Kv1.5 in the Immune System: the Good, the Bad, or the Ugly?

For the last 20 years, knowledge of the physiological role of voltage-dependent potassium channels (Kv) in the immune system has grown exponentially. Leukocytes express a limited repertoire of Kv channels, which contribute to the membrane potential. These proteins are involved in the immune response and are therefore considered good pharmacological targets. Although there is a clear consensus about the physiological relevance of Kv1.3, the expression and the role of Kv1.5 are controversial. However, recent reports indicate that certain heteromeric Kv1.3/Kv1.5 associations may provide insight on Kv1.5. Here, we summarize what is known about this issue and highlight the role of Kv1.5 partnership interactions that could be responsible for this debate. The Kv1.3/Kv1.5 heterotetrameric composition of the channel and their possible differential associations with accessory regulatory proteins warrant further investigation.


Kv1.5 in the immune system
Pioneer work conducted during the 1970s by Gallin and coworkers described the first K + currents in peritoneal macrophages (Gallin et al., 1975;Gallin and Gallin, 1977;Gallin and Livengood, 1980). Later, this K + outward conductance was characterized in both lymphocytes and macrophages (Gallin, 1981(Gallin, , 1984Ypey and Clapham, 1984;Decoursey et al., 1987). Although delayed rectifier K + currents are similar in both cell types, Kv1.5 was soon detected in microglia (brain macrophages) (Pyo et al., 1997;Jou et al., 1998). These early works suggested that Kv1.5 plays an important role in the immune system. However, elicited currents showed certain C-type inactivation, which is absent in Kv1.5. In addition,Kv1.3 blockers, such as Charybdotoxin, were used to pharmacologically characterize the current in macrophages (Kim et al., 1996). However, this apparent discrepancy could be explained by the cellular models analyzed ( Table 1). These works mostly analyzed peritoneal elicited macrophages, and under these experimental conditions, cells were isolated upon intraperitoneal injection (Gallin and Livengood, 1980;Ypey and Clapham, 1984). Currently, we know that Kv1.3 and Kv1.5 are subject to differential regulation (Vicente et al., 2006;Villalonga et al., 2010a). Under activation conditions, unlike Kv1.5,Kv1.3 is selectively activated. Any study in activated cells would underestimate the Kv1.5dependent component of the outward K + current. Later works from Eder and coworkers demonstrated that, unlike T-cells, resting bone marrow-derived macrophages express less inactivating outward K + currents, which are selectively induced by specific growth factors (Eder and Fischer, 1997). These more recent reports suggested that notable differences exist between T-lymphocytes and macrophages. In 2003, we published a complete biophysical, pharmacological and molecular characterization of the Kv1 channels present in macrophages (Vicente et al., 2003). Although pharmacological experiments mice (Koni et al., 2003). However, this protein plays a critical role during activation and proliferation of leukocytes, and several studies point to this channel as an excellent target for immunomodulation (Beeton and Chandy, 2005;Rangaraju et al., 2009). Kv1.3 is abundantly expressed in T-effector memory lymphocytes. These T-cells are key mediators in autoimmune inflammatory diseases, such as multiple sclerosis, rheumatoid arthritis, psoriasis, and type I diabetes, and Kv1.3-based therapies are effective in experimental models (Beeton and Chandy, 2005;Rangaraju et al., 2009). Although Kv1.3 activity determines the level of leukocyte activation, this may be conditioned by the presence of other Kv1 isoforms and their association with accessory subunits (Beeton and Chandy, 2005;Vicente et al., 2005a).
Lymphocytes express several Kv currents (n-, n′-and l-type channels). While the type n channel is the most commonly observed K + channel in normal T-lymphocytes, the subtype l has a larger singlechannel conductance, and the n′ channel has biophysical and pharmacological properties similar to those of the n channel but is more resistant to block by tetraethylammonium (TEA) (Grissmer et al., 1992). Whereas Kv1.3 is associated with the n-type channel and Kv3.1 seems to account for the l-type, the protein responsible for the n′-type is uncertain (Grissmer et al., 1992;Cahalan and Chandy, 1997). In addition, other channel proteins have been described in immune system cells. Kv1.1 is present in CD4 − CD8 − thymocytes, and the presence of Kv1.1, Kv1.2, and Kv1.6 has been documented in naïve CD4 + lymphocytes (Liu et al., 2002). In addition, several regulatory subunits may tune Kv currents resembling different entities. Thus, classical Kvβ subunits are present in lymphocytes and macrophages (Autieri et al., 1997;Vicente et al., 2005a). Furthermore, other types of modulatory subunits, such as KCNEs, are also present in leukocytes (Sole et al., 2009;Sole and Felipe, 2010). For example, KCNE1 was cloned from T-cells (Attali et al., 1992), and KCNE4 is expressed in macrophages (Sole et al., 2009). In this scenario deciphering the composition of channel structures that include channel subunits and regulatory proteins (channelosome) in leukocytes is worth of effort.
In addition, pharmacological studies in lymphocytes as well as in macrophages have demonstrated that the blockage of the K + outward current by K + channel-blockers, such as charybdotoxin, TEA and 4-Aminopyridine (4-AP) reveals other components. These components have more depolarized thresholds for activation, absence of inactivation under sustained depolarized pulses and negligible cumulative inactivation (Ypey and Clapham, 1984;Decoursey et al., 1987;Verheugen and Korn, 1997). Although the presence of Kv3.1 could somehow explain these features, Kv1.5 may be an alternative candidate (Grissmer et al., 1992;Verheugen and Korn, 1997;Vicente et al., 2003Vicente et al., , 2005a.
During the same time frame, Mackenzie et al. (2003) described that human alveolar macrophages only express Kv1.3, with no other Kv1 isoforms. However, because MgTx (1 nM) does not abrogate Fc receptor-mediated phagocytosis, the authors suggested that although Kv1.3 sets the resting membrane potential, it is not required for phagocytosis. The debate intensified when Park et al. (2006), using a similar experimental model, concluded that Kv1.5, but not Kv1.3, plays a pivotal role in human alveolar macrophages.
A common feature of all these studies is the sensitivity of K + currents to some blockers such as MgTx, 4-AP, TEA, or Shk-Dap 22 . Several works also tried to abrogate K + currents by incubating with antisense oligonucleotides and adenovirus, and differing results have indicated possible roles for both proteins in the immune physiology (Chung et al., 2001;Mullen et al., 2006;Pannasch et al., 2006). Therefore, for the first time, a new putative role for the Kv1.5 channel in the immune system was highlighted in a comprehensive review . In 2006, the field took an important step forward. Several laboratories identified shared expression of both Kv1.3 and Kv1.5 proteins in the myeloid lineage (Mullen et al., 2006;Pannasch et al., 2006;Park et al., 2006;Vicente et al., 2006). Thus, both channels were found to modulate distinct functions in microglia, dendritic cells, and macrophages. However, the precise mechanism was still under debate (Fordyce et al., 2005). The initial evidence of heterotetrameric associations was suggested 1 year previously (Vicente et al., 2005b). These reports led many laboratories to the hypothesis that Kv1.3 and Kv1.5 could interact, forming a heterotetrameric functional channel.
Subsequent work performed in our laboratory unequivocally demonstrated that both proteins heteromerize in macrophages (Vicente et al., 2006;Villalonga et al., 2007;Villalonga et al., 2010a). Specifically, our results revealed that the functional heteromeric complex shows different sensitivity to Kv1.3 blockers and exhibits biophysical features accounted for by a variable tetrameric protein ratio.

physiological role of the heterotetrameric channel in the immune system
With few exceptions, most laboratories find that microglia, macrophages, and dendritic cells, all from the myeloid lineage, coexpress Kv1.5 and Kv1.3. In addition, many studies demonstrated that both proteins are involved in leukocyte physiology in some way. The question of to what extent Kv1.5 controls the immune function remains. Arguments both against and in favor of a crucial role for Kv1.5 in the immune system have been presented. However, the answer could be the variable composition of a tetramer (Vicente et al., 2006;Villalonga et al., 2007Villalonga et al., , 2010a (Figure 1). Similar arguments are valid for many K + channels in the cardiovascular and nervous systems (Roden et al., 2002;Vacher et al., 2008).
Role of Kv1.5 in the immune system this channel. Later, a ternary complex formed by caveolin 3, SAP97 ( Synapse-associated protein 97) and Kv1.5 was discovered (Folco et al., 2004). However, evidence indicates that the channel localization is more complex than previously thought . The coexpression of caveolin (Cav1 and Cav3) and Kv1.5 triggers their association and places the channel within lipid rafts (McEwen et al., 2008). However, in native tissues, the presence of Kv1.5 in rafts is under debate (Eldstrom et al., 2006;Martinez-Marmol et al., 2008). Although heterologously expressed Kv1.5 is located in raft microdomains in HEK 293 and fibroblasts cells, the channel is not present in rafts in cardiomyoblasts, skeletal muscle myoblasts and macrophages (Martens et al., 2001;Eldstrom et al., 2006;Vicente et al., 2008). However, upon activation, high Kv1.3/Kv1.5 ratios in macrophage channels partially relocate Kv1.5 in rafts . In this context, experimental evidence has suggested that the putative association of Kv1.5 with Kv1.3 and other partnership interactions with accessory proteins may influence the channelosome surface expression (Figure 2). In fact, different Kv1.3/Kv1.5 ratios influence oligomeric channel trafficking and locate functional heteromeric channels in different surface microdomains ).

Kv1.5 regulatory partnership interactions
Although the Kv1.3/Kv1.5 association could be considered as a main regulatory mechanism that controls leukocyte excitability (at least in myeloid cells), alternative associations to accessory proteins must be contemplated. In this context, Kv1.5 may interact with most proteins that have been extensively studied with Kv1.3. For example, similar to Kv1.3, Kv1.5 has a PDZ domain at the C-terminus that more importantly, the threshold for activation of heteromeric channels depolarizes (Vicente et al., 2006). At physiological membrane potentials (−30 to −60 mV), Kv1.3/Kv1.5 (low Kv1.3 ratio) channel activity is impaired. Because negative potentials, contributed by Kv1.3, should open CRAC (Ca 2+ release-activated Ca 2+ ) channels leading to further activating signals (Cahalan and Chandy, 2009), this scenario triggers immunosuppression and anti-inflammatory effects (Villalonga et al., 2010a,b). A similar situation has been described in B-lymphocytes where a + 25 mV shift in the membrane potential negatively modulates the capability of B-cell producing antibodies (Freedman et al., 1992). Analogous results were obtained when two different macrophage cell lines were studied, such that macrophages that had higher Kv1.3/Kv1.5 ratios were more sensitive to Kv1.3-blockers, indicating that the physiological response is under Kv1.3 antagonist control (Villalonga et al., 2007). Similar phenotypical changes are produced during human dendritic cell maturation, which suggest that this is a general feature of Kv1.3/Kv1.5 expressing leukocytes (Zsiros et al., 2009).

Kv1.5 localization in the immune system
Another unresolved but important issue concerns to what extent the Kv1.3/Kv1.5 heterotetramerization affects the channelosome localization. Kv1.3 forms part of a macromolecular complex associated with the TCR (T-cell receptor) in lymphocytes. Kv1.3, together with K Ca 3.1, generates the membrane potential that activates CRAC (Ca 2+ -release-activated Ca 2+ ) channel upon TCR activation Cahalan and Chandy, 2009). This event occurs within the so-called immunological synapse (IS) between the T-cell and the antigen presenting cell (Panyi et al., 2003Toth et al., 2009;Krummel and Cahalan, 2010;Varga et al., 2010). The association of Kv1.3 and the TCR/CD3 receptor has been documented by FRET (fluorescence resonance energy transfer). In this vein, molecular interactions between Kv1.3 and β1-integrin, and SAP-97 (synapse-associated protein 97) and Kvβ2 regulatory subunits has been also documented (Panyi et al., 2004, Cahalan and. This macromolecular complex is located in lipid rafts . Lipid rafts are microdomain platforms, rich in sphingolipids and cholesterol, where many signaling molecules and their targets converge. Many studies demonstrate that Kv1.3 channelosome concentrates in the T-cell IS, within lipid rafts, and the raft disruption impairs the immune response (Nicolaou et al., 2007;Pottosin et al., 2007;Toth et al., 2009). In addition, immobilizing Kv1.3 channels at the IS increases the Ca 2+ signaling, raising the downstream signaling transduction pathways (Nicolaou et al., 2007). Macrophages and dendritic cells are professional antigen presenting cells, and share the expression of Kv1.3 and Kv1.5 (Vicente et al., 2003;Villalonga et al., 2007;Zsiros et al., 2009). There is no information about the specific localization of the channelosome in these cells within the IS. However, it is tempting to speculate that some molecular aggregation similar to that observed in T-cells may exist. In this context, Kv1.5 localization in lipid raft is under debate . Ten years ago, Kv1.5 was the first ion channel detected in lipid rafts and caveolae (Martens et al., 2001). However, authors argued against a physical interaction between caveolins and FIguRE 2 | Distinct voltage-dependent K + channel compositions trigger different membrane surface microdomain targeting. While Kv1.3 efficiently targets to lipid rafts (a), Kv1.5 targeting depends on partnership interactions. Kv1.5 localizes in rafts when overexpressed in heterologous systems (b). However, the channel does not target to these microdomains in native tissues, probably due to the presence of interacting subunits yet to determine (e.g., Kvβ2.1) (c). In leukocytes, heterotetrameric channels with high Kv1.3/ Kv1.5 (↑Kv1.3/Kv1.5) ratio localize in rafts (d). On the contrary, low Kv1.3/Kv1.5 ratios (↓Kv1.3/Kv1.5) impair raft localization of the channels (e). Macrophage activation, which triggers a selective increase of Kv1.3 subunits, generates high Kv1.3/Kv1.5 ratio heteromers which target back to lipid rafts (f).

conclusion
Although the voltage-dependent Kv1.3 channel is essential for the immune cell physiology increasing evidence indicate that Kv1.5 plays an important role fine tuning the immune response. Pharmacological and biophysical reports argue against homomeric Kv1.5 complexes, but hetero-oligomeric associations with Kv1.3 increase the possibilities for Kv channels in leukocytes. The identification of multiple heterotetrameric conformations and their membrane surface localization may bring to light a better use for K + channels as pharmacological targets in autoimmune diseases. Furthermore, in this scenario, the multiple regulatory subunit association to homo-and heteromeric channels in the immune system is revealed as an important mechanism for further study to enhance the current understanding of the role of Kv1.5 in the immune physiology.
interacts with SAP97 (Murata et al., 2001;Eldstrom et al., 2003;Abi-Char et al., 2008). In addition, Kv1.5 also interacts with members of KChiP (K + channel-interacting proteins) family (Li et al., 2005). These interactions are known to modulate the surface expression of several K + channels, such as Kv4.2 (Shibata et al., 2003). Other possible candidates are the classical Kvβ subunits (Martens et al., 1999). These ancillary subunits, which are tightly modulated upon proliferation and the way of activation in macrophages, regulate Kv1.3 and Kv1.5 activity and may also control the channel expression at the membrane surface (Martens et al., 1999;Vicente et al., 2005a). In fact, the presence of the Kvβ2.1 subunit impairs the location of Kv1.5 in rafts, and this has been proposed to be the mechanism of how Kv1.5 colocalizes with caveolin in heterologous expression systems that lack Kvβ expression but does not in native cells and tissues . Finally, new partnership associations with KCNE subunits have considerably increased the myriad of possibilities. For example, KCNE4 has been identified as a new dominant-negative Kv1.3 regulatory subunit that impairs channel activity, surface targeting, and traffic (Sole et al., 2009). In this context, KCNE2 seems to associate with mature forms of Kv1.5, triggering physiological consequences not yet fully understood (Roepke et al., 2008).