4.1. Endoplasmic Reticulum
After translation, KChs are located in the ER exit sites (ERES) and are packed into COPII-coated vesicles and transported via microtubules to the ER-Golgi intermediate compartment (ERGIC). Many motifs are essential for such forward trafficking. For instance, diacidic signatures at the C-terminus of Kir channels are abundant. Thus, there is a VLSEVDETD motif in Kir1 channels, FYCENE in Kir2.1 channels [
40], ELETEEEE in Kir3.2A channels, NQDMEIGV in Kir3.4 channels, and DXE in Kir6.2 channels [
21]. Similar clusters are also present in other KChs, such as K
2P3.2 (EDE) [
41], Kv1.3 (YMVIEE) [
42], and the HRETE sequence of Kv1 channels, which exert great influence in trafficking. Furthermore, an acidic extension of this HRETE in the C-terminus of the mouse Kv1.3 (ETEGE) [
43] and a cluster of acidic residues (DDXXDXXX) in a splice variant of K
Ca1.1 [
44] have also been implicated.
Another example is the VXXSL sequence, which is essential for efficient Kv1.4 anterograde traffic. Variations of this domain with other members in the same family explain the relative surface targeting efficiencies for each channel. Swapping such a domain between channels interchanges their forward trafficking efficiencies [
45]. Finally, mutations at the C-terminal cyclic nucleotide-binding domain of Kv11.1 can also cause defective anterograde trafficking of the channel with fatal consequences [
46].
The association of Kvβ subunits also has a notable effect on the ER exit of Kvs. The interaction between Kv1 α and Kvβ subunits occurs in the ER early in channel biosynthesis. This assembly sometimes increases the traffic of channels to the plasma membrane. For example, Kvβ2 and Kv1.2 associate early in channel biogenesis, interacting cotranslationally, even before Kv1.2 is completely translated and translocated into the ER. The β-subunit also affects the
N-glycosylation of the channel, mediating proper folding and traffic throughout the ER journey [
24].
In addition to forward trafficking motifs, ER quality controls prevent the anterograde traffic of misfolded channels. Defective proteins may undergo several rounds of folding before irreversibly being targeted for ER-associated degradation (ERAD). This mechanism mediates the retrotranslocation of the protein back to the cytosol and a subsequent ubiquitin-dependent proteasomal degradation. The mechanism for ER retention mostly consists of the exposure of retrieval motifs. Thus, di- or tri-basic signatures, recognized by COPI vesicle coats, retrieve the defective channel from Golgi back to the ER [
47]. An RXR motif located at the C-terminus of K
ATP channels is an example [
48]. Although many KChs also contain RXR domains, only in Kv11.1 (ERG) [
49] have the domains been definitively confirmed. A similar basic C-terminal signal (KRR) in K
2P3.2 binds to COPI and drives the channel back to the ER [
50]. Other ER retention signals might be less discrete but otherwise widespread. For instance, hydrophobic sequences situated at the N-terminus of Kv4.2 promote channel aggregation, misfolding, and subsequent degradation [
29].
ER retention can be physiological and not due to unsatisfactory quality control. For instance, pore-based retention mechanisms have been described for Kv1 and Kv7. The pore region of Kv1.1 contains two critical residues for the ER retention of this channel [
51]. Similarly, a pore residue in Kv7.3 is responsible for an inefficient forward trafficking [
52]. Finally, a splice variant of K
Ca1.1 exhibits another retention/retrieval ER mechanism, which implies a hydrophobic domain (CVLF) in the intracellular S0–S1 loop [
53].
Many ancillary proteins may alter KCh surface targeting. Thus, KCNE subunits are also key regulators of Kv traffic in the immune system. As mentioned above, KCNE4 impairs Kv1.3 membrane targeting while retaining the channel in the ER by two mechanisms [
27]. First, KCNE4 masks the forward trafficking motif (YMVIEE) at the C-terminus of Kv1.3, and second, KCNE4 contains a potent ER retention motif that further limits the surface expression of the complex [
54]. KChIPs are also involved in both membrane targeting and retention of KChs. Coexpression of KChIPs1–3 causes a dramatic redistribution of Kv4.2, releasing intrinsic ER retention and improving the cell surface expression via a mechanism that may involve masking of a cytoplasmic trafficking and/or solubility determinant in excitable cells [
29]. In contrast, KChIP2 promotes ER retention when coexpressed with Kv1.5 [
55]. On the other hand, Kv7 channels, such as Kv7.2, require CaM to properly fold and adopt an active conformation to exit the ER. Thus, CaM acting as a Ca
2+ sensor confers Ca
2+-dependence for the cell surface trafficking of the channel [
56,
57]. Similarly, the Kv7.2–Kv7.3 complex requires a Ca
2+/CaM-dependent mechanism for targeting the axonal surface in neuronal cells [
58,
59]. In this vein, K
Ca channels are monomers at this step of the anterograde traffic. C-termini of the monomers interact constitutively with CaM, which allows for the multimerization and thus promotes the surface expression of the functional channel [
60,
61]. Finally, K
ATP functional channels are formed by the association of Kir6 subunits and SUR proteins stabilized in macromolecular dynamic complexes that contain kinases, ankyrins, and other cytoskeleton and adaptor proteins. Both SUR and Kir6 contain putative ER retention motifs (RXR) that retain every subunit in the ER when expressed alone. Their association masks ER retention motifs, improving the membrane targeting of the functional complex [
48,
62].
4.2. Golgi Sorting
The Golgi apparatus is at the center of the secretory pathway. The Golgi’s structure of stacked flattened cisternae confers the proper environment for accurate protein maturation. Proteins entering the ERGIC are transferred through the
cis-,
medial-, and
trans-cisternae to be further processed. The
trans-Golgi network (TGN) organizes the sorting of proteins to the plasma membrane, where cargo proteins concentrate into specific pools of nascent clathrin-coated vesicles. The sorting signals that regulate such stages of KCh maturation remain largely unexplored.
Trans-Golgi export of Kir channels is mediated by a cluster of residues located within the confluence of the cytoplasmic N- and C-terminal domains. This creates a tertiary structure that contributes to the interaction with adaptor protein complex 1 (AP1), driving the incorporation of Kir channels into clathrin-coated vesicles. This domain includes basic residues at the N-terminus and a hydrophobic cleft at the C-terminus [
63]. Furthermore, the sorting of KChs to specific plasma membrane compartments is already defined in the TGN. For instance, neuronal Kv2.1 and Kv4.2 are sorted into distinct pools of transport vesicles specifically targeted to proximal (Kv2.1) or distal (Kv4.2) dendrite compartments. Kv2.1 vesicles traffic by a mechanism involving myosin and actin filaments, whereas Kv4.2 vesicles depend on kinesin and microtubules [
64]. Several mutations that impair the post-Golgi sorting process cause the mislocalization of these channels in the neuronal membranes. Kv2.1 localizes in large surface clusters in soma and proximal dendrites due to a serine-enriched C-terminal motif, known as the proximal restriction and clustering (PRC) signal [
65]. Disruption of this motif triggers incorrect post-Golgi sorting, causing aberrant localization of Kv2.1 to distal dendrites. Similarly, the C-terminal dileucine motif of Kv4.2 drives dendritic targeting [
66], and mutations cause impaired vesicular sorting of the channel [
64].
In addition to polarized traffic, the packing of KChs into specialized membrane microdomains occurs at the TGN. For instance, Kv1.3 lipid raft targeting depends on a caveolin interaction through its N-terminal caveolin-binding domain (QRQVWLLF). Such an interaction could be functional at the TGN, where lipid rafts begin to structure as cholesterol is concentrated [
67]. Altogether, the TGN can not only direct the journey of KCh nascent vesicles but also the channel affinity for structured plasma membrane domains.
Anterograde traffic of several proteins through the Golgi requires specific golgin molecules. Thus, Kir1.1, Kv1.5, and Kv4.3 require Golgin-160 to reach the plasma membrane, even though there is no obvious sequence similarity in the cytoplasmic tails of these channels. Kir1.1 membrane traffic is also promoted by GM130. In contrast, GM130 is responsible for the Kv11.1 retention at this traffic step [
68]. Furthermore, Kir2.1 is recognized by Golgin-97 as an export cargo to be delivered into the TGN [
69]. Consequently, some Kir2.1 N-terminal motifs are required to exit the Golgi as it reaches the membrane [
70].
4.3. Plasma Membrane Arrangement
Eventually, KChs are inserted into the plasma membrane. Mechanisms to maintain KChs at their target destination are needed in polarized cells. A major mechanism for protein retention involves KCh anchoring to the cytoskeleton. For instance, neuronal Kv7 channels are retained at the axonal initial segment by being anchored to the actin cytoskeleton through a C-terminal ankyrin-G-binding motif (I/LAXGES/TDXE/D) [
71,
72]. Likewise, cortactin immobilizes Kv1.3 through an SH3 C-terminal domain (PQTP). Such an interaction at the immunological synapse suggests that the polarization of the T cell would promote Kv1.3 trapping at active sites of the actin network [
73]. Other arrangement mechanisms include coclustering by protein–protein interactions. For instance, a PDZ-binding motif at the C-terminal end of Kv1 channels (S/TXV) facilitates Kv1 immobilization by PSD-95 interaction [
74]. The effects of protein–lipid interactions on the membrane organization of KChs remain elusive, although the effects are expected to be considerable given the number of KChs that target lipid raft microdomains [
75,
76].
Kvβ subunits modulate the cell surface expression of many Kv1 channels. Kvβ members not only promote Kv1.2 membrane targeting but also need the NADP+-binding pocket to do so. Defects in the cofactor-binding pocket reduce axonal targeting of the channel in hippocampal neurons [
77]. In arterial myocytes, Kvβ2, taking a chaperone role, facilitates Kv1.5 trafficking and membrane localization [
78].
SNARE proteins, syntaxins, and integrins help channels to reach the membrane. SNARE proteins are membrane-integrated proteins responsible for vesicle-associated exocytosis, but they can also act as a recruitment signal for proteins that must reach the membrane. KAT1, a plant Shaker-related member, shares a number of structural features with the voltage-sensitive Kv superfamily of eukaryotes. KAT1 binds the plasma membrane protein SYP121, a Q-SNARE protein, through a conserved RYXXWE motif located at the cytosolic surface of the voltage-sensor domain [
79]. K
2P and Kv2.1, as well as many other Kvs, interact with SNARE proteins and syntaxins, shaping the intracellular itinerary of the channels. However, the mechanisms by which SNARE proteins affect vesicular transport are unknown [
80,
81].
Scaffolding proteins are also very important for membrane trafficking and protein stabilization at specific locations. The C-terminal PDZ domains of Kir channels interact with several partners that regulate intracellular trafficking. Members from the MAGUK family, including SAP91, PSD-95, Chapsyn-110, SAP102, and Veli or actin-binding LIM proteins, are scaffolding proteins related to the membrane expression of Kir channels. Thus, PSD-95 facilitates Kir2.3 clustering on the plasma membrane [
82], and SAP97 regulates Kir2 traffic [
83]. In addition, dystrophin-associated complexes are also involved in the anchoring and stabilization of these channels at the plasma membrane [
84].
In addition to the conventional secretory pathway, which requires COPII vesicles to exit the ER and reach the membrane, some proteins use unconventional routes to target the cell surface. These nonclassical pathways are complex and usually comprise cargo molecules with an unknown signature [
85,
86]. For instance, Kv4.2 uses COPI vesicles, instead of COPII, but only in coexpression with KChIP1 [
30]. The CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel uses the ER–plasma membrane (PM) junctions, which bypass the Golgi apparatus to reach the membrane [
87]. Concerning KChs, Kv2.1 targets and stabilizes these domains. In fact, Kv2.1 provides platforms for delivery and retrieval of multiple membrane proteins [
88,
89]. The Golgi bypass has recently been under analysis. Thus, unknown combinations of SNARE proteins and/or specific molecules yet to be identified would drive KChs to the membrane unconventionally.
Spatially, KChs can be located in specific membrane microdomains, such as lipid rafts. Raft domains are membrane fractions enriched in sphingolipids and cholesterol that serve as scaffolding regions where signal transduction pathways interface. Proteins reach these domains via different mechanisms, such as lateral diffusion, the formation of lipid “shells” surrounding the channel, which increase the affinity for rafts, protein–lipid interactions, such as palmitoylation (see below), or protein–protein interactions [
75]. In this last scenario, caveolin acts as a scaffolding protein driving ion channels to raft domains. For instance, an N-terminal motif for the caveolin-1 interaction (FQRQVWLL) has been described in Kv1.3 [
67]. Caveolins, by impairing lateral diffusion, increase the time that KChs are located in these platforms. In addition, KChIP proteins also govern Kv4.3 targeting to lipid raft domains [
90]. Finally, PSD-95 is partially responsible for the lipid raft targeting of Kv1.4 and protects Kv1.3 against phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination and endocytosis [
33,
91]. Kv2.1, Kv1.5, or Kir3.1 are also found in these domains [
75], but no direct interaction with scaffolding or auxiliary proteins has been reported.
Mechanisms of membrane arrangement also involve channel internalization. Lysosomal endocytosis is not always associated with degradation. For instance, Kir6.x target to the plasma membrane when associated with SUR subunits. However, differential interactions between SUR members and Kir6.x channels condition the subcellular location of the complex. SUR1 targets Kir6 to the plasma membrane, while Kir6/SUR2 localizes mainly in endosomal and lysosomal compartments. In cardiomyocytes, Kir6/SUR2 complexes in endosomal compartments are in constant recycling from the membrane to endosomes. Thus, these oligomers represent a pool of available channels to dynamically regulate heart function [
92]. Similarly, K
Ca2.3, a long-life membrane protein, undergoes dynamin- and Rab5-dependent endocytosis that generates a recycling endosome compartment for a quick and dynamic K
Ca2.3 return to the cell surface. This mechanism is responsible for K
Ca2.3 long-life membrane expression [
61,
93].