Kv4.2 regulates baseline synaptic strength by inhibiting R-type channel-mediated calcium signaling in the hippocampus

Kv4.2 channels, which mediate A-type K+ current, exert significant influence on synaptic input signals and synaptic plasticity in the principal cells of the hippocampus. While their influence on activity-dependent regulation of synaptic response is well-established, the impact of Kv4.2 channels on baseline synaptic strength remains elusive. To investigate this, we selectively inhibited postsynaptic Kv4.2 by introducing Kv4.2 antibodies into the hippocampal granule cells and evaluated its impact on the baseline synaptic transmission. Our results demonstrated that Kv4.2 inhibitions led to notable increase in the amplitude of AMPA receptor (AMPAR)-mediated synaptic currents, and this effect was in parallel with the Kv4.2 expression level at dendritic regions. This Kv4.2-dependent synaptic potentiation was effectively abolished by intracellular 10 mM BAPTA or block of R-type calcium channels (RTCC) and downstream signaling molecules including protein kinase A (PKA) and protein kinase C (PKC). Importantly, Kv4.2 inhibitions did not occlude further synaptic strengthening high frequency stimulation, suggesting that synaptic strength regulation by Kv4.2 s distinct from the mechanism of long-term potentiation. Our study highlights the role of Kv4.2 in regulating the baseline synaptic strength, where Kv4.2-mediated inhibition of RTCC is crucial.

It is well established that the regulation of AMPAR densities at postsynaptic density (PSD) is a key mechanism underlying activity-dependent regulation of synaptic strength.Long-term potentiation (LTP) induced by high frequency stimulation requires NMDA receptor activation (Luscher and Malenka., 2012) that facilitate Ca2+ influx to activate Ca2+/calmodulin-dependent kinase II (CaMKII) and various downstream signaling cascades to trigger long-lasting potentiation in synaptic strength (Opazo et al., 2010, Lisman et al., 2012, Sanheuza and Lisman., 2013, Herring and Nicoll., 2016, Yasuda et al., 2022, Hell et al., 2023).However, the regulatory mechanism responsible for maintaining synaptic strength during basal synaptic transmission is not fully understood.Recent findings indicated that genetic deletion and pharmacological blockade of Kv4.2 induce an enhancement in miniature excitatory postsynaptic current (mEPSC) amplitude (Kim et al., 2007, Murphy et al., 2022).These observations suggest the possibility that Kv4.2 plays a role in controlling AMPA receptors (AMPAR) during basal synaptic transmission, although the underlying mechanisms remain to be uncovered.It is therefore crucial to investigate whether Kv4.2 can govern AMPARs to regulate basal synaptic strength, and if so, to elucidate the underlying mechanism.
To address these questions, we introduced Kv4.2-specific antibodies into the cells using whole-cell patch technique and evaluated the role of these channels on basal synaptic transmission at postsynaptic sites.Our findings revealed that Kv4.2 inhibition-induced synaptic potentiation was attributed to an increase in AMPA-mediated current through Ca +2 -dependent mechanism.Synaptic potentiation by Kv4.2 inhibition required calcium influx through R-type calcium channels (RTCC), and activation of PKC and PKA signaling pathways.Additionally, our observations suggested that Kv4.2 channels may act as input-specific synaptic regulators depending on their expression in the hippocampus.Collectively, we identified Kv4.2 as robust regulator of basal synaptic strength in the hippocampus, potentially contributing to the maintenance of level of AMPA density within physiological range by suppressing RTCC-dependent Ca 2+ signaling under basal conditions.Patch pipettes (6 -7 MΩ) and monopolar stimulator pipettes (3 -4 MΩ) were obtained from borosilicate glass capillaries with a horizontal pipette puller (P-97, Sutter Instruments).The internal pipette solution contained the following (in mM): 143 K-gluconate, 7 KCl, 15 HEPES, 4 MgATP, 0.3 NaGTP, 4 Na-ascorbate, and 0.1 EGTA, with the pH adjusted to 7.3 with KOH, and with osmolality of approximately 300 mOsml/L.To measure IPSP and IPSC amplitude, internal pipette solution contained the Cs-based internal solution in following (in mM): 120 Cs-methanesulfonate, 10 CsCl, 10 HEPES, 2 MgCl, 3 MgATP, 0.4 NaGTP, 5 Na2+ phosphocreatine, 0.1 EGTA, with pH adjusted to 7.3 with CsOH.The antibody blocking experiments included Kv4.2 antibodies (1 μg/ml) in the pipette solution.Series resistance (Rs) after establishing whole-cell configuration was between 10 and 20 MΩ.Rs was monitored by applying a short (10s) hyperpolarization (1mV) pulse during the recording.If Rs of the recorded cells changed 20% of the baseline value, the cells were discarded.
For EPSC recordings, the stimulator intensity (100 μs duration; 8-18 V) of extracellular stimulation was adjusted to evoke EPSC amplitudes between 50 pA and 150 pA for the baseline with 50ms intervals delivered every 10s or 30s between sweeps.The holding potential was maintained at -70mV.For EPSP recordings, the cells were held at its RMP.A stimulator (Stimulus Isolator A360; WPI) connected to a monopolar electrode filled with recording aCSF was placed in outer molecular layer of the DG to evoke LEC stimulation induced synaptic responses in the mature granule cells of the hippocampus.Synaptic responses at other synapses are indicated.The PPR was calculated as the ratio of the second EPSC over the first EPSC amplitude.All recordings were performed in the presence of GABA blockers (100 μM PTX, 1 μM CGP52432) to block inhibitory synaptic transmission unless otherwise indicated.
The LTP was induced by applying 10 bouts of high frequency stimulation (HFS) of perforant path synapses.HFS consists of 10 stimuli at 100 Hz under current clamp mode and the bouts were given at 5 Hz.The stimulation intensity was adjusted to evoked at least 2 AP at the first bout.
Intrinsic properties under current-clamp mode and the following parameters were obtained: (1) resting membrane potential (RMP), ( 2) input resistance (membrane potential changes at a given hyperpolarizing current input (−35 pA, 500 ms)), (3) F-I curve (firing frequencies (F) against the amplitude of injected currents from 50pA to 400pA for 500ms duration with 50pA increment (I)), (4) AP half-width (measured as the width at 50% of the spike peak amplitude) (5) AP threshold (the voltage at which dV/dt exceeded 40 mV/ms).

Immunohistochemistry analysis
For the immunofluorescence staining, 7-week old C57BL/6 mice were anesthetized with isoflurane and perfused transcardially with 1X PBS and 4% paraformaldehyde (PFA) for 10-15minutes.Brains were removed and cut into 30 μm-thick sections using a vibratome (VT1200S, Leica), and postfixed overnight at 4 °C in 4% PFA solution.The slices were washed 5 times in 1X PBS with 0.3% Triton X-100 (PBS-T) for 5 minutes.Then the sections were incubated three times in a blocking solution (2.5% donkey serum + 2.5% goat serum in PBS-T) for 1 hour at room temperature.The sections were then incubated overnight at 4 °C in blocking solutions containing primary antibodies (anti-Kv4.2antibody, APC-023, Alomone).After washing 5 times in 0.3% PBS-T for 5 minutes, sections were then incubated with secondary antibodies in blocking solution for at least 1 hr at RT.After rinsing with PBS, sections were mounted on glass slides using mounting medium containing DAPI.The immunostained sections were imaged with a confocal laser scanning microscope (FV1200, Olympus) using a 40x oilimmersion objective lens.

Data analysis
All data were presented as mean ± standard error of the mean (SEM).Statistical analysis was performed using IgorPro (Version 7.08, Wavemetrics) and Prism.Nonparametric Mann-Whitney U test was used to compare non-paired groups, and Wilcoxon signed-rank test or paired sample t test were used to compare paired groups.Fluorescence intensity was analyzed using Image J. P-values of <0.05 were considered statistically significant.

Kv4.2 channel contributes to regulating the synaptic transmission of dentate granule cells
To investigate the functional role of Kv4.2 in regulating synaptic transmission in the dentate gyrus (DG), we performed whole-cell recordings to record synaptic responses in mature granule cells (GCs) in the DG by stimulating lateral perforant pathway (LPP).Throughout the studies, inhibitory synaptic transmission was blocked by PTX (100 μM, GABAAR blocker) and CGP52432 (1 μM, GABAbR blocker) unless indicated otherwise.Mature GC, characterized by their location within the outer granule cell layer and their low input resistance (Rin < 200 MΩ), were selected for observations to avoid potential confounding factors related to the maturation stages of GCs (Kerloch et al., 2019).
It is generally thought that K + channel inhibition can amplify EPSPs by increasing input resistance or dendritic excitability.To examine whether Kv4.2 inhibitions has amplifying effects on EPSPs, we compared the magnitude of increase induced by Kv4.2 Ab on EPSP and EPSC amplitude (Fig. 2G).The results showed equivalent enhancements, indicating that the inhibition of Kv4.2 channels primarily enhanced synaptic strength and that further enhancement was not evident (Figure 2G, EPSPkv4.2 vs EPSCkv4.2, p>0.53) (Figure 2G).In spite, the effects of Kv4.2 inhibitions on the intrinsic excitability of GCs were evident.Kv4.2 Ab induced a significant increase in input resistance (219.5 ± 7.17 vs 253.97 ± 7.35, n = 67, p < 0.0001) and led to depolarization of the resting membrane potential (RMP) (-76.8 ± 0.79 vs -71.9 ± 0.93, n = 85, p < 0.0001) (Figure 2H, 2I).In contrast, AP half-width at rheobase current (0.75 ± 0.02 vs 0.74 ± 0.2, n = 16, p>0.7,) and the number of action potentials (AP) generated by +200 pA depolarizing current injections (200 pA: 7.76 ± 0.73 vs 8 ± 0.78, n = 16, p>0.5) showed no significant alteration due to Kv4.2 inhibitions, indicating that Kv4.2 had no discernible impact on GC firing properties (Figure 2J, 2K).Therefore, our findings imply that despite a substantial increase in input resistance by Kv4.2 inhibitions, this increase was not the predominant factor that induced amplification in EPSP amplitude, which contradicted to the well-known mechanism of K channel inhibition on EPSPs.Our data suggested that Kv4.2 channels exert profound influence on the AMPA-mediated currents to modulate synaptic response.
Subsequently, we investigated whether different effects of Kv4.2 inhibitions on different synapses are related to the expression patterns of Kv4.2 in dendrites by conducting immunohistochemical analysis in CA3 and DG regions of the hippocampus.While both regions exhibited rare expression of Kv4.2 in somatic regions, robust Kv4.2 expressions was observed along the dendritic regions of GCs (Figure 3C), but not in CA3 (Figure 3D).In CA3 region, Kv4.2 expressions were relatively low in stratum lucidum (SL) but substantially higher in the stratum radiatum (SR) region (Figure 3D).
When the relative fluorescence intensity of Kv4.2 expressions in different dendritic layers was plotted against their magnitude of enhancement in EPSC amplitude, it was clear that Kv4.2 channel expression correlated with the extent of EPSC enhancement (Figure 3E).Dendrites in the SR, IML and OML, which exhibited relatively similar level of Kv4.2 expressions, showed comparable enhancement of EPSC amplitudes upon Kv4.2 inhibitions.In contrast, dendrites in SL in CA3, where Kv4.2 expressions was minimal, showed no potentiation in synaptic strength by Kv4.2 inhibitions.Therefore, our findings demonstrated Kv4.2 expressions correlated with synaptic strength regulation by Kv4.2.
Given that the NMDA-EPSC was intact following Kv4.2 inhibitions, involvement of presynaptic mechanism in Kv4.2 Ab-mediated synaptic strengthening could be ruled out.In spite that changes in sEPSC frequency are regarded as an indication of presynaptic modification, recent research proposed that the trafficking of AMPARs to NMDAR-containing silent synapses could influence sEPSC frequency by modifying the number of functional synapses at postsynaptic sites (Ying Yang et al., 2013).To test this possibility, we conducted minimal stimulation experiments to examine changes in eEPSC amplitude and failure rate by stimulating LEC synapses.Kv4.2 inhibitions led to an increase in potency (EPSC amplitude without failures) and a reduction in the failure rate (Figure 6B -6D, potency: 1.42 ± 0.12, n = 16, p < 0.001; failure rate: 0.48 ± 0.05 vs 0.18 ± 0.05, n = 16, p < 0.0001).Therefore, enhanced sEPSC frequency and reduced failure rate by Kv4.2 inhibition suggest that AMPARs have been inserted into silent synapses of GCs.Overall, our results offer evidence that Kv4.2 inhibitions increase postsynaptic Ca 2+ influx through RTCC to initiate PKA and PKC signaling events that ultimately leads to the postsynaptic insertion of AMPARs.

Kv4.1 regulates dendritic excitability, while Kv4.2 regulates synaptic strength
Since Kv4.1 also regulates K + conductance, we next attempted to examine whether Kv4.1 regulates synaptic strength.As previously reported (Kim et al., 2020), immunohistochemical analysis revealed a stronger expression of Kv4.1 in perisomatic regions of GCs, gradually decreasing along the proximo-distal axis of dendrites.Furthermore, expression of Kv4.1 in the dendritic regions was lower than the perisomatic region, with gradual decrease in the intensity along the proximo-distal axis of the dendrites (Figure 7A).The contrasting expression pattern of the Kv4.1 (Figure 7A) and Kv4.2 (Figure 3A) in the dendritic layers of hippocampal dentate gyrus suggested different roles of these two channels in synaptic transmission regulation.

Distinct mechanisms involved in Kv4.2 inhibition-induced synaptic strengthening in GCs
Long-term potentiation (LTP) of synaptic strength has been known to be attributed to increased density of AMPA receptors and downregulation of voltage-gated K + channels that would synergistically produce potentiate synaptic responses.Therefore, we investigated the effect of Kv4.2 inhibitions on synaptic strengthening during LTP induction using theta burst stimulation (TBS).LTP experiments were initiated after 15 minutes of patch break-in to ensure that Kv4.2 Ab effect on AMPAR-mediated currents stabilized.After recording baseline EPSCs in LPP-GC synapses for about 5 minutes, 10 bouts of high frequency stimulation (HFS, 10 stimuli at 100 Hz) at 5 Hz was applied.EPSCs were subsequently measured for 15 minutes to observe the extent of synaptic potentiation induced by TBS.Even under conditions where Kv4.2 was inhibited, TBS still significantly increased EPSC amplitude (Figure 8A, 1.74 ± 0.15, p < 0.05).This enhancement in EPSC amplitude following LTP induction was comparable to that of the control groups, which did not include Kv4.2 Ab (Figure 8C, EPSC+Kv4.2Ab vs EPSC-kv4.2Ab:1.85 ± 0.33 vs 1.74 ± 0.15, n = 4, p>0.6).Therefore, inhibition of Kv4.2 did not occlude further synaptic strengthening induced by TBS, indicating that the regulation of AMPAR by Kv4.2 differs from that of LTP.

Discussion
In summary, as demonstrated in Figure 8D, our findings underscore that Kv4.2 channels regulate AMPARmediated currents by modulating RTCC-dependent Ca 2+ signaling.Notably, Kv4.2 channel inhibition did not occlude the expression of LTP, suggesting that Kv4.2 Ab-induced synaptic strengthening operates through mechanisms distinct from EPSC enhancement observed during LTP induction.Consistent with these observations, RTCC and PKA/PKC emerged as primary regulators of Kv4.2-dependent synaptic strengthening, a process different from LTP induction predominantly governed by NMDAR and CaMKII (Hell et al., 2023).Also, Kv4.2 exhibited input-specific regulation depending on their expression pattern in the hippocampus.Overall, our findings proposed that Kv4.2 channels play essential role in maintaining baseline AMPA density through regulatory mechanism distinct from that involved in LTP.

Kv4.2 channels affect AMPA receptor mediated current to shape synaptic response
Considerable evidence has established the role of Kv4.2-mediated A-type K + current in dendritic signaling and synaptic plasticity (Hoffman et , 1997).Inhibition of Kv4.2 mediated outward currents in dendrites have been regarded to amplify synaptic depolarization, but we could not find its amplification effect.On the contrary, our studies provide evidence that Kv4.2 channels regulate AMPAR-mediated currents to enhance EPSP amplitude.
To note, there are several shortcomings in methods that were used to assess Kv4.2 contributions in previous studies.Firstly, the commonly used transient A-type blocker, including 4-Amnopyridne (4-AP), BaCl2 or phrixotoxins (PaTx), has nonspecific effect on other potassium (Kv) channels, potentially complicating the interpretation of the obtained results.Secondly, both genetic ablation of Kv4.2 and pharmacological blockade of Kv4.2 channels fail to distinguish the effects of presynaptic and postsynaptic Kv4.2 channels.Additionally, compensatory overexpression of other K + channels cannot be ruled out in studies using genetic ablation models.(Kim et al, 2005, Nerbonne et al., 2008;Foehring., 2008;Andrasfalvy et al., 2008).Altogether, it is difficult to interpret that those results are solely attributable to the blockade of dendritic Kv4.2 channels.
To circumvent these problems and identify the role of dendritic Kv4.2 channels in the synaptic responses of the cell, we recorded the synaptic responses with the patch clamp technique by including specific antibodies to Kv4.2 in the intracellular solution.Intracellular dialysis of Kv4.2 Ab inhibited transient current without affecting sustained current in GCs (Figure 1).By observing equivalent increase in EPSP and EPSC amplitude, we were able to conclude that Kv4.2 channels act as a powerful regulator of AMPA currents rather than dendritic excitability in hippocampal GCs (Figure 1, 2).It needs to be investigated in future studies whether other K + channels known as dendritic channels can regulate AMPA currents.

Regulation of RTCC by Kv4.2 inhibitions in dendritic spine
In our study, we have demonstrated that intracellular BAPTA and Ni 2+ completely blocked potentiating effect of Kv4.2 Ab, indicating that Kv4.2-dependent regulation of AMPA currents is Ca 2+ dependent.This finding strongly suggests that under basal conditions regulation of AMPA current by Kv4.2 channels is mediated by regulating calcium level via RTCCs.In support of this idea, it was previously shown that blockade of Kv4.2 channels led to an increase in spine calcium levels and mEPSC amplitude and these enhancements were prevented when R-type calcium channels were blocked using nickel (Ni 2+ ) (Murphy et al., 2022).
Considering that L-type Ca 2+ channel blocker did not prevent Kv4.2-dependent regulation of AMPA currents (Figure 4B), these suggested that Kv4.2-dependent regulation is not towards all voltage-gated Ca 2+ channels, but specific to RTCCs.This specificity may be attributed to the fact that RTCC, not L-type calcium channel, is the major calcium source to action potential-evoked Ca 2+ influx in dendritic spines in CA1 PN (Sabatini and Svobada 2000, Yasuda et al., 2003, Bloodgood and Sabatini et al., 2007).Also, the observed close proximity of Cav2.3 to Kv4.2 channels in dendritic spines of pyramidal neurons (Murphy et al., 2022) implicated that Kv4.2mediated synaptic response regulation through RTCC is specialized function in dendritic spines.Given the considerable heterogeneity in the expression of VGCC classes across different cell types in dendritic spines, with cortical pyramidal neurons predominantly expressing L, P/Q, and T-type channels (Koester and Sakmann 2000), it is essential to explore the regulation of VGCCs by Kv4.2 channels in various cell types in the future studies.
In addition to VGCCs, NMDARs are potential calcium source at subthreshold potentials that can also be regulated by K + channels (Sobczyk et al., 2005;Higley and Sabatini., 2008).For example, small-conductance calcium-activated channels (SK) in CA1 (Ngo Anh, 2005) and large-conductance calcium-activated channels (BK) channels in GCs (Zhang et al., 2018) regulate NMDAR activity in the dendritic spines.In the context, Ca 2+ influx through NMDARs activate SK or BK channels and their activation provide hyperpolarizing current to inhibit NMDAR activation.Conversely, the inhibition of SK or BK channels enhance NMDA-mediated currents.However, we did not find any evidence that Kv4.2 inhibitions increase NMDARs-mediated currents.This may be due to higher activation threshold for NMDAR opening (Scheuss et al., 2009) than that of RTCC (Wormuth et al., 2016), suggesting that influence by Kv4.2 inhibitions at subthreshold potentials may be limited RTCC.

RTCC-mediated regulation is distinct from NMDAR-dependent regulation for AMPA receptors
Our data revealed that AMPAR enhancement induced by Kv4.2 inhibition operates through distinct and nonoverlapping mechanisms compared to that associated with LTP induction (Figure 8).LTP in PP-GC synapses is known to regulated by NMDAR- (Kim et al., 2018) or LTCC-mediated (Lopez Rojas et al 2015, Kim et al., 2022) calcium influx, subsequently initiating CaMKII to increase AMPAR density in synaptic sites.However, Kv4.2-mediated regulation of synaptic strength primarily relied on RTCC-mediated calcium influx and signaling molecules including PKA and PKC Furthermore, prior investigations have shown that inhibiting RTCC-mediated Ca 2+ signaling reduced IA current by decreasing the surface expression level of Kv4.2 in a KChIP-dependent manner (Wang et al, 2014, Murphy et al., 2022).These studies suggested that RTCC-mediated Ca 2+ influx promotes the functional expression of Kv4.2 in dendrites (Murphy et al., 2022).Considering that our data provides evidence that RTCC-mediated Ca 2+ influx contributes to enhancement in AMPAR-mediated current, RTCC-mediated Ca 2+ signaling serves to regulate both AMPA density and Kv4.2 surface expression.These suggest that Ca 2+ influx through RTCC enhances AMPA density while simultaneously increasing Kv4.2 surface expression, which may act as a mechanism to suppress further increments in AMPA density.Therefore, Kv4.2-RTCC may establish a negative feedback loop to maintain an appropriate level of synaptic Ca 2+ and AMPARs under basal conditions.This regulation of AMPAR and Kv4.2 by RTCC-mediated calcium influx differs from that by NMDAR during LTP.It is a widely established that NMDAR-mediated Ca2+influx leads to trafficking of AMPARs to surface membrane while it induces the internalization of Kv4.2, working synergistically to enhance EPSP amplitude (Kim andHoffman., 2007, Jung andHoffman., 2009).Therefore, while robust increase in AMPAR density in association with Kv4.2 inhibitions is induced by NMDAR-mediated calcium influx during LTP, AMPAR remains limited in the physiological range under baseline synaptic transmission by Kv4.2-RTCC negative feedback mechanism.In conclusion, given the broad expression of Kv4.2 in the brain and potential pathological impacts of disruptions in Kv4.2 function, our research offers a comprehensive framework for understanding the physiological and pathological roles of Kv4.2-dependent regulation of synaptic transmission.

Figure 1 .
Figure 1.Kv4.2-mediated A-type K+ current enhances EPSP amplitude in mature granule cells Aa (top) Left: Illustration of the recording configuration, demonstrating the dialysis of Kv4.2 antibodies (Kv4.2Ab) through the recording pipette in granule cells of the dentate gyrus.Right: Representative traces of outward currents evoked by a depolarizing voltage step pulse from a holding potential of -70mV to + 30mV recorded at 3 min (marked with 1, black) and 15-min (marked with 2, red) after patch break-in from dentate granule cells of the hippocampus.Scale bar, 1nA and 200ms.Inset showing superimposed current traces of 1 and 2 highlighting the specific reduction of transient A-type current due to the dialysis of Kv4.2 antibodies through intracellular solution.Scale bar, 1nA and 10ms.(bottom) The current amplitude, both transient (closed circle) or sustained (opened circle) current, were normalized to their respective initial value following patch break-in.These normalized amplitudes were plotted against the duration of the experiment