A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca2+ regulation of Kv4 channels

The Kv4 family of A-type voltage-gated K+ channels regulates the excitability in hippocampal pyramidal neuron dendrites and are key determinants of dendritic integration, spike timing-dependent plasticity, long-term potentiation, and learning. Kv4.2 channel expression is down-regulated following hippocampal seizures and in epilepsy, suggesting A-type currents as therapeutic targets. In addition to pore-forming Kv4 subunits, modulatory auxiliary subunits called K+ channel–interacting proteins (KChIPs) modulate Kv4 expression and activity and are required to recapitulate native hippocampal A-type currents in heterologous expression systems. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. As members of the EF-hand domain–containing neuronal Ca2+ sensor protein family, KChIP auxiliary proteins may convey Ca2+ sensitivity upon Kv4 channels; however, to what degree intracellular Ca2+ regulates KChIP–Kv4.2 complexes is unclear. To answer this question, we expressed KChIP2 with Kv4.2 in HEK293T cells, and, with whole-cell patch–clamp electrophysiology, measured an ∼1.5-fold increase in Kv4.2 current density in the presence of elevated intracellular Ca2+. Intriguingly, the Ca2+ regulation of Kv4 current was specific to KChIP2b and KChIP2c splice isoforms that lack a putative polybasic domain that is present in longer KChIP2a1 and KChIP2a isoforms. Site-directed acidification of the basic residues within the polybasic motif of KChIP2a1 rescued Ca2+-mediated regulation of Kv4 current density. These results support divergent Ca2+ regulation of Kv4 channels mediated by alternative splicing of KChIP2 isoforms. They suggest that distinct KChIP–Kv4 interactions may differentially control excitability and function of hippocampal dendrites.

The Kv4 3 family of voltage-gated K ϩ channels is expressed across many brain regions where they perform a unique role. Tetrameric Kv4 channels mediate a fast activating and inactivating outward K ϩ current in heterologous systems that produce the A-type current (I A ) in neurons (1). In area CA1 of the hippocampus, pyramidal neurons express primarily Kv4 channels in a graded manner that increases distal to the cell soma within the dendritic arbor (2). Within this cellular niche, Kv4 channels are key determinants of action potential repolarization, dendritic excitability and integration, spike timing-dependent plasticity, long-term potentiation, and learning (3)(4)(5)(6)(7). Down-regulation of Kv4 channel expression occurs following hippocampal seizures and in epilepsy suggesting A-type currents as targets for novel therapeutics (8,9). Recapitulation of native hippocampal A-type K ϩ currents in heterologous expression systems requires nonconducting modulatory auxiliary subunits known as K-channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) (10 -12). Both KChIPs and DPPs work in concert to modify Kv4 expression and channel kinetics. In mammals, KChIPs are expressed from four genes in specific patterns throughout the brain that encode KChIPs 1-4 (13)(14)(15). In the hippocampus, KChIPs 2-4 predominate. The majority of KChIP isoforms promote forward trafficking to increase plasma membrane expression of Kv4 channels (16,17). KChIPs also accelerate Kv4 channel opening, slow fast inactivation, shift voltage dependence of inactivation (VDI) toward more depolarized potentials, and accelerate recovery from inactivation (RFI) (13). Taken together, KChIPs regulate Kv4 channels to increase channel number and activity.
Responses to changing intracellular Ca 2ϩ involves Ca 2ϩsensing proteins that contain high-affinity Ca 2ϩ -binding sites. As members of the neuronal Ca 2ϩ sensor gene family, KChIPs contain Ca 2ϩ -binding EF hands, two each in N-and C-terminal lobules (13). Only the two C-terminal EF hands (EF-3 and EF-4) are thought to bind Ca 2ϩ under normal conditions, whereas the N-terminal EF-hands are degenerated such that EF-1 does not bind Ca 2ϩ and EF-2 binds Mg 2ϩ preferentially over Ca 2ϩ (18 -22). The EF-hands of KChIP family members are highly conserved suggesting that Ca 2ϩ binding is important for KChIP function. Indeed, in vitro withdrawal of Ca 2ϩ from KChIP results in conformational changes, including reduced rigidity and loss of helical content (20,21,23). Understanding the contribution of Ca 2ϩ to KChIP regulation of Kv4 complexes is of great interest because it suggests the potential for feedback regulation in neurons. Many studies have made mutations to the EF-hands of KChIP family members to tease apart relative functions for EF-2, EF-3, and EF-4 in Kv4 regulation (13,24). Although this approach is informative, Ca 2ϩ binding and intrinsic structural roles for the EF-hands cannot be dissociated. However, published studies examining the role of intracellular Ca 2ϩ in regulation of KChIP-Kv4.2 complexes have been performed in several different cell types, using various KChIP/Kv4 family members and differing methods for elevating or reducing intracellular Ca 2ϩ . Thus, there are inconsistencies in the literature regarding Ca 2ϩ -KChIP regulation of Kv4 function. Differing reports have shown Ca 2ϩ may either retain or disrupt KChIP-Kv binding, increase or decrease channel currents, or alter channel gating in various ways (13, 24 -27).
Kv4.2 and Kv4.3 are the most abundant Kv4 subunits expressed in excitable tissues, including the heart and brain. In area CA1 of the hippocampus Kv4.2 predominates and is responsible for the A-type current in pyramidal neuron dendrites. Significant functional diversity in A-type currents may arise through the multiple 5Ј start sites and alternative N-terminal exons in the four Kcnip genes that generates 17 different proteins with diverse tissue-specific expression patterns (14,15). In situ hybridization localization of KChIPs 1-4 suggest that KChIP2 and KChIP4 are the most abundant KChIPs in area CA1 of the hippocampus (14). RT-PCR analysis of whole-hippocampus extracts suggests that KChIP2a and KChIP4a predominate, whereas KChIP2b and -2c are present to a lesser extent (14,28). In the future, more sensitive measurements of KChIP splice isoform expression are needed to determine how spatial differences may lead to region and cell-type specificity of Kv4 regulation.
Although much research has focused on the role of the EFhands in binding Ca 2ϩ , the influence of the variable N-terminal regions on Ca 2ϩ regulation remains unclear. In this study, we show that elevated intracellular Ca 2ϩ regulates Kv4 function by increasing current density. Ca 2ϩ regulation is conserved across the Kv4 family but is specific to short isoforms of KChIP2 and to KChIP3a. A shared basic and hydrophobic N-terminal domain in KChIP2a1 and KChIP2a confers resistance to Kv4 regulation by intracellular Ca 2ϩ .

KChIP2c regulates Kv4.2 channel kinetics and inactivation
KChIPs 1-4 are all assumed to bind Ca 2ϩ under normal circumstances; however, KChIP2c has previously been reported to bind to Kv4.2 in a Ca 2ϩ -dependent manner (25). We first determined whether KChIP2c maintained regulation of Kv4.2 channels in nominal Ca 2ϩ (0 Ca 2ϩ , 10 mM EGTA). To assess KChIP2c regulation of Kv4.2 currents, we expressed Kv4.2 alone (Fig. 1a) or in the presence of KChIP2c (Fig. 1b) in HEK293T cells and performed whole-cell patch-clamp electrophysiology (Fig. 1c). KChIP2c regulation of Kv4.2 peak currents were measured using a voltage-clamp protocol. Because Kv4 channels inactivate at relatively hyperpolarized holding potentials (Ϫ70.24 Ϯ 0.91 mV; Table 1), a pre-pulse to Ϫ120 mV is used to alleviate latent inactivation. A step to ϩ60 mV elicits an outward K ϩ current carried by Kv4.2 channels (Fig.  1d). As predicted from the established literature (25), coexpression of KChIP2c increased Kv4.2 current density ϳ2.07-fold (Fig. 1e) and accelerated the 10 -90% current rise time ϳ1.53fold (Fig. 1f). When fit to a double exponential (Fig. S1), KChip2c coexpression slowed the fast rate of inactivation ϳ3-fold ( Fig. 1g) but had no effect on the second, slower component of decay (Fig. 1h). After stepping through a series of increasing voltages, we found that KChIP2c coexpression had no effect on the voltage dependence of activation (VDA) for Kv4.2; however, KChIP2c shifted the VDI to more depolarized potentials as described previously for most KChIP family members ( Fig. 1i; Table 1) (15). KChIP2c coexpression also dramatically accelerated the RFI as shown by others ( Fig. 1j; Table 1) (25). Based on these results, we can confirm that at low-intracellular free Ca 2ϩ (0 Ca 2ϩ /10 mM EGTA), KChIP2c regulation of Kv4 current is intact.

Ca 2؉ regulation of Kv4.2 current density requires KChIP2c
To assess the Ca 2ϩ dependence of Kv4.2 function, whole-cell patch-clamp recordings were performed in either nominal Ca 2ϩ (0 Ca 2ϩ and 10 mM EGTA) or ϳ10 M Ca 2ϩ (5 mM Ca 2ϩ /10 mM hydroxyethyl)ethylenediamine-N,NЈ,NЈ-triacetic acid (HEDTA)) ( Fig. 2, a and b). Although the high affinity Ca 2ϩ chelator EGTA is ideal for maintaining low levels of intracellular Ca 2ϩ , even small pipetting errors can lead to large differences in free Ca 2ϩ above 1 M. Intracellular free Ca 2ϩ is better clamped within the 10 M range using the low-affinity Ca 2ϩ buffer HEDTA (29). In cells expressing Kv4.2 alone, 10 M Ca 2ϩ affected neither K ϩ current density, VDA, VDI, or RFI ( Fig. 2c; Table 2). To assess whether Ca 2ϩ regulates Kv4.2 channels through KChIP auxiliary proteins we coexpressed KChIP2c. Coexpression of KChIP2c enhanced K ϩ current density under nominal Ca 2ϩ as described above (Fig. 1e, and Fig. 2d; Table 1); however, in the presence of KChIP2c and ϳ10 M Ca 2ϩ , K ϩ current density was increased, whereas VDA, VDI, and RFI were not affected ( Fig. 2d; Table 1). To determine whether the Ca 2ϩ regulated increase in current density was evident in the presence of another obligatory Kv4 subunit DPP6, we measured the same electrophysiological parameters in cells coexpressing Kv4.2, KChIP2c, and DPP6 (Fig. 2e). Although the trend for a Ca 2ϩ -regulated increase in peak current density remained, it did not achieve statistical significance ( Fig. 2e; Table 2). The effect of Ca 2ϩ was most pronounced on Kv4.2-KChIP2c K ϩ currents elicited at the peak of the G-V curve. To more closely examine the effects of Ca 2ϩ on peak currents, we analyzed both the magnitude and kinetics of Kv4.2 currents evoked with a single voltage step to ϩ60 mV in HEK293T cells expressing Kv4.2 and KChIP2c (Fig. 3a). As observed previously, peak K ϩ currents were larger in magnitude in the presence of 10 M Ca 2ϩ (Fig. 3, a and b). Additionally, Ca 2ϩ accelerated the 10 -90% rise time and the fast 1 of current decay while leaving the second, slower 2 component unchanged (Fig. 3, c-e). However, Ca 2ϩ regulation of 10 -90% rise time also occurred in the absence of KChIP2c suggesting this is a KChIP-independent effect ( Table 2). Taken together, elevated intracellular Ca 2ϩ regulates Kv4.2 current density and requires the presence of KChIP2c.

Ca 2؉ regulation is conserved across the Kv4 family of K ؉ channels
Although Kv4.2 is the predominant Kv4 isoform in CA1 neurons of the hippocampus, Kv4.1 and Kv4.3 transcripts and proteins are also present, albeit at significantly lower levels (12, 30 -32). Kv4.1 is expressed at relatively low levels throughout the brain, whereas Kv4.3 is more abundantly expressed in dentate gyrus, hippocampal interneurons, cerebellum, and cardiac tissues. Kv4.1, Kv4.2, and Kv4.3 share Ͼ62% sequence conservation in domains involved in binding to KChIPs and are functionally regulated by KChIP isoforms (Fig. 4a) (13,33). To determine whether Ca 2ϩ regulation through KChIP2c is conserved across the Kv4 family, we coexpressed either Kv4. 1 (Fig. 4, d-f). These results suggest that Ca 2ϩ increases K ϩ current density across the Kv4 channel family in a KChIP2c-dependent manner.

A polybasic domain in KChIP2 isoforms prevents Ca 2؉ regulation of KChIP-Kv4 complexes
The Kcnip1-4 genes contain multiple alternatively spliced 5Ј exons that generate considerable N-terminal variation and  Fig. 1. c, voltage-clamp recording configuration for measuring Kv4.2 K ϩ currents. d, representative peak outward K ϩ current mediated by Kv4.2 and evoked with a step depolarization. e, peak outward K ϩ current density. Raw current was normalized to cell size measured by whole-cell capacitance on cell break-in. f, time required for the outward K ϩ current to rise from 10 to 90% of the peak. g and h, kinetics of channel inactivation was measured by fitting the falling phase of peak outward K ϩ current with a double-exponential decay and comparing fast (g) or slow (h) components across conditions. i, normalized conductance and current versus applied test membrane potential for VDA and VDI protocols. Colored connecting lines represent Boltzman curve fits. j, time-dependent fractional recovery of peak outward K ϩ current after a VDI prepulse. Curves are fitted to a single exponential function. Error bars represent mean Ϯ S.E. *, p Ͻ 0.05; ****, p Ͻ 0.0001 by unpaired t test. Refer to Tables 1 and 2 for numerical data and replicate information.

Ca 2؉ regulation of KChIP-Kv4 channel complexes
functional diversity (Fig. 5a) (14,15). To determine whether Ca 2ϩ regulation is conserved across all isoforms of the KChIP2 family, we coexpressed either KChIP2a1, KChIP2a, KChIP2b, or KChIP2c with Kv4.2 and measured peak voltage-gated K ϩ currents. In the presence of ϳ10 M Ca 2ϩ , KChIP2b expression resulted in an increase in Kv4.2 K ϩ current density to a similar extent as KChIP2c (Fig. 5b), whereas rise time and inactivation kinetics of Kv4.2 were not affected ( Table 2). We also tested a select number of KChIP isoforms from the KChIP1, -3, and -4 gene families and found that only KChIP3a-Kv4.2 complexes responded to elevated Ca 2ϩ with increased current density equal in magnitude to the short forms of KChIP2 (Tables 2 and  3). Surprisingly, the longer KChIP2 isoforms, KChIP2a1 and KChIP2a, did not respond to ϳ10 M Ca 2ϩ ( Fig. 5b; Table 2). Sequence alignment of KChIP2 isoforms identified a 16-amino acid sequence unique to Ca 2ϩ -insensitive KChIP isoforms that consisted of basic residues flanked by hydrophobic and aliphatic amino acids that resembled a polybasic motif (Fig. 5c).
To determine whether the putative polybasic motif present in KChIP2a1 and KChIP2a was required for KChIP resistance to Ca 2ϩ regulation, we acidified this stretch by mutating RXHRXR to DXDDXD. Whole-cell voltage-clamp recordings of Kv4.2 currents revealed an ϳ1.5-fold increase in peak current density of KChIP2a1DDDD-Kv4 expressing cells in the presence of ϳ10 M intracellular Ca 2ϩ (Fig. 5d). These results support an inhibitory role for the putative polybasic motif present in longer KChIP2a1 and KChIP2a isoforms. This motif limits sensitivity of Kv4 complexes to regulation by elevated intracellular Ca 2ϩ .

Discussion
Here, we provide evidence for a subset of KChIP isoforms that enhance Kv4-type voltage-gated K ϩ channel current density in Table 1 Biophysical properties of Kv4 family members in nominal 0 M free Ca 2؉ (gray boxes) or ϳ10 M free Ca 2؉ (red boxes) I K indicates peak voltage-gated K ϩ current. All entries describe the mean Ϯ S.D.

Ca 2؉ regulation of KChIP-Kv4 channel complexes
response to elevations in intracellular Ca 2ϩ . KChIPs that transmit changes in intracellular Ca 2ϩ to Kv4 channels and those that do not share highly conserved EF-hand domains within the globular protein core; however, unique N-terminal domains generated by alternatively spliced exons differentiate Ca 2ϩ -regulated KChIPs from Ca 2ϩ -insensitive KChIP isoforms. Using whole-cell voltageclamp recordings in HEK293T cells expressing Kv4 channels and an array of KChIP isoforms, we show that elevated intracellular Ca 2ϩ has no effect on KChIP2a1 and KChIP2a. Conversely, cells expressing KChIP2b, KChIP2c, and KChIP3a show ϳ1.5-fold increase in Kv4 K ϩ current density. We have identified a novel motif shared by KChIP2a1 and KChIP2a that is absent from the Ca 2ϩ -sensitive KChIP2b, KChIP2c, and KChIP3a isoforms. Com-parisons of the variable N-terminal domains suggest that a conserved amino acid sequence containing basic amino acids limits Ca 2ϩ sensitivity of KChIP2a1 and KChIP2a. Acidification of the KChIP2a1 basic motif confers Ca 2ϩ sensitivity, increasing Kv4.2 current density in a Ca 2ϩ -dependent manner. Taken together, these data support a role for Ca 2ϩ regulation of Kv4 channel current density in neuronal populations that express a subset of KChIP isoforms.

Ca 2؉ sensing by KChIPs
In published studies of the Ca 2ϩ -dependent/independent regulation of Kv4 by KChIPs, many groups have used a series of KChIP EF-hand mutants (EFmut) to inhibit Ca 2ϩ binding. Free Ca 2ϩ was calculated using webmaxchelator (https://web.stanford.edu/~cpatton/webmaxcS.htm) (Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.). b, schematic describing intracellular dialysis of buffering solutions to clamp free Ca 2ϩ . Color scheme is used throughout Figs. 2-5 for nominal 0 M free Ca 2ϩ (black/white) and ϳ10 M free Ca 2ϩ (red). c-e, construct expression (top), VDA protocol (above-middle inset), representative VDA K ϩ currents (above-middle), VDA current density versus test potential (below-middle), and normalized conductance and current versus applied test membrane potential for VDA and VDI protocols (bottom, Kv4.2 data also appears in Fig. 4b). Curves were fitted to a Boltzmann function. Horizontal scale bar, 100 ms; vertical scale bars, 1 nA. Error bars represent mean Ϯ S.E. **, p Ͻ 0.01 by unpaired t test. Refer to Tables 1  and 2 for numerical data and replicate information.

Ca 2؉ regulation of KChIP-Kv4 channel complexes
In these experiments, an Asp to Ala mutation is made at position 1 of one or more KChIP EF-hands. EFmut KChIPs are then coexpressed with Kv4 channels, and the Ca 2ϩ -dependent effects of KChIP regulation on Kv4 currents are inferred. Each successive EF-hand mutation leads to a progressive loss of KChIP regulation of Kv4 channels with a particular requirement for EF-3 and EF-4, the EF-hands known to bind Ca 2ϩ with highest affinity (18 -24, 34). However, mutations to EF-hand domains disrupt tertiary structure, potentially leading to misinterpretation of results that may arise independent of Ca 2ϩ binding (35). It has also been reported that EFmut KChIP1 is misfolded, further suggesting that EF-hand mutations affect KChIP structure (23). With these caveats in hand, some reports have shown that EFmut KChIPs retain Kv4.2 binding suggesting a Ca 2ϩ -independent KChIP-Kv4 interaction (13,25). Other groups using similar methodologies found that KChIP-Kv4 binding is Ca 2ϩ -dependent (26,27). In the work presented here, without disrupting KChIP EF-hands, we show that KChIP and Kv4 channels remain associated after dialysis with 0 Ca 2ϩ , 10 mM EGTA intracellular solution, a low Ca 2ϩ condition that disrupts KChIP helical structure in vitro (23).  (39). Instead, increased current density is likely due to an increase in KChIP-Kv4 protein expression and plasma membrane surface trafficking (13,16,25,38,39). Therefore, our results suggest a post-ER/Golgi pool of Kv4 may be recruited to the plasma membrane by Ca 2ϩ in a KChIP-dependent manner (Fig. 6).

Ca 2؉ regulation of KChIP-Kv4 plasma membrane localization
KChIPs, like other members of the neuronal Ca 2ϩ sensor-1 family, are composed of N-and C-terminal lobules each containing two EF hands. Although the globular structures of KChIP1, KChIP3, and KChIP4 have been described (40 -42), only the N-terminal domain of KChIP4a has been captured suggesting most KChIPs have an unstructured and flexible N-terminal domain in vitro. Two separate groups have independently described identical structures for the channel complex containing KChIP1 and Kv4.3 (23,43). These KChIP1-Kv4.3 structures are likely similar to those occurring between the various KChIP-Kv4 combinations examined above; however, without crystallized KChIP N termini, the published KChIP1-Kv4.3 structure can only be generalized to globular KChIP-Kv4 interactions. When bound to Kv4.3, KChIP1 sequesters the cytoplasmic N terminus of Kv4.3 within a deep hydrophobic pocket. KChIP sequestration is thought to be responsible for slowing of Kv4 N-type inactivation (44). Although the EF-hand-containing globular core is highly conserved, the four mammalian Kcnip genes have multiple transcription start sites and alternative 5Ј exons that result in at least 17 isoforms with differing N-terminal domains (15). The variable N-terminal regions differentially regulate Kv4 channel properties, subcellular localization to lipid membranes, and Ca 2ϩ sensitivity, as described here. KChIP1a, KChIP1b, and KChIP4a are all considered membrane-associated either through N-myristoylation (KChIP1a/KChIP1b) or due to a transmembrane ␣-helix (KChIP4a) (45). These KChIP-Kv4 complexes do not respond to Ca 2ϩ suggesting Ca 2ϩ resistance may be mediated through membrane localization. KChIP2c is considered cytoplasmic, whereas KChIP2a1, KChIP2a, KChIP2b, and KChIP3a contain putative palmitoylation sites and may associate with the plasma membrane in a regulated manner dependent upon palmitoyltransferase activity. KChIP2c and KChIP3a have a primarily cytoplasmic localization when compared with KChIP2a and KChIP2b. However, upon coexpression with Kv4.3, each KChIP is effectively recruited to the plasma membrane to regulate Kv4.3 channels similarly (28). Additionally, differential regulation by KChIP isoforms may influence Kv4 surface stability, channel-recycling rate, or ratio of surface and intracellular complexes. In one study, KChIP3a was shown to bind to recycling endosome-related proteins (Rabs) in a Ca 2ϩ -independent manner (46). This study also identified the KChIP3a N terminus as a Ca 2ϩ -dependent binding site for calmodulin (CaM) and a Ca 2ϩ -independent binding site for the phosphatase calcineurin (CaN). The presence of KChIP3a was shown to increase the catalytic activity of CaM-CaN in vitro. Intriguingly, CaN is known to promote Kv4.2 surface expression in hippocampal neurons (47,48); this represents one trafficking mechanism that may explain the Ca 2ϩ dependence of membrane insertion, at least for the KChIP3a isoform. KChIP2 N-terminal regions have a notable conservation of hydrophobic and basic amino acids (66% of amino acids in KChIP2a). Such sequences resemble putative polybasic motifs. Polybasic motifs promote association with the plasma membrane through electrostatic interactions with phospholipid headgroups. An alternatively spliced polybasic motif in large conductance Ca 2ϩ -and  ). b, peak outward K ϩ current density. Raw current was normalized to cell size measured by whole-cell capacitance on cell break-in. c, time required for the outward K ϩ current to rise from 10 to 90% of the peak. d and e, kinetics of channel inactivation was measured by fitting the falling phase of peak outward K ϩ current with a double-exponential decay and comparing fast (d) or slow (e) components across conditions. Error bars, mean Ϯ S.E. *, p Ͻ 0.05; **, p Ͻ 0.01 by unpaired t test. Refer to Table 2 for numerical data and replicate information.

Ca 2؉ regulation of KChIP-Kv4 channel complexes
voltage-activated K ϩ channels (BK) has been put forward as an electrostatic switch that regulates palmitoylation state and plasma membrane channel density (49). The putative polybasic motif unique to KChIP2a1 and KChIP2a prevents Ca 2ϩ regulation of Kv4 channels (Fig. 5f). More research will be required to determine whether alternative splicing of the putative polybasic motif in KChIP2 isoforms regulates Kv4 forward trafficking.

Ca 2؉ regulation of KChIP-Kv4 complexes in hippocampal dendrites
Ca 2ϩ enhancement of Kv4 current density may function as a feedback mechanism to regulate excitability in tissues that express the appropriate KChIPs, including the heart and brain. Kcnip1, Kcnip2, Kcnip3, and Kcnip4 gene transcripts have all been detected in the hippocampus. Although KChIP1 is primarily expressed in interneurons, KChIP2, KChIP3, and KChIP4 are abundantly expressed in excitatory neurons. However, the relative expression level of alternatively spliced KChIP transcripts in the hippocampus is still unknown. It is possible that hippocampal cell types have a specific complement of KChIP isoform expression providing varying levels or subcellular distribution of Kv4 channels in response to elevated intracellular Ca 2ϩ . In the brain, alternative splicing is reg-ulated in response to synaptic activity and mutations in mRNA splice sites can result in several pathophysiological diseases (50,51). Therefore, splicing may act as a molecular switch to alter the KChIP complement to regulate Kv channel function. Putative Ca 2ϩ -sensitive KChIPs in the hippocampus have been shown to respond to voltage-gated Ca 2ϩ entry within a Kv4.2-Cav2.3 microdomain to regulate the size of excitatory postsynaptic potentials (52,53). Further study is required to determine whether the Ca 2ϩ -mediated changes in Kv4 channel density observed in this study regulate dendritic excitability and/or plasticity in hippocampal pyramidal neurons. Kv4.2 channels internalize to strengthen synaptic input in response to synaptic plasticity mechanisms (3). This process may be balanced by Ca 2ϩ regulation of Kv4 surface expression during neuronal excitation, either through local ER release or influx through voltagegated Ca 2ϩ channels. Interestingly, Kv4 channels colocalize and are regulated by voltage-gated Ca 2ϩ channels in the cerebellum (54) and hippocampus (53). Local Ca 2ϩ concentrations within 50 -100 nm of voltage-gated Ca 2ϩ channels are theorized to exceed the 10 M Ca 2ϩ concentrations used in this study supporting the idea that local Ca 2ϩ elevations in neurons could modulate Kv4 surface expression by the mechanism described in this report Proposed KChIP-binding domains (KChIP BD 1/2) and the Kv4 tetramerization domain (T1) are shown. b, normalized conductance and current versus applied test membrane potential for VDA and VDI protocols in Kv4.1, Kv4.2 (data also appears in Fig. 2d), or Kv4.3 and KChIP2c expressing HEK293T cells are shown. c, because of day-to-day variability of Kv4.1 and Kv4.3 construct expression, current density (pA/picofarad (pF)) was normalized by day of experiment. Statistical analysis by two-way ANOVA showed a significant effect of treatment (p Ͻ 0.0001) but not by Kv4 isoform expression (due to normalization) or the interaction (p ϭ 0.9076). Sidak's multiple comparisons among the Kv4 isoforms in control and treated conditions yielded significant differences within groups (Kv4.1, p ϭ 0.0026; Kv4.2, p ϭ 0.0107; and Kv4.3, p ϭ 0.0253). d and e, macroscopic channel kinetics are presented as described in Fig. 3. d, statistical analysis of 10 -90% rise times found significant differences among Kv4 isoforms (p ϭ 0.  Table 1 for numerical data and replicate information. (55). Furthermore, Kv4 channels are known to regulate spiketiming-dependent plasticity by inhibiting back-propagation of action potentials (2). Further studies are needed to determine whether Ca 2ϩ -KChIP-regulated enhancement of Kv4 current density in neuronal dendrites may affect spike-timing plasticity. In summary, we present a step forward in resolving an underlying mechanism for Ca 2ϩ regulation of KChIP-Kv4 complexes. Ongoing efforts are aimed at understanding how Ca 2ϩ regulation of KChIP-Kv4 function may mediate critical aspects of excitability and function of hippocampal dendrites.

Ca 2؉ regulation of KChIP-Kv4 channel complexes
(MC211934) (Origene). Mouse KChIP2c was generously provided by Henry Jerng and Paul Pfaffinger (Baylor College of Medicine, Houston, TX). Site-directed mutagenesis to acidify hKChIP2a1 was outsourced to Bioinnovatise, LLC (Rockville, MD). Briefly, the plasmid sequence encoding SLRPH-RPRLL was acidified to SLDPDDPDLL by substitution of the WT sequence encoding the targeted basic amino acids of hKChIP2a1 (RC213131) to GAC aspartic acid codons.

Heterologous channel expression
The day prior to transfection, cells were seeded onto 6-well cell culture-treated dishes at a concentration of 600 ϫ 10 6 cells per well. After 16 -20 h, plasmids encoding Kv4.2-WT (0.4 g) and GFP (0.2 g) were transfected using X-tremeGENE 9 (XG9; Sigma). Briefly, for each well, 5 l of XG9 was added to 100 l of minimum essential medium (MEM) and incubated Table 3 Biophysical properties of Kv4.2-KChIP1, -3, and -4 isoform complexes in 0 M free Ca 2؉ (gray) or ϳ10 M free Ca 2؉ (red) I K indicates peak voltage-gated K ϩ current. All entries describe the mean Ϯ S.D.
* represents statistical significance (0.05 Ͼ p Ͼ 0.0001) by unpaired t test. All comparisons are made within each plasmid expression condition between 0 M free Ca 2ϩ and 10 M free Ca 2ϩ .

Ca 2؉ regulation of KChIP-Kv4 channel complexes
Ն5 min at room temperature. In a separate tube, plasmid DNA was added to 100 l of MEM. After DNAs were added to MEM, this solution was mixed with the XG9/MEM solution and incubated at room temperature for 20 min. After XG9 -DNA complexes were formed, the mixture was added dropwise to one well of the 6-well dish. 24 -48 h after transfection, on the day of recording, transfected cultures were trypsinized for 2 min, washed, pelleted by centrifuge, resuspended in 5-10 ml of DMEM, and seeded at low density onto glass coverslips and allowed to adhere Ն1 h before recordings were started. Human

Electrophysiology
Coverslips were transferred to a recording chamber at room temperature and superfused (2-3 ml min Ϫ1 ) in 95% O 2 , 5% CO 2 -saturated extracellular solution (in mM: 115 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 2 CaCl 2 , 1 MgCl 2 , 25 glucose, pH 7.2-7.3, 300 mosM/liter) at 22-24°C. The patch electrodes contained (in mM): 115 KCl, 10 NaCl, 20 KOH, 10 HEPES, and either 10 EGTA or 10 HEDTA and 5 CaCl 2 . Patch-electrode solutions were brought up to pH 7.3-7.4 with KOH and adjusted to 290 -300 mosM/liter using glucose. Peak currents were elicited by voltage steps from a holding potential of Ϫ60 to Ϫ120 mV for 400 ms to relieve channel inactivation and to ϩ60 mV for 400 ms to achieve maximal channel activation. Inactivation rates were measured by fitting the falling phase of currents with a double-exponential decay (Fig. S1). Voltage dependence of activation was performed using the same holding and hyperpolarizing steps as above but with a range of intermediate activation potentials (Ϫ100, Ϫ80, Ϫ60, Ϫ40, Ϫ30, Ϫ20, and Ϫ10 and 0, ϩ10, ϩ20, ϩ30, ϩ40, and ϩ60 mV). Conductance was calculated using the equation: G ϭ I K /(V m Ϫ V rev ). Steadystate inactivation was performed using 400-ms conditioning steps from the holding potential to Ϫ140, Ϫ130, Ϫ120, Ϫ100, Ϫ80, Ϫ60, Ϫ40, Ϫ20, Ϫ10, and 0 mV immediately before a 400-ms step to the peak of current activation at ϩ60 mV. Recovery from inactivation was measured using two 400-ms voltage steps to ϩ60 mV from the holding potential separated by increasing time intervals (5,10,15,20,25,50,100,200, and 500 ms). Electrophysiological recordings were obtained using a Multiclamp 700B amplifier and PClamp 10 (Molecular, Devices, Sunnyvale, CA). Currents were normalized to cell size using whole-cell capacitance upon cell break-in; liquid junction potential error was corrected on line (Ϫ6.5 mV in the pipette), and leak currents were subtracted using a P/4 protocol. Data were analyzed using Microsoft Excel, GraphPad Prism 6, and IGOR Pro (WaveMetrics, Lake Oswego, OR). When acquiring recordings for 0 Ca 2ϩ and ϳ10 M Ca 2ϩ comparisons, all cells within a given genetic condition were performed on similar numbers of cells within the same day and also staggered by time of day. Pooled data are presented as either mean or box plots Ϯ S.E. or S.D. where indicated.