A Novel Flp Reporter Mouse Shows That TRPA1 Expression Is Largely Limited to Sensory Neuron Subsets.

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal cation channel that is activated by electrophilic irritants, oxidative stress, cold temperature, and GPCR signaling. TRPA1 expression has been primarily identified in subsets of nociceptive sensory afferents and is considered a target for future analgesics. Nevertheless, TRPA1 has been implicated in other cell types including keratinocytes, epithelium, enterochromaffin cells, endothelium, astrocytes, and CNS neurons. Here, we developed a knock-in mouse that expresses the recombinase FlpO in TRPA1-expressing cells. We crossed the TRPA1Flp mouse with the R26ai65f mouse that expresses tdTomato in a Flp-sensitive manner. We found tdTomato expression correlated well with TRPA1 mRNA expression and sensitivity to TRPA1 agonists in subsets of TRPV1 (transient receptor potential vanilloid receptor type 1)-expressing neurons in the vagal ganglia and dorsal root ganglia (DRGs), although tdTomato expression efficiency was limited in DRG. We observed tdTomato-expressing afferent fibers centrally (in the medulla and spinal cord) and peripherally in the esophagus, gut, airways, bladder, and skin. Furthermore, chemogenetic activation of TRPA1-expressing nerves in the paw evoked flinching behavior. tdTomato expression was very limited in other cell types. We found tdTomato in subepithelial cells in the gut mucosa but not in enterochromaffin cells. tdTomato was also observed in supporting cells within the cochlea, but not in hair cells. Lastly, tdTomato was occasionally observed in neurons in the somatomotor cortex and the piriform area, but not in astrocytes or vascular endothelium. Thus, this novel mouse strain may be useful for mapping and manipulating TRPA1-expressing cells and deciphering the role of TRPA1 in physiological and pathophysiological processes.

Mapping of the Sensory Innervation of the Mouse Lung by Specific Vagal and Dorsal Root Ganglion Neuronal Subsets.

The airways are densely innervated by sensory afferent nerves, whose activation regulates respiration and triggers defensive reflexes (e.g., cough, bronchospasm). Airway innervation is heterogeneous, and distinct afferent subsets have distinct functional responses. However, little is known of the innervation patterns of subsets within the lung. A neuroanatomical map is critical for understanding afferent activation under physiological and pathophysiological conditions. Here, we quantified the innervation of the mouse lung by vagal and dorsal root ganglion (DRG) sensory subsets defined by the expression of Pirt (all afferents), 5HT3 (vagal nodose afferents), Tac1 (tachykinergic afferents), and transient receptor potential vanilloid 1 channel (TRPV1; defensive/nociceptive afferents) using Cre-mediated reporter expression. We found that vagal afferents innervate almost all conducting airways and project into the alveolar region, whereas DRG afferents only innervate large airways. Of the two vagal ganglia, only nodose afferents project into the alveolar region, but both nodose and jugular afferents innervate conducting airways throughout the lung. Many afferents that project into the alveolar region express TRPV1. Few DRG afferents expressed TRPV1. Approximately 25% of blood vessels were innervated by vagal afferents (many were Tac1+). Approximately 10% of blood vessels had DRG afferents (some were Tac1+), but this was restricted to large vessels. Lastly, innervation of neuroepithelial bodies (NEBs) correlated with the cell number within the bodies. In conclusion, functionally distinct sensory subsets have distinct innervation patterns within the conducting airways, alveoli and blood vessels. Physiologic (e.g., stretch) and pathophysiological (e.g., inflammation, edema) stimuli likely vary throughout these regions. Our data provide a neuroanatomical basis for understanding afferent responses in vivo.

Functional evidence of distinct electrophile-induced activation states of the ion channel TRPA1.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, nonselective cation channel expressed on nociceptive sensory nerves whose activation elicits nocifensive responses (e.g. pain). TRPA1 is activated by electrophiles found in foods and pollution, or produced during inflammation and oxidative stress, via covalent modification of reactive cysteines, but the mechanism underlying electrophilic activation of TRPA1 is poorly understood. Here we studied TRPA1 activation by the irreversible electrophiles iodoacetamide and N-ethylmaleimide (NEM) following transient expression in HEK293 cells. We found that in Ca2+ imaging studies C621 is critical for electrophile-induced TRPA1 activation, but the role of C665 in TRPA1 activation is dependent on the size of the electrophile. We identified slower TRPA1 activation in whole-cell recordings compared to studies with intact cells, which is rescued by pipette solution supplementation with the antioxidant glutathione. Single-channel recordings identified two distinct electrophilic-induced TRPA1 activation phases: a partial activation that, in some channels, switched to full activation with continued electrophile exposure. Full activation but not the initial activation was regulated by C665. Fitting of open time distributions suggests that full activation correlated with an additional (and long) exponential component, thus suggesting the phases are manifestations of distinct activation states. Our results suggest that distinct NEM-induced TRPA1 activation states are evoked by sequential modification of C621 then C665.

Differential sensitivity of cinnamaldehyde-evoked calcium fluxes to ruthenium red in guinea pig and mouse trigeminal sensory neurons.

OBJECTIVE: Transient receptor potential ankyrin 1 (TRPA1) is an excitatory ion channel expressed on a subset of sensory neurons. TRPA1 is activated by a host of noxious stimuli including pollutants, irritants, oxidative stress and inflammation, and is thought to play an important role in nociception and pain perception. TRPA1 is therefore a therapeutic target for diseases with nociceptive sensory signaling components. TRPA1 orthologs have been shown to have differential sensitivity to certain ligands. Cinnamaldehyde has previously been shown to activate sensory neurons via the selective gating of TRPA1. Here, we tested the sensitivity of cinnamaldehyde-evoked responses in mouse and guinea pig sensory neurons to the pore blocker ruthenium red (RuR). RESULTS: Cinnamaldehyde, the canonical TRPA1-selective agonist, caused robust calcium fluxes in trigeminal neurons dissociated from both mice and guinea pigs. RuR effectively inhibited cinnamaldehyde-evoked responses in mouse neurons at 30 nM, with complete block seen with 3 µM. In contrast, responses in guinea pig neurons were only partially inhibited by 3 µM RuR. We conclude that RuR has a decreased affinity for guinea pig TRPA1 compared to mouse TRPA1. This study provides further evidence of differences in ligand affinity for TRPA1 in animal models relevant for drug development.

Development of a Mouse Reporter Strain for the Purinergic P2X2 Receptor.

The ATP-sensitive P2X2 ionotropic receptor plays a critical role in a number of signal processes including taste and hearing, carotid body detection of hypoxia, the exercise pressor reflex and sensory transduction of mechanical stimuli in the airways and bladder. Elucidation of the role of P2X2 has been hindered by the lack of selective tools. In particular, detection of P2X2 using established pharmacological and biochemical techniques yields dramatically different expression patterns, particularly in the peripheral and central nervous systems. Here, we have developed a knock-in P2X2-cre mouse, which we crossed with a cre-sensitive tdTomato reporter mouse to determine P2X2 expression. P2X2 was found in more than 80% of nodose vagal afferent neurons, but not in jugular vagal afferent neurons. Reporter expression correlated in vagal neurons with sensitivity to α,ß methylene ATP (αßmATP). P2X2 was expressed in 75% of petrosal afferents, but only 12% and 4% of dorsal root ganglia (DRG) and trigeminal afferents, respectively. P2X2 expression was limited to very few cell types systemically. Together with the central terminals of P2X2-expressing afferents, reporter expression in the CNS was mainly found in brainstem neurons projecting mossy fibers to the cerebellum, with little expression in the hippocampus or cortex. The structure of peripheral terminals of P2X2-expressing afferents was demonstrated in the tongue (taste buds), carotid body, trachea and esophagus. P2X2 was observed in hair cells and support cells in the cochlear, but not in spiral afferent neurons. This mouse strain provides a novel approach to the identification and manipulation of P2X2-expressing cell types.

Antimycin A increases bronchopulmonary C-fiber excitability via protein kinase C alpha.

Inflammation can increase the excitability of bronchopulmonary C-fibers leading to excessive sensations and reflexes (e.g. wheeze and cough). We have previously shown modulation of peripheral nerve terminal mitochondria by antimycin A causes hyperexcitability in TRPV1-expressing bronchopulmonary C-fibers through the activation of protein kinase C (PKC). Here, we have investigated the PKC isoform responsible for this signaling. We found PKCß1, PKCδ and PKCε were expressed by many vagal neurons, with PKCα and PKCß2 expressed by subsets of vagal neurons. In dissociated vagal neurons, antimycin A caused translocation of PKCα but not the other isoforms, and only in TRPV1-lineage neurons. In bronchopulmonary C-fiber recordings, antimycin A increased the number of action potentials evoked by α,ß-methylene ATP. Selective inhibition of PKCα, PKCß1 and PKCß2 with 50 nM bisindolylmaleimide I prevented the antimycin-induced bronchopulmonary C-fiber hyperexcitability, whereas selective inhibition of only PKCß1 and PKCß2 with 50 nM LY333531 had no effect. We therefore conclude that PKCα is required for antimycin-induced increases in bronchopulmonary C-fiber excitability.

Antimycin A-induced mitochondrial dysfunction activates vagal sensory neurons via ROS-dependent activation of TRPA1 and ROS-independent activation of TRPV1.

Inflammation causes activation of nociceptive sensory nerves, resulting in debilitating sensations and reflexes. Inflammation also induces mitochondrial dysfunction through multiple mechanisms. Sensory nerve terminals are densely packed with mitochondria, suggesting that mitochondrial signaling may play a role in inflammation-induced nociception. We have previously shown that agents that induce mitochondrial dysfunction, such as antimycin A, activate a subset of nociceptive vagal sensory nerves that express transient receptor potential (TRP) channels ankyrin 1 (A1) and vanilloid 1 (V1). However, the mechanisms underlying these responses are incompletely understood. Here, we studied the contribution of TRPA1, TRPV1 and reactive oxygen species (ROS) to antimycin A-induced vagal sensory nerve activation in dissociated neurons and at the sensory terminals of bronchopulmonary C-fibers. Nociceptive neurons were defined chemically and genetically. Antimycin A-evoked activation of vagal nociceptors in a Fura2 Ca2+ assay correlated with TRPV1 responses compared to TRPA1 responses. Nociceptor activation was dependent on both TRP channels, with TRPV1 predominating in a majority of responding nociceptors and TRPA1 predominating only in nociceptors with the greatest responses. Surprisingly, both TRPA1 and TRPV1 were activated by H2O2 when expressed in HEK293. Nevertheless, targeting ROS had no effect of antimycin A-evoked TRPV1 activation in either HEK293 or vagal neurons. In contrast, targeting ROS inhibited antimycin A-evoked TRPA1 activation in HEK293, vagal neurons and bronchopulmonary C-fibers, and a ROS-insensitive TRPA1 mutant was completely insensitive to antimycin A. We therefore conclude that mitochondrial dysfunction activates vagal nociceptors by ROS-dependent (TRPA1) and ROS-independent (TRPV1) mechanisms.

Improving redox sensitivity of roGFP1 by incorporation of selenocysteine at position 147.

OBJECTIVE: Redox-sensitive green fluorescent protein (roGFP) is a genetically-encoded redox-sensitive protein used to detect cellular oxidative stress associated with reactive oxygen species production. Here we replaced the cysteine at position 147 of roGFP1 (variant of roGFP) with selenocysteine in order to increase redox sensitivity of the redox reporter. RESULTS: Expression of roGFP1 selenoprotein (roGFP1-Se147) in HEK293 cells required the presence of a selenocysteine insertion sequence and was augmented by co-expression with SBP2. roGFP1-Se147 demonstrated a similar excitation and emission spectra to roGFP1. Although expression of roGFP1-Se147 was limited, it was sufficient enough to perform live cell imaging to evaluate sensitivity to oxidation and reduction. roGFP1-Se147 exhibited a 100-fold increase in sensitivity to oxidation with H2O2 in comparison to roGFP1 as well as a 20-fold decrease in the EC50 of H2O2. Furthermore, roGFP1-Se147, unlike roGFP1, was able to detect oxidation caused by the mitochondrial electron transport complex III inhibitor antimycin A. Unfortunately roGFP-Se147 exhibited a diminished dynamic range and photoinstability.

The exceptionally high reactivity of Cys 621 is critical for electrophilic activation of the sensory nerve ion channel TRPA1.

Activation of the sensory nerve ion channel TRPA1 by electrophiles is the key mechanism that initiates nociceptive signaling, and leads to defensive reflexes and avoidance behaviors, during oxidative stress in mammals. TRPA1 is rapidly activated by subtoxic levels of electrophiles, but it is unclear how TRPA1 outcompetes cellular antioxidants that protect cytosolic proteins from electrophiles. Here, using physiologically relevant exposures, we demonstrate that electrophiles react with cysteine residues on mammalian TRPA1 at rates that exceed the reactivity of typical cysteines by 6,000-fold and that also exceed the reactivity of antioxidant enzymes. We show that TRPA1 possesses a complex reactive cysteine profile in which C621 is necessary for electrophile-induced binding and activation. Modeling of deprotonation energies suggests that K620 contributes to C621 reactivity and mutation of K620 alone greatly reduces the effect of electrophiles on TRPA1. Nevertheless, binding of electrophiles to C621 is not sufficient for activation, which also depends on the function of another reactive cysteine (C665). Together, our results demonstrate that TRPA1 acts as an effective electrophilic sensor because of the exceptionally high reactivity of C621.

Thy1.2 YFP-16 transgenic mouse labels a subset of large-diameter sensory neurons that lack TRPV1 expression.

The Thy1.2 YFP-16 mouse expresses yellow fluorescent protein (YFP) in specific subsets of peripheral and central neurons. The original characterization of this model suggested that YFP was expressed in all sensory neurons, and this model has been subsequently used to study sensory nerve structure and function. Here, we have characterized the expression of YFP in the sensory ganglia (DRG, trigeminal and vagal) of the Thy1.2 YFP-16 mouse, using biochemical, functional and anatomical analyses. Despite previous reports, we found that YFP was only expressed in approximately half of DRG and trigeminal neurons and less than 10% of vagal neurons. YFP-expression was only found in medium and large-diameter neurons that expressed neurofilament but not TRPV1. YFP-expressing neurons failed to respond to selective agonists for TRPV1, P2X(2/3 and TRPM8 channels in Ca2+ imaging assays. Confocal analysis of glabrous skin, hairy skin of the back and ear and skeletal muscle indicated that YFP was expressed in some peripheral terminals with structures consistent with their presumed non-nociceptive nature. In summary, the Thy1.2 YFP-16 mouse expresses robust YFP expression in only a subset of sensory neurons. But this mouse model is not suitable for the study of nociceptive nerves or the function of such nerves in pain and neuropathies.

Sensory nerve terminal mitochondrial dysfunction induces hyperexcitability in airway nociceptors via protein kinase C.

Airway sensory nerve excitability is a key determinant of respiratory disease-associated reflexes and sensations such as cough and dyspnea. Inflammatory signaling modulates mitochondrial function and produces reactive oxygen species (ROS). Peripheral terminals of sensory nerves are densely packed with mitochondria; thus, we hypothesized that mitochondrial modulation would alter neuronal excitability. We recorded action potential firing from the terminals of individual bronchopulmonary C-fibers using a mouse ex vivo lung-vagal ganglia preparation. C-fibers were characterized as nociceptors or non-nociceptors based upon conduction velocity and response to transient receptor potential (TRP) channel agonists. Antimycin A (mitochondrial complex III Qi site inhibitor) had no effect on the excitability of non-nociceptors. However, antimycin A increased excitability in nociceptive C-fibers, decreasing the mechanical threshold by 50% and increasing the action potential firing elicited by a P2X2/3 agonist to 270% of control. Antimycin A-induced nociceptor hyperexcitability was independent of TRP ankyrin 1 or TRP vanilloid 1 channels. Blocking mitochondrial ATP production with oligomycin or myxothiazol had no effect on excitability. Antimycin A-induced hyperexcitability was dependent on mitochondrial ROS and was blocked by intracellular antioxidants. ROS are known to activate protein kinase C (PKC). Antimycin A-induced hyperexcitability was inhibited by the PKC inhibitor bisindolylmaleimide (BIM) I, but not by its inactive analog BIM V. In dissociated vagal neurons, antimycin A caused ROS-dependent PKC translocation to the membrane. Finally, H2O2 also induced PKC-dependent nociceptive C-fiber hyperexcitability and PKC translocation. In conclusion, ROS evoked by mitochondrial dysfunction caused nociceptor hyperexcitability via the translocation and activation of PKC.

Sensory nerve terminal mitochondrial dysfunction activates airway sensory nerves via transient receptor potential (TRP) channels.

Mitochondrial dysfunction and subsequent oxidative stress has been reported for a variety of cell types in inflammatory diseases. Given the abundance of mitochondria at the peripheral terminals of sensory nerves and the sensitivity of transient receptor potential (TRP) ankyrin 1 (A1) and TRP vanilloid 1 (V1) to reactive oxygen species (ROS) and their downstream products of lipid peroxidation, we investigated the effect of nerve terminal mitochondrial dysfunction on airway sensory nerve excitability. Here we show that mitochondrial dysfunction evoked by acute treatment with antimycin A (mitochondrial complex III Qi site inhibitor) preferentially activated TRPA1-expressing "nociceptor-like" mouse bronchopulmonary C-fibers. Action potential discharge was reduced by the TRPA1 antagonist HC-030031. Inhibition of TRPV1 further reduced C-fiber activation. In mouse dissociated vagal neurons, antimycin A induced Ca(2+) influx that was significantly reduced by pharmacological inhibition or genetic knockout of either TRPA1 or TRPV1. Inhibition of both TRPA1 and TRPV1 was required to abolish antimycin A-induced Ca(2+) influx in vagal neurons. Using an HEK293 cell expression system, antimycin A induced concentration-dependent activation of both hTRPA1 and hTRPV1 but failed to activate nontransfected cells. Myxothiazol (complex III Qo site inhibitor) inhibited antimycin A-induced TRPA1 activation, as did the reducing agent dithiothreitol. Scavenging of both superoxide and hydrogen peroxide inhibited TRPA1 activation following mitochondrial modulation. In conclusion, we present evidence that acute mitochondrial dysfunction activates airway sensory nerves preferentially via TRPA1 through the actions of mitochondrially-derived ROS. This represents a novel mechanism by which inflammation may be transduced into nociceptive electrical signaling.

Neuroprotective effects of phenolic antioxidant tBHQ associate with inhibition of FoxO3a nuclear translocation and activity.

The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K)-Akt signalling pathways retains FoxO3a in the cytoplasm, thereby inhibiting the transcriptional activation of death-promoting genes. We hypothesized that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K-Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localization of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and up-regulated Fas ligand expression. In contrast the phenolic antioxidant, tBHQ, caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a, prevented NMDA-induced up-regulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration.

Reductions in external divalent cations evoke novel voltage-gated currents in sensory neurons.

It has long been recognized that divalent cations modulate cell excitability. Sensory nerve excitability is of critical importance to peripheral diseases associated with pain, sensory dysfunction and evoked reflexes. Thus we have studied the role these cations play on dissociated sensory nerve activity. Withdrawal of both Mg(2+) and Ca(2+) from external solutions activates over 90% of dissociated mouse sensory neurons. Imaging studies demonstrate a Na(+) influx that then causes depolarization-mediated activation of voltage-gated Ca(2+) channels (Ca(V)), which allows Ca(2+) influx upon divalent re-introduction. Inhibition of Ca(V) (ω-conotoxin, nifedipine) or Na(V) (tetrodotoxin, lidocaine) fails to reduce the Na(+) influx. The Ca(2+) influx is inhibited by Ca(V) inhibitors but not by TRPM7 inhibition (spermine) or store-operated channel inhibition (SKF96365). Withdrawal of either Mg(2+) or Ca(2+) alone fails to evoke cation influxes in vagal sensory neurons. In electrophysiological studies of dissociated mouse vagal sensory neurons, withdrawal of both Mg(2+) and Ca(2+) from external solutions evokes a large slowly-inactivating voltage-gated current (I(DF)) that cannot be accounted for by an increased negative surface potential. Withdrawal of Ca(2+) alone fails to evoke I(DF). Evidence suggests I(DF) is a non-selective cation current. The I(DF) is not reduced by inhibition of Na(V) (lidocaine, riluzole), Ca(V) (cilnidipine, nifedipine), K(V) (tetraethylammonium, 4-aminopyridine) or TRPM7 channels (spermine). In summary, sensory neurons express a novel voltage-gated cation channel that is inhibited by external Ca(2+) (IC(50)∼0.5 µM) or Mg(2+) (IC(50)∼3 µM). Activation of this putative channel evokes substantial cation fluxes in sensory neurons.

Dietary flavonoid (-)epicatechin stimulates phosphatidylinositol 3-kinase-dependent anti-oxidant response element activity and up-regulates glutathione in cortical astrocytes.

Flavonoids are plant-derived polyphenolic compounds with neuroprotective properties. Recent work suggests that, in addition to acting as hydrogen donors, they activate protective signalling pathways. The anti-oxidant response element (ARE) promotes the expression of protective proteins including those required for glutathione synthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase and glutathione synthase). The use of a luciferase reporter (ARE-luc) assay showed that the dietary flavan-3-ol (-)epicatechin activates this pathway in primary cortical astrocytes but not neurones. We also examined the distribution of NF-E2-related factor-2 (Nrf2), a key transcription factor in ARE-mediated gene expression. We found, using immunocytochemistry, that Nrf2 accumulated in the nuclei of astrocytes following exposure to tert-butylhydroquinone (100 microM) and (-)epicatechin (100 nM). (-)Epicatechin signalling via Nrf2 was inhibited by wortmannin implicating a phosphatidylinositol 3-kinase-dependent pathway. Finally, (-)epicatechin increased glutathione levels in astrocytes consistent with an up-regulation of ARE-mediated gene expression. Together, this suggests that flavonoids may be cytoprotective by increasing anti-oxidant gene expression.

A functional role for small-conductance calcium-activated potassium channels in sensory pathways including nociceptive processes.

We investigated the role of small-conductance calcium-activated potassium (SK) and intermediate-conductance calcium-activated potassium channels in modulating sensory transmission from peripheral afferents into the rat spinal cord. Subunit-specific antibodies reveal high levels of SK3 immunoreactivity in laminas I, II, and III of the spinal cord. Among dorsal root ganglion neurons, both peripherin-positive (C-type) and peripherin-negative (A-type) cells show intense SK3 immunoreactivity. Furthermore, dorsal root-stimulated sensory responses recorded in vitro are inhibited when SK channel activity is increased with 1-ethyl-2-benzimidazolinone (1-EBIO). In vivo electrophysiological recordings show that neuronal responses to naturally evoked nociceptive and nonnociceptive stimuli increase after application of the selective SK channel blocker 8,14-diaza-1,7(1,4)-diquinolinacyclotetradecaphanedium di-trifluoroacetate (UCL 1848), indicating that SK channels are normally active in moderating afferent input. Conversely, neuronal responses evoked by mechanical stimuli are inhibited when SK channel activity is increased with 1-EBIO. These effects are reversed by the subsequent application of UCL 1848. Our data demonstrate that SK channels have an important role in controlling sensory input into the spinal cord.

The SK3 subunit of small conductance Ca2+-activated K+ channels interacts with both SK1 and SK2 subunits in a heterologous expression system.

The aim of this study was to determine whether functional heteromeric channels can be formed by co-assembly of rat SK3 (rSK3) potassium channel subunits with either SK1 or SK2 subunits. First, to determine whether rSK3 could co-assemble with rSK2 we created rSK3VK (an SK3 mutant insensitive to block by UCL 1848). When rSK3VK was co-expressed with rSK2 the resulting currents had an intermediate sensitivity to UCL 1848 (IC50 of approximately 5 nM compared with 120 pM for rSK2 and >300 nM for rSK3VK), suggesting that rSK3 and rSK2 can form functional heteromeric channels. To detect co-assembly of SK3 with SK1, we initially used a dominant negative construct of the human SK1 subunit (hSK1YP). hSK1YP dramatically reduced the SK3 current, supporting the idea that SK3 and SK1 subunits also interact. To determine whether these assemblies were functional we created rSK3VF, an rSK3 mutant with an enhanced affinity for tetraethylammonium chloride (TEA) (IC50 of 0.3 mM). Co-transfection of rSK3VF and hSK1 produced currents with a sensitivity to TEA not different from that of hSK1 alone (IC50 approximately 15 mM). These results suggest that hSK1 does not produce functional cell-surface assemblies with SK3. Antibody-staining experiments suggested that hSK1 may reduce the number of functional SK3 subunits reaching the cell surface. Additional experiments showed that co-expression of the rat SK1 gene with SK3 also dramatically suppressed SK current. The pharmacology of the residual current was consistent with that of homomeric SK3 assemblies. These results demonstrate interactions that cause changes in protein trafficking, cell surface expression, and channel pharmacology and strongly suggest heteromeric assembly of SK3 with the other SK channel subunits.

Small conductance Ca2+-activated K+ channels formed by the expression of rat SK1 and SK2 genes in HEK 293 cells.

The rat SK1 gene (rSK1) does not form functional Ca2+-activated potassium channels when expressed alone in mammalian cell lines. Using a selective antibody to the rSK1 subunit and a yellow fluorescent protein (YFP) tag we have discovered that rSK1 expression produces protein that remains largely at intracellular locations. We tested the idea that rSK1 may need an expression partner, rSK2, in order to form functional channels. When rSK1 was co-expressed with rSK2 in HEK 293 cells it increased the current magnitude by 77 +/- 34% (as compared with cells expressing rSK2 alone). Co-expression of rSK1 with rSK2 also changed the channel pharmacology. The sensitivity of SK current to block by apamin was reduced approximately 16-fold from an IC50 of 94 pM (for SK2 alone) to 1.4 nM (for SK2 and SK1 together). The sensitivity to block by UCL 1848 (a potent small molecule blocker of SK channels) was similarly reduced, approximately 26-fold, from an IC50 of 110 pM to 2.9 nM. These data clearly demonstrate that rSK1 and rSK2 subunits interact. The most likely explanation for this is that the subunits are able to form heteromeric assemblies.