INTRODUCTION TO K_2P CHANNELS

Potassium selective channels are the largest class of mammalian ion channel proteins, with over 75 genes in four distinct families that encode pore-forming subunits- diversity is further enhanced by subunit variation due to alternative splicing, alternative translation initiation (ATI), heteromeric assembly of subunits, and by co-assembly with accessory proteins. To this cavalcade of K⁺ channels, the two-pore domain (K_2P) K⁺ channel family contributes 15 genes (Figure 1A) encoding proteins notable for their unique subunit architecture, biophysical properties and roles in physiology [53].

K_2P channel subunits have a distinct body plan- each has four transmembrane domains (TMD), two re-entrant pore (P)-forming loops, and intracellular amino- and carboxy-termini (Figure 1B). Two identical K_2P subunits create a single, central K⁺ selective conduction pore (Figure 1C) and, in at least some cases, heterodimeric complexes form in vivo [31,105].

The open probability of K_2P channels is largely independent of changes in the voltage difference across the plasma membrane (ΔVm) under physiological conditions. Thus, K_2P channels are unlike voltage-gated K_V channels that show increased open probability (P_o) when changes in Vm act on charge-bearing voltage sensor domains (VSDs); inwardly rectifying K_ir channels that change P_o when changes in ΔVm drive K⁺ ions through the selectivity filter, unblocking the conduction pore; or calcium-activated K_ca channels that respond both via VSDs and by sensing local changes in intracellular calcium. Rather, K_2P channels pass K⁺ currents across the physiological voltage range that are subject to a broad array of regulatory chemical and physical influences [41,43,53,59], including neurotransmitters, post-translation modification (phosphorylation and SUMOylation), all three classes of second messengers (hydrophobic, hydrophilic and gaseous), temperature and mechanical stretch. This was expected [54-55] based on the early descriptions of native K⁺ background 'leak' currents [58,61,137]. Because K_2P channels respond to many of these regulatory influences concurrently, some have been characterized as 'polymodal signal integrators' [6,59,83].

K_2P channels are proving to be broadly impactful in the brain, peripheral nervous system, heart and muscles through control of excitability, principally, by setting the level and the stability of cellular resting Vm and the frequency and morphology of action potentials- for example, in hypoglossal motoneurons [12,123], dorsal root ganglion [63,98] and cerebellar granule neurons [103,106]- where they are implicated in thermosensation of both cold and warm, nociception and the action of volatile anesthetics. K_2P channels are also manifesting as important in non-excitable cells, including, the pancreas, vascular endothelium, immune T cells and a variety of cancers.

The first K⁺ channel subunit identified with two-P domains, TOK1, was cloned from the genome of the budding yeast Saccharomyces cerevisiae [65]. TOK1 channels are formed by two identical subunits, each with 8 TMD, and pass outwardly rectifying potassium currents through a single conduction pore. The TOK family of channels appears to be exclusive to fungi based on genomic analyses and is considered in detail elsewhere [53,100]. In 1996, K_2P subunits with 4 TMD were identified in the genomes of Drosophila melanogaster, Caenorhabditis elegans [54], and mammals [71].

The fruit fly isolate, K_2PΘ (originally called dORK, for Drosophila openly-rectifying K⁺ channel) was shown to display the functional properties predicted by Hodgkin and Huxley for a canonical background K⁺ channel [54,58,61,61,137]. K_2PΘ channels pass K⁺ selective currents that show little dependence on voltage or time, and consistent with their operation as Goldman-Hodgkin-Katz (GHK) open rectifiers, the single channels were found to operate as open, multi-ion, selective leak portals, passing ions more readily across the membrane from the side of higher ion concentration. Thus, K_2PΘ channels pass larger outward K⁺ currents than inward because intracellular K⁺ concentration is high (~140 mM) compared to normal extracellular levels (~4 mM) (Figure 1D). K_2PΘ is regulated by changes in the conformation of the K⁺ selectivity filter induced by phosphorylation of the cytoplasmic C-terminus [6,28,61,137].

Since 1996, fifteen mammalian KCNK genes have been identified in humans that are also present in rats and mice (Figure 1A). Each channel was named as it was cloned to reflect the particular physiological, pharmacological or biophysical attributes judged to be distinctive at the time (Table). The first mammalian K_2P subunit was called 'tandem of P-domains in a weak inward rectifying K⁺ channel', or TWIK1 for short [71]. As differences in channel behavior between experimental reports accrued, the utility of initial phenotypic monikers was diminished, supporting the adoption of formal IUPHAR nomenclature in 2005 [52]. Although proteins encoded by KCNK genes are now called K_2P subunits (and are numbered to match their encoding gene, i.e., K_2P1 is encoded by KCNK1) the original names remain in use, accompanied by their formal names to keep the literature clear and to speed efforts to correlate channels with the K⁺ currents they mediate in native cells.

Comparison of K_2P channels revealed that a few genes first described in other species were homologs of a human isolate. For this reason, the K_2P family contains 15 genes numbered 1-18 and there are no KCNK8, KCNK11 or KCNK14 genes or associated proteins.

Figure 1. Classification of function of two-pore domain channels.

A. A phylogenetic tree calculated to show the relatedness of the 15 K_2P subunits found in humans based on ClustalW alignments of the IUPHAR accession numbers for each clone, (see http://www.guidetopharmacology.org/GRAC/IonChannelListForward?class=VGIC). To date, functional expression has not been observed for K_2P 7, K_2P 12 and K_2P 15 (grey text).

B. K_2P subunits are integral membrane proteins with internal amino (N) and carboxy (C) termini, four transmembrane domains (M1-M4) and two pore forming (P)-loops.

C. Two K_2P subunits create a single, central K⁺ selective conduction pore. One subunit is shown in green and a second in blue. Under physiological conditions, K⁺ flow (red arrow) down a concentration gradient from the intra- to the extracellular milieu.

D. Example whole-cell current from K_2P1 channels heterologously expressed in CHO-K1 cells and studied with a deSUMOylating enzyme in the recording pipette. In both case, the inside of the cell contains 140 mM KCl. Left, The external solution contains 4 mM KCl. Right, The same cell recorded with 140 mM KCl on both sides of the membrane.

E. Mean current-voltage relationships for Chinese hamster ovary (CHO-K1) cells expressing SENP1 activated K_2P1 channels (adapted from [101]). Active K_2P1 channels show openly rectifying (GHK) behavior. Under quasi-physiologic conditions (Δ, 4 mM external KCl) the channels pass more outward current but show a linear current-voltage relationship with symmetrical 140 mM KCl (▲). Click image for full size.

IUPHAR channel name	HUGO gene name	Common name	Other names
K2P1	kcnk1	TWIK1	hOHO
K2P2	kcnk2	TREK1	TPKC1
K2P3	kcnk3	TASK1	TBAK1, OAT1
K2P4	kcnk4	TRAAK	KT4
K2P5	kcnk5		TASK2
K2P6	kcnk6	TWIK2	TOSS
K2P7	kcnk7		kcnk8
K2P9	kcnk9	TASK3
K2P10	kcnk10	TREK2
K2P12	kcnk12	THIK2
K2P13	kcnk13	THIK1
K2P15	kcnk15	TASK5	kcnk11, kcnk14
K2P16	kcnk16	TALK1
K2P17	kcnk17	TALK2	TASK4
K2P18	kcnk18	TRESK

Table: The nomenclature of mammalian K_2P channels
The 15 mammalian K_2P channels are named according to IUPHAR nomenclature and numbered to match the HUGO (Human Genome Organization) designations for each of the genes. Common and Other Names are based on biophysical or pharmacological properties in early reports on each clone. TWIK, tandem of P-domains in a weak inward rectifying K⁺ channel; TREK, TWIK-related K⁺ channel; TASK, two-pore domain, acid-sensitive K⁺ channel; TRAAK, two-pore domain related arachidonic acid activated K⁺ channel; THIK, two-pore domain halothane inhibited K⁺ channel; TALK, two-pore domain alkaline activated K⁺ channel; and TRESK, TWIK-related spinal cord potassium channel. K_2P6, 7, 12 and 15 pass little or no current when expressed in heterologous cells and were given common names based on sequence homology.

K_2P CHANNELS AT ATOMIC RESOLUTION

In 2012, X-ray structures were reported for human K_2P1 (TWIK1) [86] and K_2P4 (TRAAK) [20] (Figure 2A). Each structure was solved at ~3.8 Å with four K⁺ ions in the conduction pore. As expected, based on prior structure-function analysis and homology models [66,120], the selectivity filter has 4-fold symmetry and is located within a bilaterally symmetric channel corpus. Perhaps the most unexpected feature in both structures was an extracellular ‘cap-domain’ formed by the first external loop of each subunit; the cap-domain extends into the extracellular space ~35 Å above the outer mouth of the pore, creating two pathways for K⁺ ions to enter or exit the selectivity filter. To date, the cap-domain has not been observed in other K⁺ channels and appears to offer steric obstruction as an explanation for why classical pore-blocking venom toxin peptides fail to inhibit K_2P channels and background K⁺ currents in native cells. Other interesting features of the structures are two lateral portals that may expose the ion conduction pore to the hydrophobic span of the lipid bilayer and the variability in the end of M4 that connects to a functionally important part of many K_2Ps, the C-terminal tail that has been posited to play a role in transmission of gating signals to the selectivity filter [5,17,26,114,137-138]. This key modulatory element has yet to be resolved by any of the K_2P structures [18-21,39,74,86] and remains an element of great interest.

In 2013, the structure of K_2P4 was refined as a co-crystal with a Fab antibody fragment at 2.75 Å. The higher resolution structure [18,20] revealed that the M1 helix is domain swapped between the two subunits, a topology that was also seen in the structures of two K_2P4 gain-of-function mutants [74]and in structures of K_2P10 (TREK-2) [39]. The functional consequence of this topology is unclear but may facilitate inter-subunit communication. The cap-domain and the two lateral portals in the membrane spanning region were also observed in crystal structures of a truncated variant of the K_2P10 channel [39], suggesting that these features will be held in common throughout the K_2P channel family. The structure of K_2P10 was solved at 3.9 Å in complex with norfluoxetine, the active metabolite of the selective serotonin reuptake inhibitor Prozac, bound within the lateral portals (Figure 2B). This structure raises interesting questions. Similar to K_2P1 and K_2P4, the K_2P10 structure, was modeled with K⁺ ions in the conduction pore. However, norfluoxetine is an inhibitor of K_2P10 suggesting that the structure is a non-conductive conformation despite the apparently fully occupied selectivity filter. Moreover, a second structure at 3.4 Å, without norfluoxetine but with K⁺ ions in the conduction pore, shows an alternate arrangement with M4 occluding the two lateral portals and precluding membrane lipid access to the conduction pathway [39]. In all of the structures determined to date, there is an open path to the cytoplasm, consistent with the idea that the principal gate in K_2Ps is the selectivity filter [5-6,28,137], rather than an intracellular occlusion made by the pore-lining helices.

Figure 2. The primary structures of the subunits of the voltage-gated sodium channels. A. Ribbon representations of the x-ray structure of human K_2P4 resolved at 2.75 Å [18]. Left, The channel is viewed from the membrane plane with one subunit in red and the other in blue, K⁺ ions are green, and the boundary of the membrane is in grey. Right, A view of the channel rotated by ~45° to show architecture wherein the outer pore helix interacts with the inner helix from the other subunit rather than its own. Dashed lines suggest loop regions that were not resolved. The ~35 Å cap domain above the outer mouth of the pore that bifurcates the entrance to the K⁺ conduction pathway and the c-helix are indicated with arrows.

B. A cross section of a surface view of human K_2P10 colored by hydrophobicity where green is the most hydrophobic and blue is the least hydrophobic, [39]. The structure was solved to 3.9 Å in complex with norfluoxetine bound within both bilateral intramembrane side portals. Norfluoxetine is shown in light blue, dark blue, red, and orange for carbon, nitrogen, oxygen, and fluorine atoms, respectively. Click image for full size.

K_2P CHANNEL GATING

Although the open and closed structures of K_2P channels, and the nature of the transitions between conformations, remain to be clarified [90], a body of data supports the conclusion that K_2P ion conduction is controlled by a “C-type” gate [5-6,28,137]. First described in K_V channels, C-type gating refers to suppression of ion permeation due to conformational changes in the selectivity filter, rather than constriction of the conduction pathway below the filter or intracellular pore occlusion by the N-terminus [136]. Occupancy of the K⁺ binding site external to the filter appears to explain why high bath concentrations of K⁺ ions stabilize particular open states of K_2PΘ [137]. Similarly, heat and stretch appear to augment K_2P2 (TREK-1) channel currents in a manner mediated by changes in the activity of a C-type gate [6] and are proposed to stabilize particular open states of the channel [83]. Interestingly, conformational changes in the filter of K_2PΘ and K_2P2 depend on intracellular regulatory elements, phosphorylation [137] and temperature induced changes in thermal sensing (Ct) domain [5], respectively. A mechanistic basis for the transduction of intracellular signals through the channel corpus is suggested by recent crystallographic findings: K_2P4 channels with mutations that activate the C-type gate demonstrate tilting and straightening of M4 and buckling of the M2 helix [74].

Whereas K_V channels contain an inner gate formed by ‘bundle-crossing’ helices distinct from the selectivity filter, neither structural nor functional data support the presence of an equivalent restrictive gate in K_2P channels [6,18,39,74,99,111,137]. Thus, accessibility of quaternary ammonium ions to the inner pore of K_2P2 channels is unchanged by activation, suggesting that the inner portion of the conduction pore is constitutively open [99,111]. Nonetheless, the structures of K_2P4 and K_2P10 suggest that membrane lipid [20-21] and norfluoxetine [39], respectively, can access the inner vestibule of the channel below the potassium conduction pathway to suppress permeation. Similarly, molecular dynamics simulation (MDS) and mutagenesis suggest that dewetting of the inner pore of K_2P1 by the motion of hydrophobic residues may contribute to channel closure [2]. At present, it is unclear how these observations consistent with inner pore occlusion are related to the operation of what appears to be a controlling C-type filer gate, and if they represent mechanisms present throughout the K_2P family.

Although K_2P channels do not contain a voltage-sensing domain as found in K_V channels, intracellular phosphorylation of hippocampal K_2P2 channels has been demonstrated to produce voltage dependent operation under physiological conditions [17]. This may relate to a study showing that some K_2P channels studied in symmetrical elevated KCl and the absence of activators, open in voltage-dependent manner (ascribed to movement of K⁺ ions within the selectivity filter) and operate as openly rectifying leak channels once activate [116].

HIGHLIGHTS OF K_2P CHANNEL BY SUBTYPE

The operation of K_2P channels is subject to tight regulatory control [53]. Thus, their description must highlight the modulatory pathways that act directly on the subunits: the list includes but is not limited to phosphorylation, pH, medications and their metabolites, drugs of abuse, volatile anesthetics, second-messengers, cytoskeletal proteins, post-translational modification pathways and non-covalent assembly with protein-partners. Other regulatory influences may be direct and via interacting proteins include membrane stretch, temperature and oxygen tension. Identification of some pathways that regulate K_2P channels have revealed new cellular [104] and molecular [108] pathways for study. K_2P regulators change the density of channels at the cell surface, modify open probability, block the pore, alter single channel conductance, and change ion selectivity. Those that decrease K⁺ flux increase cellular excitability as they permit membrane depolarization whereas stimuli that increase the activity of K_2P channels dampen excitability due to membrane hyperpolarization.

Here, we highlight key functional attributes of K_2P subunits by the 15 human genes organized according to IUPHAR nomenclature and noting prevalent common names; a more complete fingerprint of each channel is found in the accompanying database while detailed appraisal of the biophysics, structure-function, pathophysiology and pharmacology of each K_2P channel can be found in exhaustive reviews [41,53,59,100] and in the citations that accompany each listing in the database.

K_2P1 Channels

K_2P1 (TWIK1) has been an enigma since it was described as an acid-insensitive weak inward rectifier on heterologous expression [71]; the debate remains vigorous. Several groups reported that they observed no channel activity in the same expression systems [55,107]. Since mRNA transcripts for the KCNK1 gene are abundant in kidney, placenta, lungs, [71,124], and throughout the central nervous system [124], and cardiac conduction pathway [47], it was apparent that the gene was transcribed. Moreover, the protein was observed at the cell surface. This suggested it operated as a receptor that was electrically silent or that a regulator of the channel had yet to be identified. Three mechanisms have been proposed to account for the low basal activity of K_2P1 on heterologous expression.

SUMO regulation. In 2005, data were published showing that K_2P1 channels at the plasma membrane were held silent when they were covalently modified on Lys274 by the small ubiquitin-like modifier protein, SUMO [108]. SUMOylation is an enzyme mediated, reversible post-translational modification pathway found in all eukaryotic cells [56]. K_2P1 channels only passed current when SUMO was removed from the channels with SUMO-specific proteases (SENPs) or when K_2P1-Lys274, a site on the intracellular C-helix of the subunits, was mutated to preclude SUMOylation [101]. Once deSUMOylated, K_2P1 channels passed open-rectifying, acid-sensitive, K⁺ selective currents in COS7 cells, CHO-K1 cells and Xenopus oocytes [101,108], (Figure 1E).

The observation that K_2P1 channels were SUMOylated was surprising at first because the SUMO pathway had not previously been shown to operate at the plasma membrane. Now, SUMOylation is recognized to regulate the activity of a growing cadre of membrane proteins, including K_V channels [11,35,102], TRP channels [67] and kainate receptor ion channels [82]. Yet, questions remain. Some groups failed to observe SUMOylated K_2P1 after detergent purification [42], however, the labile nature of SUMO-target conjugates during isolation is known in many cases where target proteins operate as if SUMOylated in the intact cells [56]. Indeed, both fluorescent resonance energy transfer (FRET) and single molecule visualization using total internal reflection microscopy were subsequently used to verify assembly of K_2P1 and SUMO1 in live cells [101,105].

Hydrophobic dewetting. Based on the crystal structure of human K_2P1 [86], molecular dynamic simulations (MDS) identified a “hydrophobic cuff” in the inner vestibule of the channel below the selectivity filter comprised of four residues, Leu146 on M2 and Leu261 on M4, from each subunit [2]. One hundred ns simulations showed stochastic motion of the cuff to restrict water access to the internal entrance of the pore, creating an energetic barrier to ion permeation. In support of this model, hydrophilic mutations of Leu146 were reported to yield K_2P1 channels currents in Xenopus oocytes that were not observed with wild type channels [2,23].

Rapid endocytosis. Others attribute the low basal activity of K_2P1 on heterologous expression to rapid, constitutive trafficking of the channel away from the plasma membrane to recycling endosomes [13,44]. Site-directed mutagenesis showed that endocytosis was dynamin dependent and required a di-isoleucine motif (Ile293Ile294) in K_2P1 [44]. Mutation of two residues yielded channels that passed measurable currents in MDCK cells, HEK293 cells and Xenopus oocytes. The channel was also reported to interact with ARF6, a small G protein that modulates endocytosis at the apical surface of epithelial cells, and forms complexes with the nucleotide exchange factor EFA6 [37]. Indeed, endocytosis has also been implicated in the surface half-life of K_2P2, K_2P3, K_2P9 and K_2P18, and in the cases of K_2P1 and K_2P3, both clathrin-mediated and clathrin-independent mechanisms appear to operate [93]. How SUMOylation, hydrophobic dewetting of the inner pore, and endocytosis contribute individually or in concert to regulation of K_2P1 in native cells remains under active study.

While K_2P1 has yielded measurable currents in experimental expression systems only after enzyme treatment or point mutation, evidence continues to accrue that the native channels are active and play key roles in plasma membrane excitability in several tissues. Thus, mice deficient in K_2P1 show altered Vm in pancreatic β cells [23] and manifest defects in renal transport of phosphate and water in their proximal tubules and medullary collecting ducts, respectively [88]. Using transgenic zebrafish, K_2P1 was demonstrated to control heart rate as well as atrial morphology [27]. In rat cerebellar granule neurons (CGN), K_2P1 subunits were shown to pass IKso, the standing outward, background K⁺ current that determines Vm and excitability of CGN (via assembly with K_2P3 or K_2P9 to form heterodimeric channels) [105]. IKso determines the response of these neurons to changes in pH [85], pO₂ [103], and volatile anesthetics subject to regulation by SUMO of K_2P1 [105]. In humans, K_2P1 mediates arrhythmogenic depolarization of myocytes exposed to low serum potassium (hypokalemia), an anomalous response the authors showed was due to enhanced permeation of the channels by Na⁺ [51,79]. Supporting this unexpected behavior for a K⁺ selective channel, K_2P2 had previously been shown in rat brain in two forms due to ATI, one manifesting a stable change in ion selectivity allowing Na⁺ flux [125].

Whereas K_2P1 does not appear to assemble with K_2P2 in rat CGN [105], the two subunits were proposed to form dimers in hippocampal astrocytes yielding a passive membrane K⁺ conductance that set Vm [133]. However, this finding has also proven to be controversial. Subsequent studies suggested that K_2P1 [133] and K_2P2 were retained inside astrocytes and that the electrical properties were unaltered in astrocytes from mice lacking both channels [40]. Yet another study found trafficking of K_2P1 and K_2P2 to the astrocyte surface to be interdependent, and that K_2P1-K_2P2 heterodimers were essential for passive K⁺ conductance and cannabinoid-induced glutamate release [60]. Indeed, glutamate has been proposed to play a role in forward trafficking of K_2P1 via a metabotropic glutamate receptor pathway [132]. Perhaps, K_2P1 trafficking and assembly with K_2P2 will prove to be differentially regulated in a cell-type dependent fashion.

K_2P2, K_2P4 and K_2P10 channels

Ready expression of K_2P2 (TREK1, TWIK-related K⁺ channel 1; Table), the closely related K_2P4 (TRAAK, TWIK-related arachidonic acid activated K⁺ channel) and K_2P10 (TREK2) channels has expedited their study. All three are expressed throughout the CNS and the myocardium [45,84,105,124,133], and are subject to regulation by a diverse array of regulators, including lipids, neurotransmitter-activated second messenger pathways, phosphorylation, anesthetics and drugs [17,39,59]. This subgroup of K_2P channels is also subject to regulation by physical stimuli, including temperature [1,92] and mechanical stretch [20,80-81]. Because physical stimuli are often coincident in biology, this group of channels are proposed to act as polymodal signal integrators [59].

The importance of K_2P2 and K_2P4 as thermal sensors in native cells remains a matter of controversy because they are ~5 times less sensitive to temperature than TRPV channels [1,62]. Thus, knock-out mice lacking Kcnk2 and Kcnk4 genes show altered responses to changes in temperature [92] but some posit this is due to close localization of K_2P2 and TRP channels in the native cells [135], allowing K_2P to tune the temperature response indirectly via regulation of neuronal excitability. However, arguing otherwise, the intracellular C-terminus of K_2P2 has been shown to operate directly as a primary heat-sensing element in the channel with a strong mechanistic rationale for linkage to the C-type gate in the filter [5].

The classification of K_2P2 and K_2P4 as mechanosensitive channels [80-81] rather than secondarily responsive to the stimuli due to changes to the fluidity and composition of the plasma membrane [41,59] has also generated disagreement. Perhaps, these controversies will prove to be semantic. All ion channels show varying degrees of responsiveness to temperature and mechanical stress; for some this is a critical defining feature, with well defined structural determinants, whereas for other channels of lower responsiveness the impact on physiology may be limited or non-essential.

The effort to correlate native currents and K_2P2 and K_2P4 channels is ongoing. In addition to polymodal regulation, both K_2P2 [125] and K_2P10 [118] are subject to ATI, a process whereby a downstream start codon is used to generate a foreshortened version of each subunit; for K_2P2, ATI is regulated in a developmentally and spatially determined manner in rat brain to yield a channel that allows increased Na⁺ ion permeability. Suggesting a role for K_2P2 in nociception and analgesia, and an in vivo effect of ATI, K_2P2 current is reversibly increased by exposure to fenamate-derived non-steroid anti-inflammatory agents [128], and while augmentation is not yet understood in full-length channels, the effect on the ATI-truncated K_2P2 channels appears to be due to an increase in K⁺ selectivity. Also of note, the truncated ATI isoform is insensitive to diclofenac, the most commonly prescribed fenamate derivative in the United States [128].

Diversity of K_2P2 function arises as well through protein kinase A-dependent phosphorylation of Ser348, a modification that endows the channels with voltage-dependence when expressed in tissue culture cells and natively in hippocampal cells [17]. Further, increased intracellular PIP2 shifts the voltage-dependence of K_2P2 channels to hyperpolarized potentials, augmenting current magnitude across the physiological voltage range [25,76]. Finally, recent reports suggest that, like K_2P1, the operation of K_2P2 and K_2P4 channels can be varied through formation of heterodimers in both heterologous expression systems [14,72] and in dorsal root ganglion neurons [70].

K_2P3 and K_2P9 Channels

K_2P3 (TASK1, Two P domain, acid-sensitive potassium channel 1), and K_2P9 (TASK3) channels are expressed throughout the central and peripheral nervous systems and are proposed to mediate currents that depolarize neurons during acidification [30,105,123]. K_2P3 is also expressed in human atrial myocytes and has been shown to mediate increased background K⁺ current that contribute to the shortening of action potential duration in patients with atrial fibrillation [117]. Levels of mRNA encoding K_2P9 are amplified in human breast cancer cells, leading to the proposal that KCNK9 operates as a proto-oncogene [87,97]. Knockout studies in mice indicate that K_2P3 and K_2P9 are required for adrenal gland development and regulated aldosterone secretion [7,57]. Missense mutations in the maternal copy of the imprinted KCNK9 gene lead to Birk Barel syndrome, presenting in patients as mental retardation, hypotonia, and facial dysmorphism [8].

Like K_2P1, K_2P3 and K_2P9 pass K⁺ selective currents that are blocked by protonation of a histidine residue in the outer mouth of the pore in the first P loop (Gly-Tyr-Gly-His) [75,77]. In addition to forming SUMO-sensitive heterodimers with K_2P1 subunits, K_2P3 and K_2P9 heterodimerize in vivo to form a channel with distinct sensitivities to acidification [12,31,105] and to pungent stimuli such as hydroxy-α-sanshool, an active ingredient in Szechuan peppers [10].

Surface levels of K_2P3 and K_2P9 are regulated by the competing actions of protein partners that bind to trafficking motifs on the channel subunit to liberate or retain the channel in the endoplasmic reticulum (ER) as well as by trafficking pathways that operate at the plasma membrane [37]. Forward trafficking to the plasma membrane requires that the channel subunits bind the soluble adapter protein 14-3-3β, in a phosphorylation dependent manner, and this suppresses the interaction with βCOP, a vesicular transport protein that acts to retain the channel in the ER [94,139]. The annexin protein p11 [50], present in some cell types, has also been found to modulate K_2P3 trafficking via this pathway, binding in a 14-3-3 dependent manner [95].

K_2P5, K_2P16 and K_2P17 Channels

K_2P5 (for a period TASK2), K_2P16 (TALK1) and K_2P17 (TALK2) pass larger currents as the extracellular pH becomes more alkaline [36,49]. The pH-dependent gating of the channels appears to arise from neutralization of basic residues near the second P loop in the primary structure that influence operation of the selectivity filter (Arg in K_2P5 and K_2P16; Lys in K_2P17). Studies with concatemeric K_2P5 subunits show the pH sensors in both subunits must be neutralized for a dimeric channel to conduct K⁺ ions [91]. Alkaline activation of these channels is proposed to play a role in secretion of biocarbonate in the lumen of the exocrine pancreas [46]. The knock-out of K_2P5 in mice has been explicitly associated with bicarbonate handling in proximal tubule cells of the kidney leading to impaired reabsorption, metabolic acidosis, hyponatremia and hypotension [134]. Supporting the relevance of these findings to human disease, Balkan Endemic Nephropathy, a chronic tubulointerstitial disease leading to renal failure, has been associated with a KCNK5 variant (T108P) [126] that produces both loss-of-function [129] and dominant negative effects on K_2P5 channels expressed in heterologous systems [112]. K_2P5 also appears to contribute to rapid control of acid-base balance by regulating the activity of central respiratory chemoreceptors and CO₂ elimination by ventilation [48,131].

K_2P5 has also been implicated in cytosolic volume regulation in Ehrlich and mouse proximal tubule cells where increased intracellular pressure yields efflux of K⁺, along with Cl^- and organic osmolytes, obliging outflow of water and normalization of cell volume [9,89]. Moreover, auditory function is lost in K_2P5 knockout mice, perhaps because the channels, expressed in the cochlea, spiral ganglion, Reissner's membrane and stria vascularis, are required for K⁺ recycling in the outer sulcus lateralis [22].

K_2P18 Channels

K_2P18 (TRESK), the final member of the family to be identified in the human genome [115], also operates as an open rectifying K⁺ selective channel [10,38,64]. K_2P18 is notable for its expression in the spinal cord, trigeminal and dorsal root ganglia where it appears to pass a significant portion of the background K⁺ current that determines the excitability of somatosensory nociceptive fibers. K_2P18 has also been implicated in normal operation of peripheral nociceptor neurons by a ~50% decrease in its mRNA upon axiotomy [127]. Correlation of K_2P18 channels and pain was provided by linkage of a frame-shift mutation (F139WfsX24) in KCNK18 and migraine with aura in a large, multigenerational family [68]. The F139WfsX24 variant is truncated at residue 162 and appears to act as a dominant-negative subunit to suppress the function of wild type K_2P18 channels. K_2P18 is also notable for being reversibly activated by the calmodulin-dependent protein phosphatase calcineurin in response to increased levels of cytosolic Ca²⁺ [32-33]. Of note, Ca²⁺ modulation of K_2P18 is suppressed by phosphorylation of Ser264 and subsequent binding of 14-3-3η or 14-3-3γ (but not 14-3-3β) to a residue on the intracellular loop of the channel [34].

K_2P6, K_2P7, and K_2P15: subunits that, thus far, remain quiet

K_2P6 (TWIK2) and K_2P7 exhibit small currents on heterologous expression slowing their characterization and delineation of their roles [16,24]. Both proteins are expressed in the eye, lung and stomach [113]. K_2P6 has been posited to play a role in regulating arterial blood pressure by determining the Vm of vascular smooth muscle cells [73]. K_2P7 has a His in the first P-domain (Gly-Tyr-Gly-His) that has been proposed to mediate proton block based observations of this role for the homologous residue in K_2P1, K_2P3 and K_2P9 [75,101,110]. In contrast, K_2P7 is notable for an unconventional signature sequence in the second P-domain, Gly-Leu-Glu, not found in other human K_2P subunits that carry Gly-Tyr-Gly, Gly-Leu-Gly, or Gly-Phe-Gly. The presence of a large, negatively-charged glutamate residue in the second P-domain, rather than a small glycine, has led some to suggest that K_2P7 may not function as a K⁺ channel [41]. K_2P15 transcripts are widely expressed in humans but it has not been reported to pass current and has been postulated to require an as yet unidentified subunit or regulator [3].

K_2P12 and K_2P13 Channels

K_2P12 (THIK2) was initially reported to traffic to the cell surface but not to pass current nor to assemble with K_2P13 (THIK1) with which it shares sequence similarity [109]; in contrast, K_2P13 passed current and was inhibited by the volatile anesthetic halothane. Subsequently, a study reported that encoding K_2P12 linked to K_2P13 to force their co-assembly as a heterodimeric concatemer produced electrically active channels at the cell surface [15]. Because homodimeric concatemers of K_2P12 appeared to be retained in the ER of MDCK cells, the heterodimeric constructs were proposed to mask ER retention or retrieval motifs [15].

THE PHARMACOLOGY OF K_2P CHANNELS

To date, K_2P channels have few recognized natural blockers and a scant pharmacopeia [78,130]. The lack of identified pore-blocking peptide neurotoxins appears to reflect shielding of the outer vestibule by the cap-domain. Medications acting on K_2P channels include the halogenated volatile anesthetics, a class of drugs known to act on background K⁺ channels before the genes were cloned. Thus, currents mediated by K_2P2, K_2P3, K_2P4, K_2P9, K_2P10 and K_2P18 are augmented by halothane, isofluorane and sevofluorane [69,105,119,122]. In contrast, these same agents suppress K_2P13 activity. Although halothane has little impact on the activity of K_2P1 channels in CGN, halothane augments the current passed by heterodimeric channels formed by K_2P1 and K_2P9 by ~300% [105]. Structure-function analysis has revealed that the effect of volatile anesthetics on K_2P2, K_2P3 and K_2P9 channels requires a domain on the proximal C-terminus of the subunits and has implicated direct binding of the Gαq signaling protein [96,122]. Because Gαq proteins diminish K_2P channel function, the volatile anesthetics may augment activity by suppressing this interaction [29]. As noted above, K_2P10 is inhibited by active metabolite of the selective serotonin reuptake inhibitor Prozac [39] and K_2P2 is activated by fenamate-derived anti-inflammatory agents [128]. Efforts are ongoing to develop K_2P pharmacology and an activator selective for the mechanosensitive subclass K_2P2/K_2P4/K_2P10 [4,130] and an inhibitor that acts on K_2P4, K_2P6, and K_2P9 [121] have recently been reported.

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