 A and B). Specifically, the ImAHP amplitude in WT CA1 pyramidal neurons is 293 ± 17 pA (n = 15 cells from six WT mice), whereas in Kcnq5dn/dn mice, it is 250 ± 36 pA (n = 16 cells from four Kcnq5dn/dn mice; P = 0.30, Student's t test). In contrast, in CA3 pyramidal neurons, the ImAHP amplitude is 201 ± 28 (n = 21 cells from six WT mice) and 107 ± 19 pA (n = 13 cells from three Kcnq5dn/dn mice; P < 0.05, Student's t test) in WT and Kcnq5dn/dn mice, respectively. Additionally, the sensitivity of the apamin-insensitive ImAHP to 10 μM XE991, a specific M-current and KCNQ2/KCNQ3 blocker, is similar between WT and Kcnq5dn/dn mice (WT: 54 ± 13%, n = 4 from two mice; Kcnq5dn/dn: 55 ± 3%, n = 3 from one mouse; P = 0.95). We also did not see any changes in the input resistance (WT: 180 ± 10 MΩ, n = 21 from six mice; Kcnq5dn/dn: 194 ± 18 MΩ, n = 14 from three mice; P = 0.47, Student's t test) or in the apamin-sensitive ImAHP (I-SK) in mice carrying the Kcnq5dn gene (I-SK WT: 162 ± 14 pA, n = 8 from four mice; I-SK Kcnq5dn: 130 ± 18 pA, n = 7 from three mice; pooled data from Kcnq5dn/dn and Kcnq5+/dn mice; P = 0.1786, Student's t test). These data then suggest that KCNQ5 can contribute to the ImAHP CA3 but not CA1 neurons of the hippocampus.

Fig. 4.

Fig. 4.

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Loss of the IsAHP and ImAHP in Kcnq5dn/dn mice is cell type-specific. (A) (Left and Center) ImAHP in either Kcnq5+/+ or Kcnq5dn/dn mice recorded from CA1 pyramidal neurons. (Right) Summary graph of the ImAHP amplitude in Kcnq5+/+ (n = 15) and Kcnq5dn/dn (n = 16) mice. No statistical difference is observed between the two groups (P = 0.3, Student's t test). (B) (Left and Center) ImAHP in either Kcnq5+/+ or Kcnq5dn/dn mice recorded from CA3 pyramidal neurons. (Right) Summary graph of the ImAHP amplitude in Kcnq5+/+ (n = 21) and Kcnq5dn/dn (n = 13) mice. Asterisk indicates P < 0.05 (Student's t test). (C) (Left and Center) IsAHP in either Kcnq5+/+ or Kcnq5dn/dn mice recorded from CA1 pyramidal neurons. (Right) Summary graph of the sAHP charge in Kcnq5+/+ (n = 15) and Kcnq5dn/dn (n = 16) mice. No statistical difference is observed between the two groups (P = 0.74, Student's t test). (D) (Left and Center) IsAHP in either Kcnq5+/+ or Kcnq5dn/dn mice recorded from CA3 pyramidal neurons. (Right) Summary graph of the sAHP charge in Kcnq5+/+ (n = 21) and Kcnq5dn/dn (n = 14) mice. Asterisk indicates P < 0.005 (Student's t test).

To quantify the role of KCNQ5 in the sAHP current, we measured both the charge transfer (QsAHP) and the amplitude of the IsAHP in CA1 and CA3 pyramidal neurons of WT and Kcnq5dn/dn mice. As shown in Fig. 4 C and D, the QsAHP and IsAHP amplitude from Kcnq5dn/dn mice recorded from CA1 pyramidal neurons is similar to the responses recorded in WT mice (IsAHP WT: 39 ± 4 pA, n = 15 cells from six mice; Kcnq5dn/dn: 40 ± 5 pA, n = 16 cells from four mice; P = 0.86, Student's t test). In contrast, the QsAHP was significantly decreased by about 25–30% in CA3 pyramidal neurons from Kcnq5dn/dn mice (QsAHP WT: 2155 ± 149 pC, n = 21 cells from six mice; Kcnq5dn/dn: 1524 ± 110 pC, n = 14 cells from three mice; P < 0.005, Student's t test). As with the QsAHP (Fig. 4D), the IsAHP amplitude is decreased to similar levels in CA3 pyramidal neurons (IsAHP WT: 457 ± 30 pA, n = 21 cells from six mice; Kcnq5dn/dn: 342 ± 26, n = 14 from three mice; P = 0.05, Student's t test). We also measured the WT IsAHP in the presence of 10 mM 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) to ensure that the current we are referring to as the IsAHP in CA3 neurons is attributable to the calcium-activated sAHP. In WT cells in which BAPTA was included in the pipette, the IsAHP is 23 ± 3 pA (n = 4 from one mouse) as opposed to the IsAHP in the absence of BAPTA, which is 457 ± 30 pA (n = 21 from six WT mice). Together, these data indicate that the IsAHP and ImAHP recorded in CA3 pyramidal neurons are partly mediated by KCNQ5 homomers or KCNQ3/5 heteromers. Currently, we cannot distinguish between these two possibilities.

Discussion

KCNQ channels have been implicated in mediating AHP conductances in neurons, but the contribution of individual KCNQ channel family members to the mAHP current has remained unknown because of the lack of subtype-selective KCNQ channel blockers. An added complicating factor is that KCNQ channels can form homomers as well as heteromers with each other (i.e., KCNQ2/KCNQ3, KCNQ3/KCNQ5, KCNQ3/KCNQ4), thus creating multiple possible combinations of KCNQ-mediated currents (23, 24). KCNQ5 is the final member of the KCNQ family without a known biological role. To elucidate its contribution to AHP currents, we used mice expressing dominant negative KCNQ5 channels [KCNQ5(G278S)] driven by their native promoter, allowing us to bypass the need for specific KCNQ5 blockers. These mice provide evidence that KCNQ5 contributes to the mAHP and sAHP currents in a cell type-selective manner.

KCNQ5 and the ImAHP.

Several studies have supported the notion that KCNQ2 and KCNQ3 are major contributors to the apamin-insensitive component of ImAHP (14, 24, 25). We have found that KCNQ5 is highly expressed in CA3 and contributes to ImAHP in CA3 pyramidal neurons, thus extending the number of KCNQ family members known to contribute to mAHP. In contrast, KCNQ5(G278S) had no effect on ImAHP in CA1 neurons. This cell type-specific contribution is most likely attributable to the differential expression of KCNQ2, KCNQ3, and KCNQ5 protein in the hippocampus. In particular, we showed that KCNQ5 protein is sparse in CA1 compared with CA3, which is consistent with the Kcnq5 mRNA level in the hippocampus (22, 26). Our results are consistent with our previous finding that KCNQ3 KO and KCNQ2 heterozygous mice have significantly reduced mAHP currents in dentate granule cells, neurons that have high mRNA and protein levels for both of these subunits, but that KCNQ5 mice do not. In contrast, the situation in CA1 pyramidal neurons appears more complicated. Like CA3 pyramidal neurons, CA1 pyramidal neurons express all three KCNQ subunits, but the mAHP current was unaffected in the Kcnq5dn/dn, Kcnq2+/− heterozygous, and Kcnq3−/− mice. Others have found that mice overexpressing dominant negative KCNQ2 from a broadly active promoter have significantly diminished mAHP in CA1 pyramidal neurons (14). In the latter mouse model, both KCNQ2 and KCNQ3 currents should be repressed. The possibility that KCNQ2 is the predominant contributor to the mAHP current in these neurons could be tested by using KCNQ2 conditional KO mice to avoid the lethal phenotype of the conventional KCNQ2 KO mice (27).

KCNQ5 and the IsAHP.

Unlike the ImAHP, the contribution of KCNQ channels to the IsAHP is controversial. KCNQ channels have properties that are both similar to and different from those of the sAHP current. Both KCNQ channels and sAHP have slow activation kinetics that are temperature-sensitive [IsAHP Q10: 3–4 (28); KCNQ2, KCNQ3 Q10: 3–4 (29)]. They also share a small single-channel conductance [IsAHP: 2–8 pS (28, 30), KCNQ5: 2–3 pS; KCNQ2/KCNQ3: 8–10 pS (31–33)], have a probability of opening comparable to that of the sAHP current (28, 31), and are sensitive to modulation by muscarinic and glutamatergic metabotropic receptors (1, 24, 34). However, the IsAHP is thought to be a voltage-independent process, whereas KCNQ channels are voltage-activated (3, 24). Additionally, protein kinase A (PKA) activation blocks the IsAHP (35), whereas PKA activation facilitates KCNQ channels in a subunit-specific manner (21). These differences may be attributable to the calcium activation mechanism of the IsAHP. We have recently shown that the NCS hippocalcin is a key component in the activation of the IsAHP and that it may partially account for the PKA sensitivity of the IsAHP (9).

Currently, the evidence that KCNQ channels also mediate sAHP consists of limited pharmacological and genetic data. Application of either XE991 or linopirdine has been reported to inhibit the IsAHP partially in some studies (12, 36), but this finding has not been reproduced by all groups (25, 37). Recently, we demonstrated that KCNQ2- and KCNQ3-deficient mice have a reduced IsAHP in some types of hippocampal neurons (12). Here, we report that the IsAHP in CA3 pyramidal cells from Kcnq5dn/dn mice is impaired, supporting the possibility that KCNQ channels participate in the generation of the IsAHP. Like the ImAHP, the IsAHP may be mediated by different KCNQ family members in different neurons. Here, we find evidence that KCNQ5 may contribute to the IsAHP in CA3 but not CA1 pyramidal neurons, correlating with the high expression levels in CA3. Conversely, our previous study found that KCNQ3 and KCNQ2 are likely to contribute to the IsAHP in dentate granule cells. These cells prominently express KCNQ3, as is evident from the strong staining of mossy fibers that represent axons of those cells (Fig. 3B). Could the changes seen in the ImAHP amplitude in KCNQ-deficient mice lead to an apparent decrease in the sAHP current? We think this is unlikely because the kinetics of the mAHP currents are much faster than the rise and decay kinetics of the IsAHP, thus making little contribution to the IsAHP. Additionally, application of apamin to the ImAHP to block SK responses does not affect the sAHP current (9), consistent with the notion that changes to the ImAHP do not translate to a decrease in the IsAHP.

If KCNQ channels mediate both the ImAHP and IsAHP, the mechanism by which KCNQs mediate two temporally distinct conductances is intriguing. We tentatively propose that the ImAHP primarily reflects voltage-dependent activation and deactivation of KCNQ channels, whereas the IsAHP reflects delayed activation of KCNQ channels as a result of dependence on NCS activity. Recently, Tzingounis et al. (9) have shown that the diffusable NCS protein hippocalcin is involved in activating sAHP currents in the brain. The participation of the NCSs imposes a short time delay on the activation of sAHP because the NCSs must first bind cytosolic calcium and translocate to the membrane before gating sAHP current. Consequently, this will give rise to a delay between the ImAHP and IsAHP time course. However, the mechanism by which the NCSs activate the IsAHP is unknown. We speculate that NCS activation may occur through either (i) the direct binding of NCSs to KCNQ channels or (ii) relief of tonic kinase inhibition of KCNQ channels. Several groups have previously shown that the sAHP channels are under tonic inhibition by various kinases, including PKA and MAPK (38–40), and it is possible that NCS proteins induce a signal transduction cascade leading to removal of tonic kinase activity. Further experiments are needed to explore these possibilities.

The search for the components of sAHP is currently at a crossroads. It has been over 20 years since the first description of sAHP (41, 42), but a molecular description of the channel and its calcium-sensitive gating mechanism has remained elusive (11). Together, our data show that KCNQ5 channels contribute both to the mAHP and sAHP currents in the hippocampus. Our work raises the exciting possibility that KCNQ channels in general participate in the IsAHP. Our current and previous work using KCNQ3- and KCNQ2-deficient mice suggests that different cell types in the brain rely on a different set of KCNQ subunits for the AHP currents. Therefore, different cell types might express specific complements of KCNQ subunits tailored to fit their function in the brain. Finally, the differential cell-specific contribution of individual KCNQ isoforms to neuronal K+ currents is likely to underlie the different phenotypes of mice without functional KCNQ2, KCNQ3 (12, 14, 27, 43), and KCNQ5 channels (this work).

Materials and Methods

Generation of Kcnq5dn/dn Mice.

A 129/SvJ genomic λ-phage library (Lambda FIX II Library; Stratagene) was screened with a kcnq5 cDNA probe spanning the pore region. Inserts from hybridizing λ-clones were subcloned into the NotI site of the pKO901-DTA vector (Lexicon Genetics) and analyzed by PCR and Southern blotting for the presence of exon 6. A positive clone was identified and fully sequenced. A 6.6-kb BamHI/BstBI fragment was subcloned into the BamHI/ClaI sites of the pKO901-DTA vector, and a floxed NEO resistance cassette from pKO800 (Lexicon Genetics) was inserted in the BstEII site of intron 5. The dominant negative G278S point mutation was introduced into the WT pore motif by PCR-based mutagenesis. This change also included a silent BsiWI restriction site for easy genotyping, and the resulting construct was verified by sequencing. The vector was linearized with NotI and electroporated into MPI-2 mouse ES cells. ES cells surviving the G418 selection were screened for correct targeting by Southern blot analysis of EcoRV-digested DNA. ES cell clones having undergone homologous recombination were transfected with a plasmid expressing Cre-recombinase to remove the NEO cassette, thereby leaving a single loxP site. Cells were injected into C57BL/6 blastocysts, and highly chimeric mice were mated to produce the first filial generation.

Genotyping.

Genotyping of KCNQ5 mice was performed by PCR using the following primer pair: 5′-acccataaattctccccaacatgatcacc-3′, 5′-attacttgtttgtttctagggttgaccgtttgg-3′. This resulted in a fragment of 231 bp for WT and 265 base pairs for the dominant negative allele.

Quantitative Real-Time RT-PCR.

Total RNA was prepared from brains of WT and Kcnq5dn/dn mice using TRIZOL (Invitrogen), digested with RNase-free DNase (Ambion) and purified with RNeasy columns (Qiagen). cDNA synthesis used SuperScript II reverse transcriptase (Invitrogen), oligo(dT)15 primer, first-strand reaction buffer, 0.1 M DTT, 10 mM dNTP mix, and RNaseOUT (Invitrogen). The quantitative RT-PCR assay was run on an ABI PRISM 7700 Sequence detection System (SDS 2.1 software) using SYBR green PCR master mix (Applied Biosystems). Primers used were 5′-gggcacaatcacactgacaacc-3′ and 5′-gaaagaaatgccaaggagtgcg-3′. For quantification, we used the 2−ΔΔCt method, taking HPRT and GAPDH as reference genes.

Antibodies.

Polyclonal antisera were raised in rabbits against peptides corresponding to amino acids 16–37 of KCNQ2 (GEKKLKVGFVGLDPGAPDSTRD), 858–873 of KCNQ3 (GDGISDSIWTPSNKPT), and 813–828 of KCNQ5 (SESSGSRGSQDFYPK). These peptides contained an additional carboxy terminal cysteine for coupling to a carrier protein (keyhole limpet hemocyanin). Antisera were affinity-purified against the corresponding peptide coupled to Sulfolink-Sepharose (Pierce) and tested by Western blotting (Fig. 1F) and immunocytochemistry (Fig. S3) of tsA or HeLa cells transiently transfected with the corresponding pcDNA3+-KCNQ vector DNA. The ankyrin G antibody was from Zymed, and the antisynaptophysin antibody was from Synaptic Systems.

Western Blotting.

Brain membrane proteins (50–100 μg per lane) were separated by SDS/PAGE (8.5%); blotted to PVDF membranes; and probed with affinity-purified anti-KCNQ2 (1:1,000), anti-KCNQ3 (1:200), anti-KCNQ5 (1:200), and antisynaptophysin (1:5,000) antibodies. Detection used ECL Western blotting substrate. Details are provided in SI Materials and Methods.

Histology.

Nissl staining, immunostaining using DAB, and immunofluorescence analysis were performed on brain sections from perfusion-fixed mice, as detailed in SI Materials and Methods. Sections were examined with a Zeiss Axiophot or Zeiss Stemi-2000-c microscope or by confocal laser-scanning microscopy (LSM510; Zeiss) for immunofluorescence.

Expression in Xenopus Oocytes and Two-Electrode Voltage Clamping.

Xenopus oocytes were obtained and injected as described elsewhere (15). A final amount of 20 ng of cRNA was injected per oocyte for each condition. Oocytes were incubated for 3–4 days at 17 °C and then examined by two-electrode voltage clamping using a Turbo Tec03 (NPI Electronics) amplifier and pClamp 8.0 software (Molecular Devices). The protocol consisted of 5-s steps from −110 to +50 mV in 10-mV increments from a holding potential of −80 mV.

Slice Electrophysiology.

Transverse hippocampal slices (300 μm) were prepared from either 12- to 19-day-old WT or Kcnq5dn/dn mice. The ImAHP and IsAHP were determined following a 100-ms voltage step to +25 mV from a holding membrane potential of −55 mV. To determine the QsAHP, we integrated the sAHP current for 19 s starting 450 ms after the depolarizing voltage step. All recordings were made in the presence of 0.5 μM TTX (more details are provided in SI Materials and Methods).

Supplementary Material

Supporting Information
supp_107_22_10232__index.html (899B, html)

Acknowledgments

We thank Vitya Verdanyan for breeding Kcnq5dn mice and testing antibodies and Patrick Seidler for technical assistance. This work was partially supported by grants from the European Union (Grant FP6, KCNQpathies) and the Leibniz Gesellschaft to T.J.J. R.A.N. and A.V.T. are supported by National Institutes of Health grants. G.S. is a fellow of the AvHumboldt Foundation.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1004644107/-/DCSupplemental.

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