KChIP2 Isoforms Research Articles

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.

Calcium Regulation of Kv4-Kchip Ion Channel Complexes

The Kv4 family of A-type voltage-gated K+ channels play a unique role in the dendrites of hippocampal pyramidal neurons. In particular, Kv4.2 channels are key determinants of dendritic excitability and integration, spike timing-dependent plasticity, long-term potentiation, and learning. Downregulation of Kv4.2 channel expression occurs following hippocampal seizures and in epilepsy suggesting A-type currents as targets for novel therapeutics. In addition to pore forming Kv4 subunits, native hippocampal A-type currents require modulatory auxiliary subunits known as K-channel interacting proteins (KChIPs). KChIPs function to enhance Kv4 expression and activity. KChIP auxiliary proteins may convey Ca2+ sensitivity upon Kv4 channels as they are members of the EF-hand domain containing neuronal Ca2+ sensor protein family; however, it is currently unclear to what degree intracellular Ca2+ regulates Kv4.2-KChIP complexes. To assess this possibility, we expressed KChIP2 with Kv4.2 in HEK293T cells and performed whole-cell patch clamp recordings in either nominal or 10 μM Ca2+. In the presence of 10 μM Ca2+, we measured a 1.5 fold increase in Kv4.2 current density. Intriguingly, Ca2+ regulation was specific to short splice isoforms of KChIP2, KChIP2b and 2c. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. Sequence alignment of the variable N-termini of KChIP2 isoforms suggest a novel domain limits Ca2+ sensitivity of the longer KChIP2 isoforms, KChIP2a1 and KChIP2a.

Regulation of Kv4.3 and hERG potassium channels by KChIP2 isoforms and DPP6 and response to the dual K+ channel activator NS3623

Transient outward potassium current (Ito) contributes to early repolarization of many mammalian cardiac action potentials, including human, whilst the rapid delayed rectifier K+ current (IKr) contributes to later repolarization. Fast Ito channels can be produced from the Shal family KCNDE gene product Kv4.3s, although accessory subunits including KChIP2.x and DPP6 are also needed to produce a near physiological Ito. In this study, the effect of KChIP2.1 & KChIP2.2 (also known as KChIP2b and KChIP2c respectively), alone or in conjunction with the accessory subunit DPP6, on both Kv4.3 and hERG were evaluated. A dual Ito and IKr activator, NS3623, has been recently proposed to be beneficial in heart failure and the action of NS3623 on the two channels was also investigated. Whole-cell patch-clamp experiments were performed at 33 ± 1 °C on HEK293 cells expressing Kv4.3 or hERG in the absence or presence of these accessory subunits. Kv4.3 current magnitude was augmented by co-expression with either KChIP2.2 or KChIP2.1 and KChIP2/DPP6 with KChIP2.1 producing a greater effect than KChIP2.2. Adding DPP6 removed the difference in Kv4.3 augmentation between KChIP2.1 and KChIP2.2. The inactivation rate and recovery from inactivation were also altered by KChIP2 isoform co-expression. In contrast, hERG (Kv11.1) current was not altered by co-expression with KChIP2.1, KChIP2.2 or DPP6. NS3623 increased Kv4.3 amplitude to a similar extent with and without accessory subunit co-expression, however KChIP2 isoforms modulated the compound’s effect on inactivation time course. The agonist effect of NS3623 on hERG channels was not affected by KChIP2.1, KChIP2.2 or DPP6 co-expression.

KChIP2 Serves Multiple Functions in Cardiac Myocytes in Splice Isoform-Dependent Manner

K-channel-interacting-proteins (KChIPs) are EF-hand-motif-bearing Ca sensors that serve diverse functions. KChIP2 is the major KChIP expressed in the heart, that can modulate key cardiac channels (Itof, Nav, CaV and IKur) by directly interacting with their pore-forming subunits (Kv4, Nav1.5, Cav1.2, and Kv1.5, respectively) on the cell surface. More recent data suggest that KChIP2 can traffic to nuclei and exert transcriptional activity. Nine KChIP2 splice variants have been identified, that share C-terminal EF-hand-motifs but diverge in N-termini. Based on N-terminal sequences, KChIP2 splice variants can be broadly divided as palmitoylation-capable and palmitoylation-incapable. Questions: (1) Does palmitoylation impact on KChIP2 distribution and function?(2) Is KChIP2 splicing pattern altered in diseased heart? Methods: (1) Tag KChIP2a and 2c (palmitoylation-capable and -incapable) with mCherry, (2) Express KChIP2 isoforms in COS-7 cells alone or with GFP-Kv4.3, (3) Track KChIP2a/c distribution and relationship with Kv4.3 with confocal microscopy, (4) Quantify Itof channel function and subcellular distribution with patch clamp and subcellular fractionation/immunoblots, (5) Quantify native KChIP2 and Kv4 proteins and Itof channel function in ventricular myocytes from young and old spontaneously hypertensive rats (SY and SO, 4-6 and 22-24 months respectively; the latter suffers from severe hypertrophy/heart failure). Results: (1) When expressed alone, KChIP2a is strong in vesicles, Golgi and plasma membrane (PM), while KChIP2c is strong in nuclei and weak in PM. Pharmacological inhibition of palmitoylation directs KChIP2a from PM to nuclei, but does not alter nuclei localization of KChIP2c. (2) Kv4.3 coexpression leads to Kv4.3/KChIP2a overlap in Golgi, post-Golgi vesicles and PM. KChIP2c and Kv4.3 overlap on PM. While both KChIP2a and KChIP2c accelerate Kv4.3 recovery from inactivation, KChIP2a but not KChIP2c increases the current amplitude. (3) Relative to SY, Itof density is markedly reduced in epicardial myocytes (Epi VMs) of SO hearts. Kv4.2 and Kv4.3 proteins are modestly or not lowered in Epi VMs. While KChIP2a protein is markedly reduced, KChIP2c protein is significantly increased. Conclusions: Reversible palmitoylation confers dynamic KChIP2a localization between PM and nuclei, while KChIP2c has a more static distribution. Chronic hypertension/hypertrophy differentially impacts the expression of KChIP2a and 2c.

The "structurally minimal" isoform KChIP2d modulates recovery of Kv4.3 N-terminal deletion mutant Δ2-39

Mechanisms underlying Kv4 (Shal type) potassium channel macroscopic (open state) inactivation and recovery are unknown, as are mechanisms by which KChIP2 isoforms modulate these two processes. In a recent study (Xenopus oocytes, 2 microelectrode voltage clamp) we demonstrated that: i) Partial deletion of the Kv4.3 proximal N-terminal domain (Δ2-39; deletes N-terminal amino acids 2-39) not only slowed macroscopic inactivation, but also slowed the net rate of recovery; and ii) Co-expression of KChIP2b significantly accelerated the rate Δ2-39 recovery from inactivation. The latter effect demonstrated that an intact N-terminal domain was not obligatorily required for KChiP2b-mediated modulation of Kv4.3 recovery. To extend these prior observations, we have employed identical protocols to determine effects of KChiP2d on Δ2-39 macroscopic recovery. KChiP2d is a “structurally minimal” isoform (consisting of only the last 70 amino acids of the common C-terminal domain of larger KChIP2 isoforms) that exerts functional modulatory effects on native Kv4.3 channels. We demonstrate that KChiP2d also accelerates Δ2-39 recovery from macroscopic inactivation. Consistent with our prior Δ2-39 + KChIP2b study, these Δ2-39 + KChIP2d results: i) Further indicate that KChIP2 isoform-mediated acceleration of Kv4.3 macroscopic recovery is not obligatorily dependent upon an intact proximal N-terminal; and ii) Suggest that the last 70 amino acids of the common C-terminal of KChiP2 isoforms may contain the domain(s) responsible for modulation of recovery.

Ancillary Subunits Regulate PKC Mediated Effects on Closed-State Inactivation of Kv4.3

Kv4 channels are expressed with a variety of ancillary subunits in vivo. The most prominent of these proteins are the KChIPs, a family of cytoplasmic proteins that modulate channel gating and act as chaperones. We have previously shown that heterologously expressed Kv4.3 is regulated by PKC. After induction of PKC by PMA, current expression level is reduced. PKC also influences closed-state inactivation (CSI) in an isoform dependent manner; CSI is decreased upon PKC induction in Kv4.3-S, and increased in PKC-L. To understand the role of PKC on modulation of Kv4-based currents, we expressed channels in the presence of three KChIP2 isoforms and compared their responses to PKC. Two KChIP2 isoforms, KChIP2a and 2b, negated PKC influence on channel gating, kinetics, and expression levels in both Kv4.3-S and Kv4.3-L. In contrast, 70-amino acid KChIP2d was permissive for PKC modulation with respect to both CSI and current expression, while allowing the fast recovery from open-state inactivation characteristic of other KChIP2 isoforms. Additionally, the KChIP2d effects on CSI in Kv4.3-L were dependent on the presence of a putative PKC phosphorylation site in the C terminus. These data suggest a different physiological role for KChIP2d than the other KChIP2 isoforms, and suggest that the longer forms of KChIP2 interact with the regions of Kv4.3 affected by PKC, while KChIP2d interacts with the channel in a manner that allows PKC modulation while still accelerating recovery.

Regulation of Kv4.3 Closed-State Inactivation and Recovery by Extracellular Potassium and Intracellular KChIP2b

Mechanisms underlying Kv4 channel inactivation and recovery are presently unclear, although there is general consensus that the basic characteristics of these processes are not consistent with Shaker (Kv1) N- and P/C-type mechanisms. Kv4 channels also differ from Shaker in that they can undergo significant inactivation from pre-activated closed-states (closed-state inactivation, CSI), and that inactivation and recovery kinetics can be regulated by intracellular KChIP2 isoforms. To gain insight into the mechanisms regulating Kv4.3 CSI and recovery, we have analyzed the effects of increasing [K+]o from 2 mM to 98mM in the absence and in the presence of KChIP2b, the major KChIP2 isoform expressed in the mammalian ventricle. In the absence of KChIP2b, high [K+]o promoted Kv4.3 inactivated closed-states and significantly slowed the kinetics of recovery from both macroscopic and closed-state inactivation. Coexpression of KChIP2b in 2 mM [K+]o promoted non-inactivated closed-states and accelerated the kinetics of recovery from both macroscopic and CSI. In high [K+]o, KChIP2b eliminated or significantly reduced the slowing effects on recovery. Attenuation of CSI by the S4 charge-deletion mutant R302A, which produced significant stabilization of non-inactivated closed-states, effectively eliminated the opposing effects of high [K+]o and KChiP2b on macroscopic recovery kinetics, confirming that these results were due to alterations of CSI. Elevated [K+]o therefore slows Kv4.3 recovery by stabilizing inactivated closed-states, while KChIP2b accelerates recovery by destabilizing inactivated closed-states. Our results challenge underlying assumptions of presently popular Kv4 gating models and suggest that Kv4.3 possesses novel allosteric mechanisms, which are absent in Shaker, for coupling interactions between intracellular KChIP2b binding motifs and extracellular K+-sensitive regulatory sites.

Spatial distributions of Kv4 channels and KChip2 isoforms in the murine heart based on laser capture microdissection

Regional differences in repolarizing K(+) current densities and expression levels of their molecular components are important for coordinating the pattern of electrical excitation and repolarization of the heart. The small size of hearts from mice may obscure these interventricular and/or transmural expression differences of K(+) channels. We have examined this possibility in adult mouse ventricle using a technology that provides very high spatial resolution of tissue collection. Conventional manual dissection and laser capture microdissection (LCM) were utilized to dissect tissue from distinct ventricular regions. RNA was isolated from epicardial, mid-myocardial and endocardial layers of both the right and left ventricles. Real-time RT-PCR was used to quantify the transcript expression in these different regions. LCM revealed significant interventricular and transmural gradients for both Kv4.2 and the alpha-subunit of KChIP2. The expression profile of a second K(+) channel transcript, Kir2.1, which is responsible for the inwardly rectifying K(+) current I(k1), showed no interventricular or transmural gradients and therefore served as a negative control. Our findings are in contrast to previous reports of a relatively uniform left ventricular transmural pattern of expression of Kv4.2, Kv4.3 and KChIP2 in adult mouse heart, which appear to be different than that in larger mammals. Specifically, our results demonstrate significant epi- to endocardial differences in the patterns of expression of both Kv4.2 and KChIP2.

C-terminal Domain of Kv4.2 and Associated KChIP2 Interactions Regulate Functional Expression and Gating of Kv4.2

The Kv4.2 transient voltage-dependent potassium current contributes to the morphology of the cardiac action potential as well as to neuronal excitability and firing frequency. Here we report profound effects of the Kv4.2 C terminus on the surface expression and activation gating properties of Kv4.2 that are modulated by the direct interaction between KChIP2, an auxiliary regulatory subunit, and the C terminus of Kv4.2. We show that increasingly large truncations of the C terminus of rat Kv4.2 (wild type) cause a progressive decrease of Kv4.2 current along with a shift in voltage-dependent activation that is closely correlated with negative charge deletion. Co-expression of more limited Kv4.2 C-terminal truncation mutants (T588 and T528) with KChIP2 results in a doubling of Kv4.2 protein expression and up to an 8-fold increase in Kv4.2 current amplitude. Pulsechase experiments show that co-expression with KChIP2 slows Kv4.2 wild type degradation 8-fold. Co-expression of KChIP2 with an intermediate-length C-terminal truncation mutant (T474) shifts Kv4.2 activation voltage dependence and enhances expression of Kv4.2 current. The largest truncation mutants (T417 and DeltaC) show an intracellular localization with no measurable currents and no response to KChIP2 co-expression. Co-immunoprecipitation and competitive glutathione S-transferase-binding assays indicate a direct interaction between KChIP2 and the Kv4.2 C terminus with a relative binding affinity comparable with that of the N terminus. Overall, these results suggest that the C-terminal domain of Kv4.2 plays a critical role in voltage-dependent activation and functional expression that is mediated by direct interaction between the Kv4.2 C terminus and KChIP2.

Transmural Gradients of Repolarization and Excitation–Contraction Coupling in Mouse Ventricle

See related article, pages 1306–1313 The effects of changes in action potential waveform on excitation–contraction coupling in mammalian ventricle were first identified almost 40 years ago.1 Many important details concerning the relationship between action potential duration and tension development have been elucidated.2 Recent work has demonstrated significant effects on the intracellular Ca transient and excitation–contraction coupling of early repolarization of the action potential, which is strongly regulated by a Ca2+-independent transient outward K+ current denoted I to.3–6 The molecular physiology and some aspects of the pharmacology of this current are now quite well understood, based, in part, on a series of comprehensive articles from the Nerbonne et al,7,8 Strauss and Campbell,9 and others.6 Interestingly, a somewhat similar transient outward K+ current is expressed in neurons10 and in selected regions near the intraventricular septum of mammalian hearts.11–13 One of the most striking features of the transient outward K+ current in mammalian ventricle is the difference in its transmural expression, with significantly higher expression levels in epi- than in endocardium14 (see also references 8, 9, 11). The basis for this transmural heterogeneity, and also for the higher levels of expression in the right ventricle compared with the left, continues to be a topic of intense investigation. Significant new information and plausible working hypotheses for the way in which this heterogeneity …

Transient outward potassium current, 'Ito', phenotypes in the mammalian left ventricle: underlying molecular, cellular and biophysical mechanisms.

At least two functionally distinct transient outward K(+) current (I(to)) phenotypes can exist across the free wall of the left ventricle (LV). Based upon their voltage-dependent kinetics of recovery from inactivation, these two phenotypes are designated 'I(to,fast)' (recovery time constants on the order of tens of milliseconds) and 'I(to,slow)' (recovery time constants on the order of thousands of milliseconds). Depending upon species, either I(to,fast), I(to,slow) or both current phenotypes may be expressed in the LV free wall. The expression gradients of these two I(to) phenotypes across the LV free wall are typically heterogeneous and, depending upon species, may consist of functional phenotypic gradients of both I(to,fast) and I(to,slow) and/or density gradients of either phenotype. We review the present evidence (molecular, biophysical, electrophysiological and pharmacological) for Kv4.2/4.3 alpha subunits underlying LV I(to,fast) and Kv1.4 alpha subunits underlying LV I(to,slow) and speculate upon the potential roles of each of these currents in determining frequency-dependent action potential characteristics of LV subepicardial versus subendocardial myocytes in different species. We also review the possible functional implications of (i) ancillary subunits that regulate Kv1.4 and Kv4.2/4.3 (Kvbeta subunits, DPPs), (ii) KChIP2 isoforms, (iii) spider toxin-mediated block of Kv4.2/4.3 (Heteropoda toxins, phrixotoxins), and (iv) potential mechanisms of modulation of I(to,fast) and I(to,slow) by cellular redox state, [Ca(2)(+)](i) and kinase-mediated phosphorylation. I(to) phenotypic activation and state-dependent gating models and molecular structure-function relationships are also discussed.

Contribution of N‐ and C‐terminal channel domains to Kv channel interacting proteins in a mammalian cell line

Association of Shal gene‐related voltage‐gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C‐terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine‐scanning mutagenesis within the proximal 40 amino acid portion and a structure‐based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C‐terminus was truncated at various positions. Wild‐type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2‐induced modulation of Kv4.2 currents was studied with whole‐cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N‐terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C‐terminal deletions also had marked effects on KChIP2‐dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C‐terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose ‘anchoring’ at T1‐ and C‐terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.

Novel KChIP2 isoforms increase functional diversity of transient outward potassium currents.

Kv4.3 channels conduct transient outward K(+) currents in the human heart and brain where they mediate the early phase of action potential repolarization. KChIP2 proteins are members of a new class of calcium sensors that modulate the surface expression and biophysical properties of Kv4 K(+) channels. Here we describe three novel isoforms of KChIP2 with an alternatively spliced C-terminus (KChIP2e, KChIP2f) or N-terminus (KChIP2g). KChIP2e and KChIP2f are expressed in the human atrium, whereas KChIP2g is predominantly expressed in the brain. The KChIP2 isoforms were coexpressed with Kv4.3 channels in Xenopus oocytes and currents recorded with two-microelectrode voltage-clamp techniques. KChIP2e caused a reduction in current amplitude, an acceleration of inactivation and a slowing of the recovery from inactivation of Kv4.3 currents. KChIP2f increased the current amplitude and slowed the rate of inactivation, but did not alter the recovery from inactivation or the voltage of half-maximal inactivation of Kv4.3 channels. KChIP2g increased current amplitudes, slowed the rate of inactivation and shifted the voltage of half-maximal inactivation to more negative potentials. The biophysical changes induced by these alternatively spliced KChIP2 proteins differ markedly from previously described KChIP2 proteins and would be expected to increase the diversity of native transient outward K(+) currents.

Regulation of Kv4.3 voltage‐dependent gating kinetics by KChIP2 isoforms

We conducted a kinetic analysis of the voltage dependence of macroscopic inactivation (tau(fast), tau(slow)), closed-state inactivation (tau(closed,inact)), recovery (tau(rec)), activation (tau(act)), and deactivation (tau(deact)) of Kv4.3 channels expressed alone in Xenopus oocytes and in the presence of the calcium-binding ancillary subunits KChIP2b and KChIP2d. We demonstrate that for all expression conditions, tau(rec), tau(closed,inact) and tau(fast) are components of closed-state inactivation transitions. The values of tau(closed,inact) and tau(fast) monotonically merge from -30 to -20 mV while the values of tau(closed,inact) and tau(rec) approach each other from -60 to -50 mV. These data generate classic bell-shaped time-constant-potential curves. With the KChIPs, these curves are distinct from that of Kv4.3 expressed alone due to acceleration of tau(rec) and slowing of tau(closed,inact) and tau(fast). Only at depolarized potentials where channels open is tau(slow) detectable suggesting that it represents an open-state inactivation mechanism. With increasing depolarization, KChIPs favour this open-state inactivation mechanism, supported by the observation of larger transient reopening currents upon membrane hyperpolarization compared to Kv4.3 expressed alone. We propose a Kv4.3 gating model wherein KChIP2 isoforms accelerate recovery, slow closed-state inactivation, and promote open-state inactivation. This model supports the observations that with KChIPs, closed-state inactivation transitions are [Ca(2+)](i)-independent, while open-state inactivation is [Ca(2+)](i)-dependent. The selective KChIP- and Ca(2+)-dependent modulation of Kv4.3 inactivation mechanisms predicted by this model provides a basis for dynamic modulation of the native cardiac transient outward current by intracellular Ca(2+) fluxes during the action potential.

Palmitoylation of KChIP Splicing Variants Is Required for Efficient Cell Surface Expression of Kv4.3 Channels

The Ca(2+)-binding proteins KChIP1-4 (KChIP3 is also known as DREAM and calsenilin) act as auxiliary subunits for voltage-gated K(+) channels in the Kv4 family. Here we identify three splicing isoforms of rat KChIP2 with variable N-terminal peptides. The two longer isoforms, which contain the 32-amino acid peptide, produce larger increases in Kv4.3 protein level and current density and more effectively localize themselves and their associated channels at the plasma membrane than the shortest variant. The 32-amino acid peptide contains potential palmitoylation cysteines. Metabolic labeling demonstrates that these cysteines in the KChIP2 isoforms, as well as the corresponding sites in KChIP3, are palmitoylated. Mutating these cysteines reduces their plasma membrane localization and the enhancement of Kv4.3 current density. Thus, palmitoylation of the KChIP auxiliary subunits controls plasma membrane localization of their associated channels.

Heterogeneous expression of KChIP2 isoforms in the ferret heart.

Kv4 channels are believed to underlie the rapidly recovering cardiac transient outward current (I(to)) phenotype. However, heterologously expressed Kv4 channels fail to fully reconstitute the native current. Kv channel interacting proteins (KChIPs) have been shown to modulate Kv4 channel function. To determine the potential involvement of KChIPs in the rapidly recovering I(to), we cloned three KChIP2 isoforms (designated fKChIP2, 2a and 2b) from the ferret heart. Based upon immunoblot data suggesting the presence of a potential endogenous KChIP-like protein in HEK 293, CHO and COS cells but absence in Xenopus oocytes, we coexpressed Kv4.3 and the fKChIP2 isoforms in Xenopus oocytes. Functional analysis showed that while all fKChIP2 isoforms produced a fourfold acceleration of recovery kinetics compared to Kv4.3 expressed alone, only fKChIP2a produced large depolarizing shifts in the V(1/2) of steady-state activation and inactivation as seen for the native rapidly recovering I(to). Analysis of RNA and protein expression of the three fKChIP2 isoforms in ferret ventricles showed that fKChIP2b was most abundant and was expressed in a gradient paralleling the rapidly recovering I(to) distribution. Ferret KChIP2 and 2a were expressed at very low levels. The ventricular expression distribution suggests that fKChIP2 isoforms are involved in modulation of the rapidly recovering I(to); however, additional regulatory factors are also likely to be involved in generating the native current.