Heterodimeric Channels Research Articles

Regulation of the two-pore domain potassium channel, THIK-1 and THIK-2, by G protein coupled receptors.

A member of THIK (two pore domain halothane-inhibited K+) channels, THIK-1, was reported as a target of Gi/o-coupled receptors (Gi/o-Rs) in neurons and microglia. We confirmed that in HEK293T cells the THIK-1 channel is activated by Gi/o-Rs and found that Gq-coupled receptors (Gq-Rs) also activates the channel. The effects of Gi/o-Rs and Gq-Rs were inhibited by the Gi/o inhibitor pertussis toxin and phospholipase C (PLC) inhibitor, respectively. The effects of Gi/o-Rs were attenuated when consensus Gβγ binding motif at the C-tail of the THIK-1 channel was mutated, suggesting that Gβγ serves as a THIK-1 channel activator upon the stimulation of Gi/o-Rs. As to the effects of Gq-Rs on the THIK-1 channel, a protein kinase C inhibitor and calcium chelators failed to inhibit the effect of a Gq coupled muscarinic M1R. Neither the hydrolysis of phosphatidyl inositol bisphosphate induced by voltage sensitive phosphatase nor the application of a diacylglycerol analogue, OAG, increased the channel current. The mediator of Gq-dependent activation of the THIK-1 channel remained unsolved. The effects of Gi/o- and Gq-Rs on the THIK-2 channel were also investigated, by using a THIK-2 mutant channel whose N-terminal domain is deleted to improve the surface membrane expression. We observed that Gi/o- and Gq-Rs activate the mutated THIK-2 channel, similarly to the THIK-1 channel. Interestingly, heterodimeric channels of THIK-1 and THIK-2 responded to Gi/o-R and Gq-R stimulation. Taken together, Gi/o- or Gq-Rs activates the THIK-1 and THIK-2 channels in a Gβγ or PLC dependent manner, respectively.

β-COP Regulates TWIK1/TREK1 Heterodimeric Channel-Mediated Passive Conductance in Astrocytes.

Mature astrocytes are characterized by a K+ conductance (passive conductance) that changes with a constant slope with voltage, which is involved in K+ homeostasis in the brain. Recently, we reported that the tandem of pore domains in a weak inward rectifying K+ channel (TWIK1 or KCNK1) and TWIK-related K+ channel 1 (TREK1 or KCNK2) form heterodimeric channels that mediate passive conductance in astrocytes. However, little is known about the binding proteins that regulate the function of the TWIK1/TREK1 heterodimeric channels. Here, we found that β-coat protein (COP) regulated the surface expression and activity of the TWIK1/TREK1 heterodimeric channels in astrocytes. β-COP binds directly to TREK1 but not TWIK1 in a heterologous expression system. However, β-COP also interacts with the TWIK1/TREK1 heterodimeric channel in a TREK1 dependent manner and enhances the surface expression of the heterodimeric channel in astrocytes. Consequently, it regulates TWIK1/TREK1 heterodimeric channel-mediated passive conductance in astrocytes in the mouse brain. Taken together, these results suggest that β-COP is a potential regulator of astrocytic passive conductance in the brain.

Allosteric coupling between transmembrane segment 4 and the selectivity filter of TALK1 potassium channels regulates their gating by extracellular pH

Opening of two-pore domain K+ channels (K2Ps) is regulated by various external cues, such as pH, membrane tension, or temperature, which allosterically modulate the selectivity filter (SF) gate. However, how these cues cause conformational changes in the SF of some K2P channels remains unclear. Herein, we investigate the mechanisms by which extracellular pH affects gating in an alkaline-activated K2P channel, TALK1, using electrophysiology and molecular dynamics (MD) simulations. We show that R233, located at the N-terminal end of transmembrane segment 4, is the primary pHo sensor. This residue distally regulates the orientation of the carbonyl group at the S1 potassium-binding site through an interacting network composed of residues on transmembrane segment 4, the pore helix domain 1, and the SF. Moreover, in the presence of divalent cations, we found the acidic pH-activated R233E mutant recapitulates the network interactions of protonated R233. Intriguingly, our data further suggested stochastic coupling between R233 and the SF gate, which can be described by an allosteric gating model. We propose that this allosteric model could predict the hybrid pH sensitivity in heterodimeric channels with alkaline-activated and acidic-activated K2P subunits.

Spadin Modulates Astrocytic Passive Conductance via Inhibition of TWIK-1/TREK-1 Heterodimeric Channels.

Astrocytes, the most abundant cell type in the brain, are non-excitable cells and play critical roles in brain function. Mature astrocytes typically exhibit a linear current–voltage relationship termed passive conductance, which is believed to enable astrocytes to maintain potassium homeostasis in the brain. We previously demonstrated that TWIK-1/TREK-1 heterodimeric channels mainly contribute to astrocytic passive conductance. However, the molecular identity of astrocytic passive conductance is still controversial and needs to be elucidated. Here, we report that spadin, an inhibitor of TREK-1, can dramatically reduce astrocytic passive conductance in brain slices. A series of gene silencing experiments demonstrated that spadin-sensitive currents are mediated by TWIK-1/TREK-1 heterodimeric channels in cultured astrocytes and hippocampal astrocytes from brain slices. Our study clearly showed that TWIK-1/TREK-1-heterodimeric channels can act as the main molecular machinery of astrocytic passive conductance, and suggested that spadin can be used as a specific inhibitor to control astrocytic passive conductance.

TRESK and TREK-2 two-pore-domain potassium channel subunits form functional heterodimers in primary somatosensory neurons

Two-pore-domain potassium channels (K2P) are the major determinants of the background potassium conductance. They play a crucial role in setting the resting membrane potential and regulating cellular excitability. These channels form homodimers; however, a few examples of heterodimerization have also been reported. The K2P channel subunits TRESK and TREK-2 provide the predominant background potassium current in the primary sensory neurons of the dorsal root and trigeminal ganglia. A recent study has shown that a TRESK mutation causes migraine because it leads to the formation of a dominant negative truncated TRESK fragment. Surprisingly, this fragment can also interact with TREK-2. In this study, we determined the biophysical and pharmacological properties of the TRESK/TREK-2 heterodimer using a covalently linked TRESK/TREK-2 construct to ensure the assembly of the different subunits. The tandem channel has an intermediate single-channel conductance compared with the TRESK and TREK-2 homodimers. Similar conductance values were recorded when TRESK and TREK-2 were coexpressed, demonstrating that the two subunits can spontaneously form functional heterodimers. The TRESK component confers calcineurin-dependent regulation to the heterodimer and gives rise to a pharmacological profile similar to the TRESK homodimer, whereas the presence of the TREK-2 subunit renders the channel sensitive to the selective TREK-2 activator T2A3. In trigeminal primary sensory neurons, we detected single-channel activity with biophysical and pharmacological properties similar to the TRESK/TREK-2 tandem, indicating that WT TRESK and TREK-2 subunits coassemble to form functional heterodimeric channels also in native cells.

TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

Two-pore domain K+ (K2P) channels have been shown to modulate neuronal excitability. The physiological role of TWIK-1, the first identified K2P channel, in neuronal cells is largely unknown, and we reported previously that TWIK-1 contributes to the intrinsic excitability of dentate gyrus granule cells (DGGCs) in mice. In the present study, we investigated the coexpression of TWIK-1 and TASK-3, another K2P member, in DGGCs. Immunohistochemical staining data showed that TASK-3 proteins were highly localized in the proximal dendrites and soma of DGGCs, and this localization is similar to the expression pattern of TWIK-1. TWIK-1 was shown to associate with TASK-3 in DGGCs of mouse hippocampus and when both genes were overexpressed in COS-7 cells. shRNA-mediated gene silencing demonstrated that TWIK-1/TASK-3 heterodimeric channels displayed outwardly rectifying currents and contributed to the intrinsic excitability of DGGCs. Neurotensin–neurotensin receptor 1 (NT–NTSR1) signaling triggered the depolarization of DGGCs by inhibiting TWIK-1/TASK-3 heterodimeric channels, causing facilitated excitation of DGGCs. Taken together, our study clearly showed that TWIK-1/TASK-3 heterodimeric channels contribute to the intrinsic excitability of DGGCs and that their activities are regulated by NT–NTSR1 signaling.

Characterization of an insect heterodimeric voltage-gated sodium channel with unique alternative splicing mode.

Recent discovery of the heterodimeric voltage-gated sodium channels (Nav) in two aphid species, Acyrthosiphon pisum and Myzus persicae, aroused interest in exploring whether this kind of channel is conserved for aphids. Herewith, we aim to provide evidence for the conservation of heterodimeric Navs in aphids and investigate whether they have unique splicing patterns. We found that the only identifiable Nav from Toxoptera citricida consisted of two subunits, forming a heterodimeric Nav, which carried an atypical "DENS" ion selectivity filter and a conventional "MFM" inactivation gate, confirming the heterodimeric Navs' conservation within aphids. These unique heterodimeric channels may form a new Nav subfamily, specific to aphids. A more ancient member of four-domain Nav homolog was well preserved in T. citricida, carrying a typical "DEEA" and "MFL" motif. The presence of "DENS" in mammalian Naxs and "DEKT" in a fungus Nav suggested that the heterodimeric Navs may still preserve Na+ permeability. Sequencing 46 clones from nymphs and adults exposed unique splicing patterns for this heterodimeric Nav from T. citricida, revealing 7 alternatively spliced exons, evidencing that exon 5 was no longer unique to Bombyx mori, and exon k/l was semi-mutually exclusive. Two previously undescribed optional exons and a SNP site seemingly unique to aphids were identified. In conclusion, the dimeric Navs might form a new aphids-specific heterodimeric Nav subfamily. This dimeric Nav from T. citricida was characterized with distinguishable alternative splicing modes, exemplified by the discovery of two novel alternative exons and unique usage patterns of alternative exons.

Intersubunit Concerted Cooperative and cis-Type Mechanisms Modulate Allosteric Gating in Two-Pore-Domain Potassium Channel TREK-2.

In response to diverse stimuli, two-pore-domain potassium channel TREK-2 regulates cellular excitability, and hence plays a key role in mediating neuropathic pain, mood disorders and ischemia through. Although more and more input modalities are found to achieve their modulations via acting on the channel, the potential role of subunit interaction in these modulations remains to be explored. In the current study, the deletion (lack of proximal C-terminus, ΔpCt) or point mutation (G312A) was introduced into TREK-2 subunits to limit K+ conductance and used to report subunit stoichiometry. The constructs were then combined with wild type (WT) subunit to produce concatenated dimers with defined composition, and the gating kinetics of these channels to 2-Aminoethoxydiphenyl borate (2-APB) and extracellular pH (pHo) were characterized. Our results show that combination of WT and ΔpCt/G312A subunits reserves similar gating properties to that of WT dimmers, suggesting that the WT subunit exerts dominant and positive effects on the mutated one, and thus the two subunits controls channel gating via a concerted cooperative manner. Further introduction of ΔpCt into the latter subunit of heterodimeric channel G312A-WT or G312A-G312A attenuated their sensitivity to 2-APB and pHo alkalization, implicating that these signals were transduced by a cis-type mechanism. Together, our findings elucidate the mechanisms for how the two subunits control the pore gating of TREK-2, in which both intersubunit concerted cooperative and cis-type manners modulate the allosteric regulations induced by 2-APB and pHo alkalization.

Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties

The tandem of pore domain in a weak inwardly rectifying K(+) channel (Twik)-related acid-arachidonic activated K(+) channel (TRAAK) and Twik-related K(+) channels (TREK) 1 and TREK2 are active as homodimers gated by stretch, fatty acids, pH, and G protein-coupled receptors. These two-pore domain potassium (K2P) channels are broadly expressed in the nervous system where they control excitability. TREK/TRAAK KO mice display altered phenotypes related to nociception, neuroprotection afforded by polyunsaturated fatty acids, learning and memory, mood control, and sensitivity to general anesthetics. These channels have emerged as promising targets for the development of new classes of anesthetics, analgesics, antidepressants, neuroprotective agents, and drugs against addiction. Here, we show that the TREK1, TREK2, and TRAAK subunits assemble and form active heterodimeric channels with electrophysiological, regulatory, and pharmacological properties different from those of homodimeric channels. Heteromerization occurs between all TREK variants produced by alternative splicing and alternative translation initiation. These results unveil a previously unexpected diversity of K2P channels that will be challenging to analyze in vivo, but which opens new perspectives for the development of clinically relevant drugs.

Abstract 17301: Functional Characterization of KCNK3 Mutants Associated With Pulmonary Arterial Hypertension Under Physiologically Relevant Heterozygous Conditions

KCNK3 encodes a two-pore domain K+ channel, TASK-1, which is inhibited by extracellular acidity and hypoxia. Expressed in a variety of tissues, including human pulmonary artery smooth muscle cells (hPASMCs), the central nervous system, pancreas, and adrenal glands, TASK-1 contributes to the resting membrane potential of cells in which it is expressed. Recently, our group reported mutations in KCNK3 underlying idiopathic pulmonary arterial hypertension (PAH), resulting from loss of TASK-1 function, partially pharmacologically rescuable with ONO-RS-082. TASK-1 dimerizes in vivo, forming functional channels with another TASK-1 subunit or with the related TASK-3 channel. TASK-1 and TASK-3 often are expressed in the same cells, although it has been reported that TASK-1 alone is expressed in the lung. Our initial study examined mutant and wildtype (WT) homodimeric TASK-1 channels expressed heterologously in COS-7 cells. Here we further characterize PAH-linked TASK-1 mutations in physiologically relevant heterozygous conditions in COS-7 and hPASMC cell lines. We engineered heterodimeric channels consisting of one mutant and one WT subunit; compared this with co-expression of mutant and WT channels; and measured channel activity with whole cell patch clamp procedures. We found a mutation specific impact of heterozygosity on channel activity. One mutation, V221L, produces a shift in pH dependence accounting for loss of function at physiological pH 7.4, partially rescued by dimerization with a WT subunit, while another, G203D, produces near complete loss of function as a homo- or hetero-dimer. The presence of TASK-3 results in greater rescue of V221L TASK-1 activity at pH 7.4 than does WT TASK-1. Additionally, under current clamp we found that ONO-RS-082 hyperpolarizes the membrane potential in hPASMCs expressing WT or V221L TASK-1, reversible by selective block of TASK-1 with ML365. Together, our results suggest (a) TASK-1 mutant heterodimers exhibit loss of function with mutation specific severity; (b) TASK-3 may rescue mutant TASK-1 and underlie a tissue specific impact of the TASK-1 mutations observed clinically; and (c) PAH TASK-1 mutants can be pharmacologically modulated in hPASMCs and alter the critically important resting membrane potential.

TASK channels in arterial chemoreceptors and their role in oxygen and acid sensing.

Arterial chemoreceptors play a vital role in cardiorespiratory control by providing the brain with information regarding blood oxygen, carbon dioxide, and pH. The main chemoreceptor, the carotid body, is composed of sensory (type 1) cells which respond to hypoxia or acidosis with a depolarising receptor potential which in turn activates voltage-gated calcium entry, neurosecretion and excitation of adjacent afferent nerves. The receptor potential is generated by inhibition of Twik-related acid-sensitive K+ channel 1 and 3 (TASK1/TASK3) heterodimeric channels which normally maintain the cells’ resting membrane potential. These channels are thought to be directly inhibited by acidosis. Oxygen sensitivity, however, probably derives from a metabolic signalling pathway. The carotid body, isolated type 1 cells, and all forms of TASK channel found in the type 1 cell, are highly sensitive to inhibitors of mitochondrial metabolism. Moreover, type1 cell TASK channels are activated by millimolar levels of MgATP. In addition to their role in the transduction of chemostimuli, type 1 cell TASK channels have also been implicated in the modulation of chemoreceptor function by a number of neurocrine/paracrine signalling molecules including adenosine, GABA, and serotonin. They may also be instrumental in mediating the depression of the acute hypoxic ventilatory response that occurs with some general anaesthetics. Modulation of TASK channel activity is therefore a key mechanism by which the excitability of chemoreceptors can be controlled. This is not only of physiological importance but may also offer a therapeutic strategy for the treatment of cardiorespiratory disorders that are associated with chemoreceptor dysfunction.

ClC-1 and ClC-2 form hetero-dimeric channels with novel protopore functions

CLC-type chloride channels exhibit a unique double-barreled architecture with two independently functioning ion conduction pathways, the so-called protopores. There exist gating processes that open and close individual protopores as well as common processes that jointly mediate slow opening and closing of both protopores. Different isoforms exhibit distinct voltage dependences and kinetics of gating. Whereas opening of the individual and common gate of homo-dimeric ClC-1 is promoted by membrane depolarization, ClC-2 is closed at positive potentials and opens only at negative voltages. To characterize the functional interaction of protopores we engineered a concatameric construct linking the coding regions of ClC-1 and ClC-2 in an open reading frame, expressed it in mammalian cells and measured anion currents through whole-cell and single channel patch clamping. In the hetero-dimeric assembly, each protopore displayed two kinetically distinct gating processes. Fast gating of the ClC-1 protopore closely resembled fast protopore gating of homo-dimeric channels. The voltage dependence of ClC-2 fast gating was shifted to more positive potentials by the adjacent ClC-1 protopore, resulting in open ClC-2 protopores at positive voltages. We observed two slow gating processes individually acting on ClC-1 and ClC-2 protopores, with distinct time and voltage dependences. Single channel recordings demonstrated that hetero-dimerization additionally modified the unitary conductance of ClC-2 protopores. Our findings suggest that inter-subunit interactions do not only affect common gating, but also ion permeation and gating of individual protopores in hetero-dimeric ClC channels.

A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes

TWIK-1 is a member of the two-pore domain K(+) (K2P) channel family that plays an essential part in the regulation of resting membrane potential and cellular excitability. The physiological role of TWIK-1 has remained enigmatic because functional expression of TWIK-1 channels is elusive. Here we report that native TWIK-1 forms a functional channel at the plasma membrane of astrocytes. A search for TWIK-1-binding proteins led to the identification of TREK-1, another member of the K2P family. The TWIK-1/TREK-1 heterodimeric channel is formed via a disulphide bridge between residue C69 in TWIK-1 and C93 in TREK-1. Gene silencing demonstrates that surface expression of TWIK-1 and TREK-1 are interdependent. TWIK-1/TREK-1 heterodimers mediate astrocytic passive conductance and cannabinoid-induced glutamate release from astrocytes. Our study sheds new light on the diversity of K2P channels.

Disease‐causing mutations C277R and C277Y modify gating of human ClC‐1 chloride channels in myotonia congenita

Myotonia congenita is a genetic condition that is caused by mutations in the muscle chloride channel gene CLCN1 and characterized by delayed muscle relaxation and muscle stiffness. We here investigate the functional consequences of two novel disease-causing missense mutations, C277R and C277Y, using heterologous expression in HEK293T cells and patch clamp recording. Both mutations reduce macroscopic anion currents in transfected cells. Since hClC-1 is a double-barrelled anion channel, this reduction in current amplitude might be caused by altered gating of individual protopores or of joint openings and closing of both protopores. We used non-stationary noise analysis and single channel recordings to separate the mutants' effects on individual and common gating processes. We found that C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates resulting in decreases of absolute open probabilities of homodimeric channels to values below 3%. In heterodimeric channels, C277R and C277Y also reduce open probabilities and shift the common gate activation curve towards positive potentials. Moreover, C277Y modifies pore properties of hClC-1. It reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence to I(-) = NO(3)(-) >Br(-)>Cl(-). Our findings predict a dramatic reduction of the muscle fibre resting chloride conductance and thus fully explain the disease-causing effects of mutations C277R and C277Y. Moreover, they provide additional insights into the function of C277, a residue recently implicated in common gating of ClC channels.

Optical Control of Endogenous Proteins with a Photoswitchable Conditional Subunit Reveals a Role for TREK1 in GABAB Signaling

Selective ligands are lacking for many neuronal signaling proteins. Photoswitched tethered ligands (PTLs) have enabled fast and reversible control of specific proteins containing a PTL anchoring site and have been used to remote control overexpressed proteins. We report here a scheme for optical remote control of native proteins usinga "photoswitchable conditional subunit" (PCS), which contains the PTL anchoring site as well as a mutation that prevents it from reaching the plasma membrane. In cells lacking native subunits for the protein, the PCS remains nonfunctional internally. However, in cells expressing native subunits, the native subunit and PCS coassemble, traffic to the plasma membrane, and place the native protein under optical control provided by the coassembled PCS. We apply this approach to the TREK1 potassium channel, which lacks selective, reversible blockers. We find that TREK1, typically considered to be a leak channel, contributes to the hippocampal GABA(B) response.

The Membrane Interface Dictates Different Anchor Roles for “Inner Pair” and “Outer Pair” Tryptophan Indole Rings in Gramicidin A Channels

We investigated the effects of substituting two of the four tryptophans (the "inner pair" Trp(9) and Trp(11) or the "outer pair" Trp(13) and Trp(15)) in gramicidin A (gA) channels. The conformational preferences of the doubly substituted gA analogues were assessed using circular dichroism spectroscopy and size-exclusion chromatography, which show that the inner tryptophans 9 and 11 are critical for the gA's conformational preference in lipid bilayer membranes. [Phe(13,15)]gA largely retains the single-stranded helical channel structure, whereas [Phe(9,11)]gA exists primarily as double-stranded conformers. Within this context, the (2)H NMR spectra from labeled tryptophans were used to examine the changes in average indole ring orientations, induced by the Phe substitutions and by the shift in conformational preference. Using a method for deuterium labeling of already synthesized gAs, we introduced deuterium selectively onto positions C2 and C5 of the remaining tryptophan indole rings in the substituted gA analogues for solid-state (2)H NMR spectroscopy. The (least possible) changes in orientation and overall motion of each indole ring were estimated from the experimental spectra. Regardless of the mixture of backbone folds, the indole ring orientations observed in the analogues are similar to those found previously for gA channels. Both Phe-substituted analogues form single-stranded channels, as judged from the formation of heterodimeric channels with the native gA. [Phe(13,15)]gA channels have Na(+) currents that are ~50% and lifetimes that are ~80% of those of native gA channels. The double-stranded conformer(s) of [Phe(9,11)]gA do not form detectable channels. The minor single-stranded population of [Phe(9,11)]gA forms channels with Na(+) currents that are ~25% and single-channel lifetimes that are ~300% of those of native gA channels. Our results suggest that Trp(9) and Trp(11), when "reaching" for the interface, tend to drive both monomer folding (to "open" a channel) and dimer dissociation (to "close" a channel). Furthermore, the dipoles of Trp(9) and Trp(11) are relatively more important for the single-channel conductance than are the dipoles of Trp(13) and Trp(15).

Polar Groups in Membrane Channels: Consequences of Replacing Alanines with Serines in Membrane-Spanning Gramicidin Channels

To explore the consequences of burying polar, hydrogen-bonding hydroxyl groups within the hydrocarbon core of lipid bilayer membranes, we examined the structural and functional effects of alanine-to-serine substitutions in bilayer-spanning gramicidin channels. A native Ala was replaced by Ser at position 3 or 5 in the gramicidin A (gA) sequence: formyl-VG(2)A(3)LA(5)VVVWLWLWLW-ethanolamide (d-residues underlined). In the head-to-head dimers that form the conducting, membrane-spanning gA channels, these sequence positions are located near the lipid bilayer center (and subunit interface). The sequence substitutions at positions 3 and 5 were tested within the context of having either Gly or d-Ala at position 2, because d-Ala(2) causes the channel lifetimes to increase 3-fold relative to Gly(2) [Mattice et al. (1995) Biochemistry 34, 6827]. Size-exclusion chromatograms and circular dichroism spectra show that the Ala --> Ser replacements are well tolerated and have little effect on channel structure. In planar bilayers, the Ser-substituted gramicidins form well-defined channels, with cation conductances that are approximately 60% of those of the reference channels. The Ser-substituted channels are structurally equivalent to native gramicidin channels, as demonstrated by the formation of heterodimeric channels between a Ser-containing subunit and a native gramicidin subunit. These hybrid channels exhibit rectification, attributable to asymmetric placement of the single Ser hydroxyl group with respect to the bilayer center. Compared to the corresponding Ala-containing reference channels, the polar Ser residues decrease the analogues' channel-forming potency by 3 orders of magnitude, indicating a substantial energetic penalty ( approximately 15 kJ/mol) for burying the polar Ser side chain in the bilayer hydrophobic core.

Biochemical Characterization of κM-RIIIJ, a Kv1.2 Channel Blocker: EVALUATION OF CARDIOPROTECTIVE EFFECTS OF κM-CONOTOXINS

Conus snail (Conus) venoms are a valuable source of pharmacologically active compounds; some of the peptide toxin families from the snail venoms are known to interact with potassium channels. We report the purification, synthesis, and characterization of kappaM-conotoxin RIIIJ from the venom of a fish-hunting species, Conus radiatus. This conopeptide, like a previously characterized peptide in the same family, kappaM-RIIIK, inhibits the homotetrameric human Kv1.2 channels. When tested in Xenopus oocytes, kappaM-RIIIJ has an order of magnitude higher affinity (IC(50) = 33 nm) to Kv1.2 than kappaM-RIIIK (IC(50) = 352 nm). Chimeras of RIIIK and RIIIJ tested on the human Kv1.2 channels revealed that Lys-9 from kappaM-RIIIJ is a determinant of its higher potency against hKv1.2. However, when compared in a model of ischemia/reperfusion, kappaM-RIIIK (100 mug/kg of body weight), administered just before reperfusion, significantly reduces the infarct size in rat hearts in vivo without influencing hemodynamics, providing a potential compound for cardioprotective therapeutics. In contrast, kappaM-RIIIJ does not exert any detectable cardioprotective effect. kappaM-RIIIJ shows more potency for Kv1.2-Kv1.5 and Kv1.2-Kv1.6 heterodimers than kappaM-RIIIK, whereas the affinity of kappaM-RIIIK to Kv1.2-Kv1.7 heterodimeric channels is higher (IC(50) = 680 nm) than that of kappaM-RIIIJ (IC(50) = 3.15 mum). Thus, the cardioprotection seems to correlate to antagonism to heteromultimeric channels, involving the Kv1.2 alpha-subunit rather than antagonism to Kv1.2 homotetramers. Furthermore, kappaM-RIIIK and kappaM-RIIIJ provide a valuable set of probes for understanding the underlying mechanism of cardioprotection.

K2P1 Assembles with K2P3 or K2P9 to Form Sumo-Regulated Task Background Channels

TASK subunits K2P3 and K2P9 form homo- and hetero-dimeric channels in neurons with varied sensitivities to anesthetics, flavors and pH (Berg et al. J Neurosci. 2004. 24:6693-702; Bautista et al. Nat. Neurosci. 2008. 11:772-9). Here, K2P1 subunits (Rajan et al. 2005. Cell 121, 37-47) are shown to confer SUMO-regulation on TASK channels in rat cerebellar granule neurons (CGN). First, CFP (C) or YFP (Y)-tagged subunits studied in CHO cells by donor-decay Forster resonance energy transfer (FRET) confirm biochemical evidence for assembly of K2P2 and its native isoform (Thomas et al., Neuron 2008 58:859-70). Next, FRET shows association of C-K2P1 and Y-K2P1, Y-K2P3 or Y-K2P9 but not Y-K2P2 or Kv2.1. As expected from Rajan et al, FRET registers association of Y-SUMO and C-K2P1 but not C-K2P3 or C-K2P9. In contrast, Y-SUMO and C-K2P3 or C-K2P9 FRET when untagged K2P1 is co-expressed. Consistent with electrophysiological studies (EP) showing one SUMO per channel is sufficient to silence K2P channels (Plant et al. this meeting), channels with two linked K2P1 subunits (WT-WT), WT and SUMO-insensitive K2P1 (WT-K274Q), WT and K2P3 (WT-K2P3) or WT and K2P9 (WT-K2P9), pass currents when membrane patches are exposed to SUMO-protease (SENP1) and silenced by SUMO1. In contrast, K2P3-K2P3, K2P9-K2P9, K2P3-K274Q or K2P9-K274Q are constitutively active and insensitive to SENP1 and SUMO1. Finally, in CGN, immunochemistry shows K2P1, K2P3, K2P9, SUMO, SUMO E1 conjugase and SUMO E2 ligase in plasma membrane; EP reveals IKSO regulation by SENP1 and SUMO, and transfection with mutant subunits demonstrate assembly of K2P1 with K2P3 or K2P9 by FRET and EP.

The neuronal background K2P channels: focus on TREK1

Two-pore-domain K(+) (K(2P)) channel subunits are made up of four transmembrane segments and two pore-forming domains that are arranged in tandem and function as either homo- or heterodimeric channels. This structural motif is associated with unusual gating properties, including background channel activity and sensitivity to membrane stretch. Moreover, K(2P) channels are modulated by a variety of cellular lipids and pharmacological agents, including polyunsaturated fatty acids and volatile general anaesthetics. Recent in vivo studies have demonstrated that TREK1, the most thoroughly studied K(2P) channel, has a key role in the cellular mechanisms of neuroprotection, anaesthesia, pain and depression.