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TOPICAL REVIEW |
1 Smilow Neuroscience Program, Department of Physiology and Neuroscience, New York University School of Medicine, Smilow Research Center, 522 First Avenue, 6th Floor, New York, NY 10016, USA
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Abstract |
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 Top Abstract A-type K+ currents in...Structure of ISA channelsAuxiliary subunits of Kv4...Summary and perspectives: the...References |
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(Received 14 August 2008;
accepted after revision 30 September 2008;
first published online 9 October 2008)
Corresponding author B. Rudy: Smilow Neuroscience Program, Department of Physiology and Neuroscience, New York University School of Medicine, Smilow Research Center, 522 First Avenue, 6th Floor, New York, NY 10016, USA. Email: rudyb01{at}med.nyu.edu
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A-type K+ currents in brain and heart |
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TopAbstractA-type K+ currents in...Structure of ISA channelsAuxiliary subunits of Kv4...Summary and perspectives: the...References |
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Later, starting with the seminal paper by Hoffman et al. (1997), the function of the ISA in dendritic integration and plasticity has been increasingly appreciated. ISAs have been implicated in several aspects of signal processing in dendrites, including the modulation of general dendritic excitability, the regulation of action potential back propagation into the dendritic tree, the filtering and integration of synaptic potentials, distance-dependent synaptic scaling, the propagation of dendritic Ca2+ plateau potentials, the establishment and expression of long-term potentiation and the branch-specific compartmentalization of excitability and information storage (Hoffman et al. 1997; Schoppa & Westbrook, 1999; Johnston et al. 2000; Ramakers & Storm, 2002; Watanabe et al. 2002; Cai et al. 2004; Frick et al. 2004; Magee & Johnston, 2005; Chen et al. 2006; Losonczy & Magee, 2006; Kim et al. 2007).
More recently, evidence is starting to accumulate that this current is also important in disease. For example, a number of observations have suggested a link between ISA and epilepsy, both in animal models and in humans (e.g. Beck et al. 1997; Bernard et al. 2004; Ruschenschmidt et al. 2004). Direct evidence was recently found when a truncation in the gene encoding Kv4.2 (KCND2) producing a protein lacking the last 44 amino acids in the carboxyl terminal region was identified in a patient with temporal lobe epilepsy (TLE) (Singh et al. 2006). Studies in mice have also suggested an involvement of ISA channels in pain mechanisms (Hu et al. 2006).
The physiological significance of ISA channels in health and disease underlies the effort to elucidate the molecular composition of these channels. A current similar to the ISA in neurons, known as Ito (for transient outward current) is found in cardiac ventricular myocyes where it has an important role in the repolarization of the cardiac action potential.
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Structure of ISA channels |
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TopAbstractA-type K+ currents in...Structure of ISA channelsAuxiliary subunits of Kv4...Summary and perspectives: the...References |
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subunits), arranged with a fourfold symmetry around a central pore (Doyle et al. 1998; Long et al. 2005) (Fig. 1).
This tetramer forms the infrastructure of the channel and in most cases is sufficient to form a functional channel. However, in addition, many voltage-gated K+ channels contain auxiliary subunits (sometimes referred to as β subunits) that have primary sequences not resembling pore-forming subunits.
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Auxiliary subunits can modify the properties of the channels, often significantly, and in some cases might be essential for the efficient expression of functional channels in the plasma membrane. Other voltage-gated ion channels, such as sodium and calcium channels also contain auxiliary subunits with crucial roles in determining the localization and function of the channels (Isom, 2001; Tseng et al. 2007).
There is a large number of Kv subunits, the pore-forming subunits of voltage-gated K+ channels, which are classified in 12 groups or subfamilies based on sequence similarities. This diversity allows for the generation of many subtypes of voltage-gated K+ channels. It is now well established that proteins of the Kv4 subfamily of K+ channel proteins are the pore-forming subunits of the channels mediating most of the somato-dendritic ISA in neurons (Jerng et al. 2004a; Rudy et al. 2008). A total of three genes encoding Kv4 subunits have been identified in mammals: Kv4.1, Kv4.2 and Kv4.3 (Baldwin et al. 1991; Pak et al. 1991; Salkoff et al. 1992; Serodio et al. 1996). The Kv4.3 gene encodes two variants via alternative splicing: Kv4.3S and Kv4.3L (short and long). Two of these genes Kv4.2 and Kv4.3, but not Kv4.1 are prominently expressed in brain (Serodio & Rudy, 1998).
A large number of proteins, probably more than for any other channel group, have been suggested to associate with Kv4 pore forming subunits and function as auxiliary subunits. This work presents a critical review of the evidence supporting a role for these proteins as auxiliary subunits of Kv4 channels.
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Auxiliary subunits of Kv4 channels |
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TopAbstractA-type K+ currents in...Structure of ISA channelsAuxiliary subunits of Kv4...Summary and perspectives: the...References |
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Early efforts to reconstitute the channels mediating the ISA showed that coexpression in Xenopus oocytes of Kv4 proteins with brain mRNAs lacking Kv4 transcripts could restore many of the properties seen in native A-type K+ currents (Rudy, 1988; Chabala et al. 1993; Serodio et al. 1994, 1996). The first Kv4 channel associated proteins found, the so-called K+ channel interacting proteins or KChIPs were identified in 2000.
K+ channel interacting proteins
K+ channel interacting proteins (KChIPs) were identified in yeast-two hybrid screens for Kv4 associated proteins using the Kv4.3 N-terminal region as bait (An et al. 2000). Four KChIP genes (KChIP1–4) are now known, each producing multiple products via alternative splicing. At least 12 different KChIPs, spliced products of the KChIP1–4 genes, have been isolated to date from brain and heart tissue (An et al. 2000; Bahring et al. 2001; Holmqvist et al. 2002; Patel et al. 2002; Takimoto & Ren, 2002; Boland et al. 2003; Van Hoorick et al. 2003; Jerng et al. 2004a; Burgoyne, 2007).
KChIPs are small proteins (200–250 amino acids long), of the recoverin neuronal calcium sensor (NCS-1) family. They consist of a variable N-terminal region and a highly conserved C-terminal ‘core’ with four EF hand-like Ca2+-binding motifs (An et al. 2000).
KChIPs modify Kv4 currents in heterologous cells. KChIPs facilitate the trafficking, subunit assembly, stability and surface expression of Kv4 channel complexes (An et al. 2000; Shibata et al. 2003). Hence, coexpression of Kv4 proteins and KChIPs results in large increases (as much as 10-fold) in the amplitude of the expressed Kv4 current. In mammalian heterologous expression systems transfected only with Kv4 cDNAs, the expressed Kv4 proteins are retained in the endoplasmic reticulum and poorly transported to the plasma membrane. Additionally, KChIPs modify the electrophysiological properties of the channels expressed by Kv4 proteins in heterologous expression systems, which in general inactivate more slowly and recover from inactivation with faster kinetics in the presence of KChIPs (An et al. 2000; Rosati et al. 2001; Holmqvist et al. 2002) (Fig. 2). KChIPs also produce small shifts in the conductance–voltage curve, although the reported magnitude and direction of this effect varies between different studies. All KChIP isoforms have similar effects on Kv4 channels except for the alternative spliced version KChIP4a, which does not promote surface expression and prevents channel inactivation (An et al. 2000; Holmqvist et al. 2002).
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Although other than these differences all KChIP proteins appear to have similar effects on Kv4 channels in heterologous cells, each is expressed in specific neuronal populations in the CNS, suggesting specific, yet to be determined requirements for Kv4-KChIP combinations in different neurons (Rhodes et al. 2004). Many neuronal populations express two or more KChIPs; however, it is not known whether the same channel complex includes more than one type of KChIP.
Evidence that KChIPs are components of native Kv4 channels. A large and diverse series of experimental observations strongly support the notion that KChIP proteins are associated with Kv4 proteins in brain and heart and function as auxiliary subunits of native Kv4 channels in these tissues. These include the following.
Mechanism of KChIP modulation of Kv4 channels. The primary effects of KChIPs on Kv4 channels in heterologous cells are to facilitate trafficking of Kv4 channels to the plasma membrane, to slow macroscopic inactivation and to accelerate the recovery from inactivation of Kv4 channels. Wang et al. (2007) and Pioletti et al. (2006) recently described a cocrystal structure of the complex formed between the Kv4.3 N-terminus and KChIP1. The structure shows that each KChIP1 molecule (4 in the complex) interacts with two neighbouring Kv4.3 N-termini in a 4: 4 manner, forming a cross-shaped octamer. The proximal N-terminal peptide of one Kv4.3 N-terminus is sequestered (‘clamped’) by its binding to an elongated hydrophobic groove on the surface of KChIP1. At the same time, each KChIP1 binds to the T1 domain of an adjacent Kv4.3 subunit to stabilize the tetrameric Kv4.3 complex. T1 or tetramerization domains are present in the N-terminal region of Kv proteins and determine subfamily-specific association of pore-forming subunits. The stabilization of the tetrameric Kv4 complex by KChIPs is likely to contribute to facilitating the trafficking of channels to the plasma membrane (Cui et al. 2008). In addition, Shibata et al. (2003) have suggested that by binding to the N-terminus of Kv4 proteins, KChIPs may mask a cytoplasmic retention/solubility motif.
The sequestration of the N-terminus is also likely to explain how KChIPs slow down inactivation (Pioletti et al. 2006; Wang et al. 2007), by preventing a fast open-state inactivation resembling the N-type inactivation in other transient K+ channels (Gebauer et al. 2004). As a result, the channels inactivate more slowly, and predominantly from closed states (Jerng et al. 2004a). The mechanism by which KChIPs accelerate inactivation recovery is less clear.
Dipeptidyl peptidase like proteins
DPP6 and DPP10 – new auxiliary subunits of Kv4 channels: discovering a new function for dipeptidyl peptidase like proteins. Dipeptidyl peptidases (DPPs) are an important family of proteolytic enzymes that hydrolyse dipeptides from a number of protein targets resulting in the activation or inactivation of the substrate (Lambeir et al. 2003; Gorrell et al. 2006; Maes et al. 2007). Unexpectedly, purification of Kv4 channel complexes from rat cerebellar membranes suggested that a member of this protein family known as DPP6 or DPPX was an integral component of Kv4 channels in neurons (Nadal et al. 2003).
Kv4.2–KChIP complex produces incomplete ISA channels. The association of DPP6 with Kv4 proteins was discovered as a result of continuing efforts to reconstitute the ISA in heterologous expression systems. Evidence from studies in neurons and cardiac tissue accumulated supporting the conclusion that KChIPs were a component of native Kv4 channels functioning as auxiliary subunits. KChIPs significantly facilitated Kv4 channel expression at the plasma membrane in heterologous cells. Furthermore, one of the effects of KChIPs on Kv4 currents in heterologous cells, the acceleration of the kinetics of recovery from inactivation, was in the right direction to increase resemblance with native currents (An et al. 2000; Jerng et al. 2004a).
However, in the presence of KChIPs other properties become less similar to those of native currents. In particular, the kinetics of inactivation are slowed by KChIP coexpression whereas the kinetics of inactivation of native ISA channels in many populations of neurons is faster, not slower, than the inactivation of the channels produced by Kv4 expressed alone (Figs 2 and 3). This raised the possibility that native ISA channels may include additional factors that were missing in the heterologously expressed channels.
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Nadal et al. (2003) used classical biochemical techniques to immunopurify Kv4 channel complexes from solubilized rat cerebellar membranes. The complex consisted of three major polypetides, including Kv4.2, KChIP and an unidentified component of 
115 kDa present in the complex in apparent similar abundance. Sequencing revealed that this polypeptide corresponded to a previously identified integral membrane protein for which no function was previously known, the DPP-like protein DPP6.
DPP6 is a member of the dipeptidyl aminopeptidase family of proteins, with approximately 30% identity and 50% similarity to a prominent member of this family known as DPPIV or CD26 (Nadal et al. 2003). However, in contrast to DPPIV, and all other members of the family known at the time, DPP6 has mutations in the active site and lacks enzymatic activity (Wada et al. 1992; Kin et al. 2001). The effects of DPP6 on the currents expressed by Kv4.2 in heterologous cells and knockdown experiments demonstrated that DPP6 was KAF, the unidentified factor that accelerated Kv4 channel kinetics.
Effects of DPP6 on Kv4 channels in heterologous expression systems. Like KChIPs, DPP6 facilitates the trafficking of Kv4 channels to the plasma membrane in heterologous cells, resulting in up to 20-fold increases in current density (Nadal et al. 2003, 2006). This dramatic effect is largely due to an increase in the trafficking of channel complexes to the plasma membrane. Also like KChIPs, DPP6 accelerates the rate of recovery from inactivation, even to a larger degree than KChIP (Fig. 3). DPP6 also produces substantial hyperpolarizing shifts in the voltage dependence of activation and inactivation of Kv4 channels, whether they include KChIPs or not, and increases the rate of activation and inactivation (Nadal et al. 2003, 2006; Zagha et al. 2005; Amarillo et al. 2008) (Fig. 3). All these are effects that can profoundly affect neuronal properties, particularly at subthreshold membrane potentials.
DPP10: another DPP that lacks enzymatic activity and modifies Kv4 channels. Following the report that DPP6 associates with Kv4 channels, a new member of the DPP family of proteins, named DPP10, was identified (Qi et al. 2003). DPP10 was found to be prominently expressed in brain and like DPP6 to have mutations in the catalytic triad and lack DPP activity. Phylogenetic analysis showed that DPP10 and DPP6, which we collectively call DPP-like proteins or DPPLs, form an evolutionarily divergent subfamily within the extended DPPIV-like family, sharing 51% amino acid sequence identity, compared to 32% identity between DPP10 and DPPIV. DPP6 and DPP10 have nearly identical transmembrane and juxtamembrane domains, sharing 92% similarity compared with 41% similarity between DPP10 and DPPIV. The extracellular domains of the three proteins also have similar structure. However, DPP6 and DPP10 are more similar to each other than either is to DPPIV (Zagha et al. 2005).
Jerng et al. (2004b), Zagha et al. (2005) and Ren et al. (2005) showed that DPP10, like DPP6, facilitated Kv4 protein trafficking to the plasma membrane and had similar effects on the voltage dependence and kinetic properties of Kv4 currents, which were not shared by DPPIV. Similar to KChIPs, there might be a certain degree of division of labour between DPP6 and DPP10 in brain. Zagha et al. (2005) observed that DPP6 mRNAs were preferentially expressed in neurons that contain predominantly Kv4.2 (hippocampal pyramidal neurons, striatal medium spiny neurons and cerebellar granule cells) while neurons that express predominantly Kv4.3 (for example Purkinje cells and hippocampal interneurons) preferentially expressed DPP10. Some neurons may express both genes.
Evidence for a ternary Kv4–KChIP–DPPL complex in neurons: critical evaluation of DPPLs as auxiliary subunits of neuronal Kv4 channels. Nadal et al. (2003) found that DPP6 proteins copurified with Kv4.2 proteins from rat cerebellar membranes, suggesting that they are associated in the same molecular complex. However, at least in detergent solution this interaction seemed to be weak, requiring cross-linking prior to solubilization in order to optimize recovery of a stable complex in detergent solution. Furthermore, the finding that DPP6 was an associated protein of Kv4 channels was without precedent in the DPP family of proteins, none of which had been shown to function as an associated protein of other membrane proteins. It has therefore been critical to validate the association between DPPLs and native Kv4 channels and their role as auxiliary subunits.
Evidence that DPPLs are auxiliary subunits of Kv4 channels in neurons and that these channels are likely to be ternary complexes including KChIPs and DPPLs is now very strong and includes the following.
70% from 4.0 to 7.0 pS (Rocha et al. 2004; Kaulin, Santiago-Custillo, Rocha, Nadal, Rudy and Covarrubias unpublished observations). This results in a unitary conductance close to that reported (6–8.5 pS) for ISA channels in hippocampal CA1 and neocortical pyramidal neurons and cerebellar granule cells (Hoffman et al. 1997; Bekkers, 2000; Chen & Johnston, 2004). These observations suggest that DPPX is necessary and sufficient to set the unitary conductance of neuronal Kv4 channels.
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Alternative splicing of the N-terminus of DPP6 and DPP10 produces a large number of distinct isoforms which differ only in short intracellular domains following common extracellular, transmembrane and juxtamembrane domains. Five DPP6 isoforms (DPP6-S, DPP6-L, DPP6-E, DPP6-D, and DPP6-K) and four DPP10 splice variants (DPP10-A to DPP10-D) have been described (Nadal et al. 2006; Takimoto et al. 2006; Jerng et al. 2007). DPP6-S, L and K have similar effects on Kv4 channel properties, small differences were observed on the effects of these isoforms on the rate of recovery from inactivation and on the shift in the voltage dependence of inactivation (Nadal et al. 2006). On the other hand, Jerng et al. (2007) found that DPP10-A produced currents that inactivated much faster than those produced by other DPP10 isoforms and lacked the slow down of the inactivation rate during large depolarizations induced by KChIPs. The N-terminus of DPP6-E shows sequence similarity to the N-terminus of DPP10-A and was found to induce similar effects as DPP10-A (Maffie et al. 2008).
In situ hybridization with spliced version-specific probes revealed that distinct neuronal populations express combinations of different DPP6 and DPP10 isoforms, suggesting that DPPLs can contribute to the diversity of ISA in different neuronal populations (Nadal et al. 2006; Jerng et al. 2007). Strongly supporting this hypothesis, DPP6-E was found to be expressed in neurons in intermediary layers of the superior colliculus, which have ISAs that inactivate much faster than in other neurons and lack the slowing of inactivation with increasing depolarization (Maffie et al. 2008). DPPLs form dimers and each channel complex is likely to include two DPPL dimers (Strop et al. 2004; Soh & Goldstein, 2008) (Fig. 1). Since different DPPLs can form heterodimers (Strop et al. 2004) there are a large number of possible DPPL combinations which can contribute to ISA diversity.
Mechanisms of action of DPPLs. One the most fundamental effects of DPPLs appears to be facilitating the voltage-dependent gating transitions that activate the channels, resulting in large hyperpolarizing shifts in voltage dependence. Other effects of DPPLs might be consequences, at least in part, of this shift in voltage dependence. Recently Dougherty & Covarrubias (2006) found that DPP6-S causes a –26 mV shift in the gating charge–voltage (Q–V) relation, suggesting that DPP6 remodels the channel's voltage sensor. This is interesting given evidence suggesting transmembrane interactions between DPPL proteins and Kv4 subunits, possibly directly with the voltage sensor.
Zagha et al. (2005) found that chimeric DPPL proteins with extracellular domains replaced by a series of Myc tags were able to reproduce many of the effects of DPP10 and DPP6 on Kv4 channels, suggesting a transmembrane interaction between DPPLs and Kv4 proteins. This is supported by mutagenesis studies by Ren et al. (2005) suggesting that the S1 and S2 domains in the Kv4.3 voltage sensor determine association with DPP10.
Kv4-unrelated functions of DPPLs and KChIPs?. KChIP3 has been independently identified three times and assigned two additional roles besides its function as an auxiliary subunit of Kv4 channels. It was identified as DREAM (downstream regulatory element antagonist modulator), a Ca2+ regulated transcription repressor shown to bind the dynorphin response element (Cheng et al. 2002), and as calsenilin, a protein that binds and modulates some of the effects of the Alzheimer's disease-related protein presenilin. Observations with KChIP3 knockouts have provided evidence supporting these Kv4-unrelated functions. Knockouts of the KChIP3 locus produce animals with decreased pain sensitivity as a result of enhanced trascription of dynorphin, an opiate neuropeptide (Cheng et al. 2002). Another study found that KChIP3 knockouts had reduced Aβ levels and enhanced long-term potentiation consistent with its presenilin interaction and channel modulatory roles (Lilliehook et al. 2003).
DPP6 may also have Kv4-unrelated functions. It has been shown, as described earlier, that overall, DPP6 proteins are expressed in the same neuronal populations, and within these in the same neuronal compartments as Kv4 proteins. However, in one brain structure, the hippocampal mossy fibre axons, which lack Kv4.2 or Kv4.3 proteins, there is prominent expression of DPP6. The function of DPP6 in these axons remains to be investigated; however, the finding suggests that DPP6 may also have Kv4-unrelated functions in brain (Clark et al. 2008).
Other putative Kv4 channel-associated proteins
A surprisingly large number of additional proteins have been suggested to associate with Kv4 subunits. Most of these have not been pursued by workers in the field as enthusiastically as KChIPs and DPPLs, and additional validation that they interact and modulate Kv4 channels in native tissue, and therefore that the association is physiologically relevant is required. The list includes the following.
Validating channel modulation by auxiliary proteins. Essentially two experimental approaches have been used to suggest that Kvβs, KChAP and KCNE proteins are auxiliary subunits of Kv4 channels, coexpression with Kv4 proteins in heterologous expression systems and coimmunoprecipitation from solubilized extracts of native tissue.
Changes in channel properties produced by the presence of the putative associated subunits suggests that the two types of protein interact, and raises the possibility that the same interaction occurs in native cells. However, in over-expressing heterologous cells, interactions could take place between proteins that never see each other in native cells or at similar concentrations. For example Kvβs are typically found in axons in many neurons (Rhodes et al. 1997; Monaghan et al. 2001), but Kv4 proteins are found in dendrites. Moreover, the putative auxiliary protein could have effects on channel function that are not the result of physical interaction with the pore forming subunits. When the putative auxiliary protein changes channel properties in such a way that the reconstituted channels better resemble native channels, the heterologous expression experiment becomes a good argument in support of a physiological association. Yet, even for a physiologically relevant interaction the reconstituted channels may not resemble native channels due to the existence of additional binding partners or other factors. However, when this is the case, obtaining independent evidence that the association takes place in native systems becomes particularly critical. This is the case with the proteins discussed in this section. They all enhance Kv4 current levels, albeit to a much smaller degree than KChIPs and DPPLs, but most tend to produce currents that increasingly depart from native currents in heart or brain, by slowing the kinetics of activation, inactivation and/or recovery from inactivation and by producing depolarizing shifts in voltage dependence.
The observations suggest that is easy to modify Kv4 channel expression in heterologous cells. This might be related to the fact, discussed earlier, that Kv4 proteins express poorly at the cell surface. A number of proteins, especially when they are over-expressed, may non-specifically affect Kv4 channel structure or the mechanisms that retain channels in intracellular compartments. Proteins with a single transmembrane sequence are of particular concern given the evidence that the membrane spanning domain of DPPLs interacts with the Kv4 voltage sensor. This indicates that the voltage sensor of Kv4 proteins has a sufficiently ‘flexible’ or ‘open’ structure that easily accommodates an extrinsic hydrophobic sequence. It is worth mentioning in this regard that Deschenes & Tomaselli (2002) found that the Na+ channel β1 subunit, a protein with a single membrane spanning domain as in KCNE and DPPL proteins, had effects on Kv4.3 channels in heterologous cells that resembled in magnitude the effects of many of the putative Kv4 channel auxiliary proteins discussed in this section.
Co-immunoprecipitation (co-IP) from native membranes can in principle provide a very powerful argument supporting association in vivo. However, the uncertainties regarding which of the associations discussed in this section are physiologically relevant suggest to us that, in the absence of independent evidence of the association, a more strict approach to co-IP is necessary.
Typically, investigators first immunoprecipitate tissue extracts solubilized with a non-denaturing detergent using an antibody to the pore-forming subunit (sometimes of dubious specificity; Rhodes & Trimmer 2006). A denaturing detergent is then added to the precipitate to separate the polypeptides present in the immunoprecipitated complex and these are separated by electrophoresis. Immunoblots of these polypeptides are then used to show that the immunoprecipitate not only contains the pore-forming subunit but also the putative auxiliary protein(s). Often no controls are performed, and rarely a quantification of the immunoprecipitated proteins relative to their abundance in the extract is presented.
However, proteins other than those truly associated with the pore forming subunits in the native membranes prior to solubilization may appear in the immunoprecipitate. Non-specific interactions may take place once the membranes are solubilized. Hydrophobic proteins might be particularly ‘sticky’. The auxiliary proteins may not even interact with the pore-forming subunits in the solubilized material, but may bind non-specifically to the reagents used for immunoprecipitation or to the beads typically used to bring down the immunoprecipitate. A small amount of the protein might just be ‘trapped’ in the immunoprecipitate. This is why quantification could be very useful. Controls for these and other possible artifacts of the procedure will go a long way to increase the value of coimmunoprecipitation assays.
Another protein that has been suggested to associate with Kv4 channels in cardiac and nervous tissue is the neuronal calcium sensor protein-1 (NCS-1 or frequenin), a member of the EF-hand family of Ca2+-sensing proteins that includes KChIPs. NCS-1 was reported to have effects on Kv4 channels in heterologous cells similar to (but apparently less effective than) those of KChIPs (Nakamura et al. 2001; Guo et al. 2002). The evidence that has been used to support NCS-1 association to Kv4 subunits is similar to that used initially to support KChIP association. However, this evidence has not been corroborated by many research groups or strengthened by independent evidence as in the case of KChIPs. Moreover, it is not clear whether NCS-1 associates with the channels in the presence of KChIPs, since both bind to the same site.
Interactions of Kv4 subunits with cytoskeletal proteins
K+ channel proteins also interact with cytoskeletal proteins. These associations are important in the localization of channels to specific neuronal compartments and in the formation of microdomains with signalling molecules. The C terminus of Kv4.2 was found in a yeast two-hybrid screen to interact with filamin, an actin-binding protein (Petrecca et al. 2000). Filamin was also shown to coimmunoprecipitate with Kv4.2 from brain extracts. To investigate possible functional consequences of this interaction, Kv4.2 was expressed in filamin-containing and filamin-lacking cells. Kv4.2 was found to colocalize with filamin at filopodial roots in filamin-containing cells, but had a uniform expression pattern in cells lacking filamin or in filamin-containing cells transfected with a Kv4.2 mutant lacking the filamin-binding region. Petrecca et al. (2000) proposed that filamin functions as a scaffold protein in the postsynaptic density, mediating a direct link between Kv4 proteins and the actin cytoskeleton. While this hypothesis still needs to be confirmed it is of interest in that it may contribute to the highly specific and dynamic localization of Kv4 channels within dendritic spines (Burkhalter et al. 2006; Kim et al. 2007).
Kv4.2 was also found to interact with PSD-95 via Kv4.2's C-terminal putative PDZ-binding domain (VSAL) (Wong et al. 2002). PSD-95, another scaffolding protein present in the postsynaptic density, is involved in the localization of other K+ channels and glutamate receptors to synapses.
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Summary and perspectives: the impact of DPPLs and KChIPs on Kv4 channel modulation |
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TopAbstractA-type K+ currents in...Structure of ISA channelsAuxiliary subunits of Kv4...Summary and perspectives: the...References |
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750 kDa, probably one of the largest K+ channel complexes known. Research can now move from validating the associations to investigating the molecular mechanisms by which these auxiliary subunits regulate channel function, which is necessary to understand how Kv4 channels work. Future research can now also address how the association of the channels with these proteins affects neuronal function and how mutations in the proteins produce disease. Given unexplored and intriguing structural features of these proteins, including Ca2+ binding motifs in KChIPs and a DPP-like extracellular domain in DPPLs, this research is likely to lead to the discovery of novel aspects of Kv4 channel function in neurons. Of interest for example are the contributions of these proteins to the localization of Kv4 channels in specific microdomains (Burkhalter et al. 2006), as well as their modulation by extracellular and intracellular signals and their activity-dependent dynamic expression at the plasma membrane (Kim et al. 2007).
The extracellular domain of DPPLs constitutes most of the mass of these proteins (Fig. 1). As in other DPPs the extracellular domain consists of a β propeller and an /β hydrolase domain (Strop et al. 2004). β propellers provide excellent platforms for protein–protein interactions, and in DPPIV the extracellular domain binds to components of the extracellular matrix and mediates roles in cell adhesion, cellular trafficking and T cell activation (De Meester et al. 1999; Hildebrandt et al. 2000; Gorrell et al. 2001). DPPLs may confer targeting or cell adhesion properties to Kv4 channels through its homologous extracellular domain (Fig. 1). Protein–protein associations mediated by the DPPL extracellular domain could be important in determining the organization of Kv4 channels in the dendritic plasma membrane. Alternatively, cell–cell interactions or interactions with extracellular matrix components may modulate the function of ISA channels.
The other putative associated proteins of Kv4 channels that were evaluated here are unlikely to be part of the channel complex throughout the CNS given that they produce changes in channel properties not seen in neurons. Furthermore, it is unlikely, if not physically impossible, that Kv4 channels are interacting with all these proteins simultaneously. However some of these may associate with channels in specific neurons or specific neuronal structures, where the channels could have biophysical properties that are different from those known so far. However, convincing evidence that this occurs still needs to be obtained. Furthermore, the available evidence does not exclude the possibility that the interactions of Kv4 channels with KChIPs and DPPLs are dynamic, or that in some neuronal populations or in specific neuronal compartments channels may exist associated with only one of these proteins.
Less is known about the composition of Kv4 channels in other tissues. In ventricular myocytes, where Kv4 channels mediate the current known as Ito (or sometimes fast Ito), the channels contain KChIPs, and specifically KChIP2. However, these cells seem to lack DPPLs (at least in rodents, see Radicke et al. 2005). Data suggesting that MiRPs are components of cardiac Kv4 channels were discussed; however, these don't seem to reconstitute the properties of native Ito.
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TopAbstractA-type K+ currents in...Structure of ISA channelsAuxiliary subunits of Kv4...Summary and perspectives: the...References |
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), Kv4.2 and DPP6-S (
), Kv4.2 and KChIP1 (
) and Kv4.2 + KChIP1 and DPP6-S (
). Note that the relation between inactivation rate and membrane potential is distinct for channels only containing KChIP or DPP6 and channels containing both auxiliary subunits. B, half-inactivation time (t1/2) as a function of membrane potential of ISA isolated from cerebellar granule cells in Kv4.2-containing anterior cerebellar lobules (black) and Kv4.3-containing posterior lobules (red). Note that the profile of the voltage dependence of the inactivation rate of the ISA in CGNs is remarkably similar to that observed for the currents expressed by ternary channel complexes containing Kv4.2, KChIP1 and DPP6-S. From 