KCNQ2 Channels: Dynamic Molecular Interactions and Functional Role in Learning and Memory

Voltage-gated ion channels encoded by the members of KCNQ gene family (KCNQ2-5) conduct the M-type potassium current. Several neurotransmitters and signaling events have been shown to regulate the activity of the M-channel, including Ca2+ and muscarinic receptor-mediated suppression. We found that a change in the configuration of the KCNQ2 channel complex triggered by elevated intracellular Ca2+ lowers channel sensitivity to an essential co-factor, phosphatidylinositol 4,5-bisphosphate, which shuts down the channel. We also identified that a classical mechanism of M-current regulation mediated by muscarinic receptor activation requires KCNQ2 phosphorylation by PKC and dissociation of calmodulin, an auxiliary subunit of KCNQ2 channel complex. Based on these findings, we generated a knock-in mouse line that carries an alanine mutation at the key phosphorylation site of KCNQ2, KCNQ2(S559A). These mice show attenuated response to muscarinic-mediated M-current suppression, which enables us to address the role of physiological M-current suppression in vivo. Functionally, the M-current is one of the key modulators of synaptic plasticity with a proposed role in learning and memory. Thus, we aimed to identify the effects of M-current inhibition on memory processing. To discriminate between the memory processes mediated by different brain regions, we conducted perirhinal cortex-dependent and hippocampus-dependent memory tasks. KCNQ2(S559A) mice showed normal spatial memory as evidenced by successful performance with a 24 h retention interval. However, we observed a significant long-term recognition memory impairment in KCNQ2(S559A) mice with a 24 h retention interval. Inhibition of the M-current with XE991 during memory consolidation phase rescued memory deficit in KCNQ2(S559A) mice. Our mutant mice also showed deficits in long-term social odor memory, while maintaining normal olfactory responses, further implicating the M-current in memory processes mediated by perirhinal cortex. Finally, our behavioral findings were mirrored by a lower level of neuronal activation in perirhinal cortex of KCNQ2(S559A) mice compared to the wild-type during memory consolidation, as measured by c-fos expression 2 h after novel object recognition training. Our findings provide evidence for the proposed importance of M-current suppression during memory processing and offer a novel perspective on its role in recognition memory consolidation

A change in configuration of the calmodulin-KCNQ channel complex underlies Ca2+-dependent modulation of KCNQ channel activity.

All subtypes of KCNQ channel subunits (KCNQ1-5) require calmodulin as a co-factor for functional channels. It has been demonstrated that calmodulin plays a critical role in KCNQ channel trafficking as well as calcium-mediated current modulation. However, how calcium-bound calmodulin suppresses the M-current is not well understood. In this study, we investigated the molecular mechanism of KCNQ2 current suppression mediated by calcium-bound calmodulin. We show that calcium induced slow calmodulin dissociation from the KCNQ2 channel subunit. In contrast, in homomeric KCNQ3 channels, calcium facilitated calmodulin binding. We demonstrate that this difference in calmodulin binding was due to the unique cysteine residue in the KCNQ2 subunit at aa 527 in Helix B, which corresponds to an arginine residue in other KCNQ subunits including KCNQ3. In addition, a KCNQ2 channel associated protein AKAP79/150 (79 for human, 150 for rodent orthologs) also preferentially bound calcium-bound calmodulin. Therefore, the KCNQ2 channel complex was able to retain calcium-bound calmodulin either through the AKPA79/150 or KCNQ3 subunit. Functionally, increasing intracellular calcium by ionomycin suppressed currents generated by KCNQ2, KCNQ2(C527R) or heteromeric KCNQ2/KCNQ3 channels to an equivalent extent. This suggests that a change in the binding configuration, rather than dissociation of calmodulin, is responsible for KCNQ current suppression. Furthermore, we demonstrate that KCNQ current suppression was accompanied by reduced KCNQ affinity toward phosphatidylinositol 4,5-bisphosphate (PIP2) when assessed by a voltage-sensitive phosphatase, Ci-VSP. These results suggest that a rise in intracellular calcium induces a change in the configuration of CaM-KCNQ binding, which leads to the reduction of KCNQ affinity for PIP2 and subsequent current suppression

A change in configuration of the calmodulin-KCNQ channel complex underlies Ca2+-dependent modulation of KCNQ channel activity.

All subtypes of KCNQ channel subunits (KCNQ1-5) require calmodulin as a co-factor for functional channels. It has been demonstrated that calmodulin plays a critical role in KCNQ channel trafficking as well as calcium-mediated current modulation. However, how calcium-bound calmodulin suppresses the M-current is not well understood. In this study, we investigated the molecular mechanism of KCNQ2 current suppression mediated by calcium-bound calmodulin. We show that calcium induced slow calmodulin dissociation from the KCNQ2 channel subunit. In contrast, in homomeric KCNQ3 channels, calcium facilitated calmodulin binding. We demonstrate that this difference in calmodulin binding was due to the unique cysteine residue in the KCNQ2 subunit at aa 527 in Helix B, which corresponds to an arginine residue in other KCNQ subunits including KCNQ3. In addition, a KCNQ2 channel associated protein AKAP79/150 (79 for human, 150 for rodent orthologs) also preferentially bound calcium-bound calmodulin. Therefore, the KCNQ2 channel complex was able to retain calcium-bound calmodulin either through the AKPA79/150 or KCNQ3 subunit. Functionally, increasing intracellular calcium by ionomycin suppressed currents generated by KCNQ2, KCNQ2(C527R) or heteromeric KCNQ2/KCNQ3 channels to an equivalent extent. This suggests that a change in the binding configuration, rather than dissociation of calmodulin, is responsible for KCNQ current suppression. Furthermore, we demonstrate that KCNQ current suppression was accompanied by reduced KCNQ affinity toward phosphatidylinositol 4,5-bisphosphate (PIP2) when assessed by a voltage-sensitive phosphatase, Ci-VSP. These results suggest that a rise in intracellular calcium induces a change in the configuration of CaM-KCNQ binding, which leads to the reduction of KCNQ affinity for PIP2 and subsequent current suppression

Attenuating M‐current suppression in vivo by a mutant Kcnq2 gene knock‐in reduces seizure burden and prevents status epilepticus–induced neuronal death and epileptogenesis

ObjectivesThe M-current is a low-threshold voltage-gated potassium current generated by Kv7 subunits that regulates neural excitation. It is important to note that M-current suppression, induced by activation of Gq-coupled neurotransmitter receptors, can dynamically regulate the threshold of action-potential firing and firing frequency. Here we sought to directly examine whether M-current suppression is involved in seizures and epileptogenesis.MethodsKv7.2 knock-in mice lacking the key protein kinase C (PKC) phosphorylation acceptor site for M-current suppression were generated by introducing an alanine substitution at serine residue 559 of mouse Kv7.2, mKv7.2(S559A). Basic electrophysiologic properties of the M-current between wild-type and Kv7.2(S559A) knock-in mice were analyzed in primary cultured neurons. Homozygous Kv7.2(S559A) knock-in mice were used to evaluate the protective effect of mutant Kv7.2 channel against chemoconvulsant-induced seizures. In addition, pilocarpine-induced neuronal damage and spontaneously recurrent seizures were evaluated after equivalent chemoconvulsant-induced status epilepticus was achieved by coadministration of the M-current-specific channel inhibitor, XE991.ResultNeurons from Kv7.2(S559A) knock-in mice showed normal basal M-currents. Knock-in mice displayed reduced M-current suppression when challenged by a muscarinic agonist, oxotremorine-M. Kv7.2(S559A) mice were resistant to chemoconvulsant-induced seizures with no mortality. Administration of XE991 transiently exacerbated seizures in knock-in mice equivalent to those of wild-type mice. Valproate, which disrupts neurotransmitter-induced M-current suppression, showed no additional anticonvulsant effect in Kv7.2(S559A) mice. After experiencing status epilepticus, Kv7.2(S559A) knock-in mice did not show seizure-induced cell death or spontaneous recurring seizures.SignificanceThis study provides evidence that neurotransmitter-induced suppression of M-current generated by Kv7.2-containing channels exacerbates behavioral seizures. In addition, prompt recovery of M-current after status epilepticus prevents subsequent neuronal death and the development of spontaneously recurrent seizures. Therefore, prompt restoration of M-current activity may have a therapeutic benefit for epilepsy

Ionomycin treatment and PIP2 depletion.

<p>TIRF analysis indicating membrane localization of the CFP-PH probe. Top panels show epifluorescent cell images (epi) indicating total fluorescence, and TIRF images (TIRF) showing plasma membrane localization. The lower panel shows pooled data from TIRF analyses. 10 µM ionomycin induced translocation of CFP-PH, which indicates depletion of PIP2. In contrast, 1 µM or 3µM ionomycin did not alter plasma membrane localization of CFP-PH. TIRF signal is normalized to that at t  =  0. Black box indicates the presence of ionomycin. Error bars show S.E.</p

HoloCaM was retained in the KCNQ2 channel complex via AKAP150.

<p><b>A,</b> Calcium (100 µM) dependent binding of CaM to AKAP79. <b>B, </b><i>In vitro</i> binding of KCNQ2 and CaM with or without AKAP150. KCNQ2-FLAG was immunopurified by anti-FLAG conjugated resin. CaM was co-purified in calcium (+) condition only in the presence of AKAP150. <b>C,</b> Top – the summary of quantification of relative KCNQ2-bound CaM from five independent experiments shown in B<b>.</b> Bottom – the summary of quantification of relative KCNQ2-bound AKAP150 from five independent experiments shown in B. Bars are labeled corresponding to the lane numbers on the immunoblot. *<0.05 non-parametric ANOVA followed by Dunn’s multiple comparisons test, ** < 0.01 by Mann-Whitney test. Error bars show S.E.</p

Interaction of CaM with KCNQ subunits required distinct calcium conditions.

<p><b>A</b>, Distinct calcium requirement for CaM binding to the KCNQ2, KCNQ3 and KCNQ2/KCNQ3 channels. Representative immunoblots (top) of immunoprecipitation experiments and summary histogram (bottom) are shown. <b>B,</b> Time course of CaM dissociation from the KCNQ2 subunit upon exposure to 500 µM Ca²⁺. <b>C,</b> Molecular structure from the PDB file (4GOW) depicting the interaction between calcium-bound CaM(E88) and KCNQ4(R538). <b>D,</b> Sequence alignment of Helix B, the distal CaM binding domain, of KCNQ1-5 subunits. Arrow shows the cysteine residue on KCNQ2, rKCNQ2(C527), that corresponds to the conserved arginine residue in other KCNQ subtypes, such as KCNQ4(R538); r – rat, h – human. <b>E,</b> KCNQ2(C527R) mutation regained CaM binding in the presence of Ca²⁺. * < 0.05, ** < 0.01 by Mann-Whitney test. Error bars show S.E.</p

Ionomycin-induced suppression of KCNQ2 current was accompanied by a lower PIP2 affinity.

<p><b>A,</b> Representative current traces showing a voltage dependent KCNQ2 current decay due to the activation of Ci-VSP. A brief voltage step to –60 mV was applied to calculate the linear leak. <b>B,</b> Scaled KCNQ2 current traces at +10 mV showing an identical Ci-VSP-mediated current decay without ionomycin at 2-min interval. Gray and shaded red areas show S.E. <b>C,</b> Scaled KCNQ2 current traces showing the facilitation of KCNQ2 current decay 1 min after the application of 1 µM ionomycin. Control traces were obtained 1 min before ionomycin application. <b>D,</b> Ci-VSP-mediated current decay of the KCNQ2(C527R) current. Scaled current traces indicate the facilitation of current decay by 1 µM ionomycin at +10 mV. <b>E,</b> Summary of the Ci-VSP-induced current decay at control (t  =  –1 min) and t  =  1 min for indicated conditions. KCNQ2(wt) and KCNQ2(C527R) showed an equivalent Ci-VSP-mediated current decay both with and without 1µM ionomycin. **<0.01 by paired t-test. Error bars show S.E.</p