Small-conductance Potassium Channels Research Articles

UBE3A Regulates Synaptic Plasticity and Learning and Memory by Controlling SK2 Channel Endocytosis.

Gated solely by activity-induced changes in intracellular calcium, small-conductance potassium channels (SKs) are critical for a variety of functions in the CNS, from learning and memory to rhythmic activity and sleep. While there is a wealth of information on SK2 gating, kinetics, and Ca(2+) sensitivity, little is known regarding the regulation of SK2 subcellular localization. We report here that synaptic SK2 levels are regulated by the E3 ubiquitin ligase UBE3A, whose deficiency results in Angelman syndrome and overexpression in increased risk of autistic spectrum disorder. UBE3A directly ubiquitinates SK2 in the C-terminal domain, which facilitates endocytosis. In UBE3A-deficient mice, increased postsynaptic SK2 levels result in decreased NMDA receptor activation, thereby impairing hippocampal long-term synaptic plasticity. Impairments in both synaptic plasticity and fear conditioning memory in UBE3A-deficient mice are significantly ameliorated by blocking SK2. These results elucidate a mechanism by which UBE3A directly influences cognitive function.

Calcium-Induced Calcium Release during Action Potential Firing in Developing Inner Hair Cells

In the mature auditory system, inner hair cells (IHCs) convert sound-induced vibrations into electrical signals that are relayed to the central nervous system via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium-based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarization, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium-induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, interspike interval, or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behavior is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data show that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights into the calcium signaling mechanisms involved in early developmental processes.

SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx

Neutrophils cast neutrophil extracellular traps (NETs) to defend the host against invading pathogens. Although effective against microbial pathogens, a growing body of literature now suggests that NETs have negative impacts on many inflammatory and autoimmune diseases. Identifying mechanisms that regulate the process termed "NETosis" is important for treating these diseases. Although two major types of NETosis have been described to date, mechanisms regulating these forms of cell death are not clearly established. NADPH oxidase 2 (NOX2) generates large amounts of reactive oxygen species (ROS), which is essential for NOX-dependent NETosis. However, major regulators of NOX-independent NETosis are largely unknown. Here we show that calcium activated NOX-independent NETosis is fast and mediated by a calcium-activated small conductance potassium (SK) channel member SK3 and mitochondrial ROS. Although mitochondrial ROS is needed for NOX-independent NETosis, it is not important for NOX-dependent NETosis. We further demonstrate that the activation of the calcium-activated potassium channel is sufficient to induce NOX-independent NETosis. Unlike NOX-dependent NETosis, NOX-independent NETosis is accompanied by a substantially lower level of activation of ERK and moderate level of activation of Akt, whereas the activation of p38 is similar in both pathways. ERK activation is essential for the NOX-dependent pathway, whereas its activation is not essential for the NOX-independent pathway. Despite the differential activation, both NOX-dependent and -independent NETosis require Akt activity. Collectively, this study highlights key differences in these two major NETosis pathways and provides an insight into previously unknown mechanisms for NOX-independent NETosis.

SK Channel Distribution on Neuronal Axons Revealed by Single Molecule Atomic Force Microscopy

Introduction: The axon initial segment (AIS) is a specialized cellular compartment where action potentials (APs) initiate. Calcium-activated small conductance potassium (SK) channels reduce excitability by mediating the afterhyperpolarizing (AHP) current and thus modulate the intervals between spikes during a burst of action potentials. SK channels have a polarized distribution in neurons with high density in dendrites and lower density in the soma. However, it is not clear if and which SK channels are expressed on the axon and their spatial distribution. Here, we employ single molecule atomic force microscopy (AFM) combined with natural toxins to determine the localization of SK channels on neuronal axons.Methods and Results: We have previously demonstrated that integration of single molecule AFM and toxin pharmacology allows for high resolution mapping of native SK2 channels on soma and dendrites of living neurons. AFM tip functionalized with SK channel blocker-apamin, was used to detect the distribution of SK channels by measuring the unbinding forces between apamin and SK channels. The axon was identified by transfecting rat pyramidal neurons with tau-gfp which localizes at the axon. Apamin-functionalized AFM tips were used to probe 1µm∧2 scan area on the axon of transfected pyramidal neurons. We detected unbinding forces with a mean of µ=20± 9pN and a surface density of 5.9% (n=8). To determine the specificity of the detected unbinding forces, the experiments were repeated with neurons pretreated with apamin. Along with a lower frequency of unbinding events, we found that in neurons pretreated with apamin, there was a decrease in the unbinding forces. The results above indicate that apamin sensitive SK channels reside on the axon of pyramidal neurons.Conclusion: Employing single molecule AFM, we have revealed that SK channels reside on axonal surfaces of neurons.

Bifurcation diagram globally underpinning neuronal firing behaviors modified by SK conductance

Neurons in the brain utilize various firing trains to encode the input signals they have received. Firing behavior of one single neuron is thoroughly explained by using a bifurcation diagram from polarized resting to firing, and then to depolarized resting. This explanation provides an important theoretical principle for understanding neuronal biophysical behaviors. This paper reports the novel experimental and modeling results of the modification of such a bifurcation diagram by adjusting small conductance potassium (SK) channel. In experiments, changes in excitability and depolarization block in nucleus accumbens shell and medium-spiny projection neurons are explored by increasing the intensity of injected current and blocking the SK channels by apamin. A shift of bifurcation points is observed. Then, a Hodgkin—Huxley type model including the main electrophysiological processes of such neurons is developed to reproduce the experimental results. The reduction of SK channel conductance also shifts the bifurcations, which is in consistence with experiment. A global bifurcation paradigm of this shift is obtained by adjusting two parameters, intensity of injected current and SK channel conductance. This work reveals the dynamics underpinning modulation of neuronal firing behaviors by biologically important ionic conductance. The results indicate that small ionic conductance other than that responsible for spike generation can modify bifurcation points and shift the bifurcation diagram and, thus, change neuronal excitability and adaptation.

Disruption of Dopamine Neuron Activity Pattern Regulation through Selective Expression of a Human KCNN3 Mutation

The calcium-activated small conductance potassium channel SK3 plays an essential role in the regulation of dopamine neuron activity patterns. Here we demonstrate that expression of a human disease-related SK3 mutation (hSK3Δ) in dopamine neurons of mice disrupts the balance between tonic and phasic dopamine neuron activity. Expression of hSK3Δ suppressed endogenous SK currents, reducing coupling between SK channels and NMDA receptors (NMDARs) and increasing permissiveness for burst firing. Consistent with enhanced excitability of dopamine neurons, hSK3Δ increased evoked calcium signals in dopamine neurons in vivo and potentiated evoked dopamine release. Specific expression of hSK3Δ led to deficits in attention and sensory gating and heightened sensitivity to a psychomimetic drug. Sensory-motor alterations and psychomimetic sensitivity were recapitulated in a mouse model of transient, reversible dopamine neuron activation. These results demonstrate the cell-autonomous effects of a human ion channel mutation on dopamine neuron physiology and the impact of activity pattern disruption on behavior.

Loss of NR1 Subunit of NMDARs in Primary Sensory Neurons Leads to Hyperexcitability and Pain Hypersensitivity: Involvement of Ca2+-Activated Small Conductance Potassium Channels

It is well established that activation of NMDARs plays an essential role in spinal cord synaptic plasticity (i.e., central sensitization) and pain hypersensitivity after tissue injury. Despite prominent expression of NMDARs in DRG primary sensory neurons, the unique role of peripheral NMDARs in regulating intrinsic neuronal excitability and pain sensitivity is not well understood, in part due to the lack of selective molecular tools. To address this problem, we used Advillin-Cre driver to delete the NR1 subunit of NMDARs selectively in DRG neurons. In NR1 conditional knock-out (NR1-cKO) mice, NR1 expression is absent in DRG neurons but remains normal in spinal cord neurons; NMDA-induced currents are also eliminated in DRG neurons of these mice. Surprisingly, NR1-cKO mice displayed mechanical and thermal hypersensitivity compared with wild-type littermates. NR1-deficient DRG neurons show increased excitability, as indicated by increased frequency of action potentials, and enhanced excitatory synaptic transmission in spinal cord slices, as indicated by increased frequency of miniature EPSCs. This hyperexcitability can be reproduced by the NMDAR antagonist APV and by Ca(2+)-activated slow conductance K(+) (SK) channel blocker apamin. Furthermore, NR1-positive DRG neurons coexpress SK1/SK2 and apamin-sensitive afterhyperpolarization currents are elevated by NMDA and suppressed by APV in these neurons. Our findings reveal the hitherto unsuspected role of NMDARs in controlling the intrinsic excitability of primary sensory neurons possibly via Ca(2+)-activated SK channels. Our results also call attention to potential opposing effects of NMDAR antagonists as a treatment for pain and other neurological disorders.

Abstract 507: G Proteins are Required for Lipoxygenase EDHF Activity of Rabbit Arterial Smooth Muscle Cells

Arachidonic acid 15-lipoxygenase (15-LO) metabolites function as endothelium-derived hyperpolarizing factors in rabbit and human arteries. In rabbit arteries, LO metabolites mediate nitric-oxide and prostaglandin-independent relaxations to acetylcholine and AA. Previously, we characterized 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) as a major vasoactive 15-LO metabolite in rabbit arteries. 11,12,15-THETA requires a specific structure for vascular activity. 11(R),12(S),15(S)-THETA causes concentration-related relaxation whereas 11(R),12(R),15(S)-THETA is without activity. The specific structure requirement suggests a role for a receptor. Therefore, we examined the role of G proteins in 11(R),12(S),15(S)-THETA vascular activity. Western immunoblot verified protein expression of Gαs, Gαi and a Gαo in rabbit endothelial and smooth muscle cells. 11(R),12(S),15(S)-THETA increased GTPγ35S binding to rabbit arterial membranes 280±25% while 11(R),12(S),15(S)-THETA was without effect. In cell-attached patches of rabbit smooth muscle, 11(R),12(S),15(S)-THETA (100 nM) increased mean open time of apamin-sensitive, calcium-activated, small conductance potassium (SK) channels from 0.0001±0.0001 to 0.0015±0.0006. In inside-out patches, 11(R),12(S),15(S)-THETA did not increase channel opening (0.0001±0.0001) unless GTP was present (0.0051±0.0023). In the presence of GTP, an antibody against Gαs and a Gαs inhibitory peptide inhibited 11(R),12(S),15(S)-THETA SK channel activation (0.0007±0.0005, 0.0013±0.0012, respectively) whereas an antibody against Gαi was without effect (0.0042±0.0018). A cell-permeant, penetratin-linked Gαs inhibitory peptide also inhibited 11(R),12(S),15(S)-THETA SK channel activation in cell-attached patches (0.0005±0.0002) and blocked 11(R),12(S),15(S)-THETA relaxations in rabbit aorta (max relaxations = 74±6%, 23±7% for control and permeant peptide, respectively). These studies indicate that 11,12,15-THETA-induced SK channel activation and vascular relaxation are mediated by a Gs-coupled mechanism and that 11,12,15-THETA acts via a stereo-specific G protein coupled receptor/binding site.

Topography of Native SK Channels Revealed by Force Nanoscopy in Living Neurons

The spatial distribution of ion channels is an important determinant of neuronal excitability. However, there are currently no quantitative techniques to map endogenous ion channels with single-channel resolution in living cells. Here, we demonstrate that integration of pharmacology with single-molecule atomic force microscopy (AFM) allows for the high-resolution mapping of native potassium channels in living neurons. We focus on calcium-activated small conductance (SK) potassium channels, which play a critical role in brain physiology. By linking apamin, a toxin that specifically binds to SK channels, to the tip of an AFM cantilever, we are able to detect binding events between the apamin and SK channels. We find that native SK channels from rat hippocampal neurons reside primarily in dendrites as single entities and in pairs. We also show that SK channel dendritic distribution is dynamic and under the control of protein kinase A. Our study demonstrates that integration of toxin pharmacology with single-molecule AFM can be used to quantitatively map individual native ion channels in living cells, and thus provides a new tool for the study of ion channels in cellular physiology.

Increasing SK2 channel activity impairs associative learning

Enhanced intrinsic neuronal excitability of hippocampal pyramidal neurons via reductions in the postburst afterhyperpolarization (AHP) has been hypothesized to be a biomarker of successful learning. This is supported by considerable evidence that pharmacologic enhancement of neuronal excitability facilitates learning. However, it has yet to be demonstrated that pharmacologic reduction of neuronal excitability restricted to the hippocampus can retard acquisition of a hippocampus-dependent task. Thus, the present study was designed to address this latter point using a small conductance potassium (SK) channel activator NS309 focally applied to the dorsal hippocampus. SK channels are important contributors to intrinsic excitability, as measured by the medium postburst AHP. NS309 increased the medium AHP and reduced excitatory postsynaptic potential width of CA1 neurons in vitro. In vivo, NS309 reduced the spontaneous firing rate of CA1 pyramidal neurons and impaired trace eyeblink conditioning in rats. Conversely, trace eyeblink conditioning reduced levels of SK2 channel mRNA and protein in the hippocampus. Therefore, the present findings indicate that modulation of SK channels is an important cellular mechanism for associative learning and further support postburst AHP reductions in hippocampal pyramidal neurons as a biomarker of successful learning.

Thalamic Burst Firing Propensity: A Comparison of the Dorsal Lateral Geniculate and Pulvinar Nuclei in the Tree Shrew

Relay neurons in dorsal thalamic nuclei can fire high-frequency bursts of action potentials that ride the crest of voltage-dependent transient (T-type) calcium currents [low-threshold spike (LTS)]. To explore potential nucleus-specific burst features, we compared the membrane properties of dorsal lateral geniculate nucleus (dLGN) and pulvinar nucleus relay neurons using in vitro whole-cell recording in juvenile and adult tree shrew (Tupaia) tissue slices. We injected current ramps of variable slope into neurons that were sufficiently hyperpolarized to de-inactivate T-type calcium channels. In a small percentage of juvenile pulvinar and dLGN neurons, an LTS could not be evoked. In the remaining juvenile neurons and in all adult dLGN neurons, a single LTS could be evoked by current ramps. However, in the adult pulvinar, current ramps evoked multiple LTSs in >70% of recorded neurons. Using immunohistochemistry, Western blot techniques, unbiased stereology, and confocal and electron microscopy, we found that pulvinar neurons expressed more T-type calcium channels (Ca(v) 3.2) and more small conductance potassium channels (SK2) than dLGN neurons and that the pulvinar nucleus contained a higher glia-to-neuron ratio than the dLGN. Hodgkin-Huxley-type compartmental models revealed that the distinct firing modes could be replicated by manipulating T-type calcium and SK2 channel density, distribution, and kinetics. The intrinsic properties of pulvinar neurons that promote burst firing in the adult may be relevant to the treatment of conditions that involve the adult onset of aberrant thalamocortical interactions.

Hydrogen Sulfide as Endothelium-Derived Hyperpolarizing Factor Sulfhydrates Potassium Channels

Nitric oxide, the classic endothelium-derived relaxing factor (EDRF), acts through cyclic GMP and calcium without notably affecting membrane potential. A major component of EDRF activity derives from hyperpolarization and is termed endothelium-derived hyperpolarizing factor (EDHF). Hydrogen sulfide (H(2)S) is a prominent EDRF, since mice lacking its biosynthetic enzyme, cystathionine γ-lyase (CSE), display pronounced hypertension with deficient vasorelaxant responses to acetylcholine. The purpose of this study was to determine if H(2)S is a major physiological EDHF. We now show that H(2)S is a major EDHF because in blood vessels of CSE-deleted mice, hyperpolarization is virtually abolished. H(2)S acts by covalently modifying (sulfhydrating) the ATP-sensitive potassium channel, as mutating the site of sulfhydration prevents H(2)S-elicited hyperpolarization. The endothelial intermediate conductance (IK(Ca)) and small conductance (SK(Ca)) potassium channels mediate in part the effects of H(2)S, as selective IK(Ca) and SK(Ca) channel inhibitors, charybdotoxin and apamin, inhibit glibenclamide-insensitive, H(2)S-induced vasorelaxation. H(2)S is a major EDHF that causes vascular endothelial and smooth muscle cell hyperpolarization and vasorelaxation by activating the ATP-sensitive, intermediate conductance and small conductance potassium channels through cysteine S-sulfhydration. Because EDHF activity is a principal determinant of vasorelaxation in numerous vascular beds, drugs influencing H(2)S biosynthesis offer therapeutic potential.

Small conductance potassium channels drive ATP-activated chloride secretion in the oviduct

The molecular mechanisms controlling fluid secretion within the oviduct have yet to be determined. As in other epithelia, both secretory and absorptive pathways are likely to work in tandem to drive appropriate ionic movement to support fluid movement across the oviduct epithelium. This study explored the role of potassium channels in basolateral extracellular ATP (ATP(e))-stimulated ion transport in bovine oviduct epithelium using the Ussing chamber short-circuit current (I(SC)) technique. Basal I(SC) in bovine oviduct epithelium comprises both chloride secretion and sodium absorption and was inhibited by treatment with basolateral K(+) channel inhibitors tetrapentlyammonium chloride (TPeA) or BaCl(2). Similarly, ATP-stimulated chloride secretion was significantly attenuated by pretreatment with BaCl(2,) tetraethylammonium (TEA), tolbutamide, and TPeA. Basolateral K(+) current, isolated using nystatin-perforation technique, was rapidly activated by ATP(e), and pretreatment of monolayers with thapsigargin or TPeA abolished this ATP-stimulated K(+) current. To further investigate the type of K(+) channel involved in the ATP response in the bovine oviduct, a number of specific Ca(2+)-activated K(+) channel inhibitors were tested on the ATP-induced ΔI(SC) in intact monolayers. Charbydotoxin, (high conductance and intermediate conductance inhibitor), or paxilline, (high conductance inhibitor) did not significantly alter the ATP(e) response. However, pretreatment with the small conductance inhibitor apamin resulted in a 60% reduction in the response to ATP(e). The presence of small conductance family member KCNN3 was confirmed by RT-PCR and immunohistochemistry. Measurements of intracellular calcium using Fura-2 spectrofluorescence imaging revealed the ability of ATP(e) to increase intracellular calcium in a phospholipase C-inositol 1,4,5-trisphosphate pathway-sensitive manner. In conclusion, these results provide strong evidence that purinergic activation of a calcium-dependent, apamin-sensitive potassium conductance is essential to promote chloride secretion and thus fluid formation in the oviduct.

Recognition of Voltage-Dependent Sodium Channels by Calmodulin

Voltage-dependent or voltage-gated sodium channels (VDSC, VGSC, NaV1.x) control essential processes in muscle cells and neurons. Associated channelopathies include Dravet syndrome, epilepsy, Long QT syndrome 3, ventricular fibrillation, familial autism, pain insensitivity, and defects in the generation and propagation of action potentials. Calmodulin (CaM), a eukaryotic calcium sensor, regulates sodium channels by binding to the intracellular C-terminal region of the pore-forming alpha subunit. Thermodynamic analysis and high resolution structural (NMR) studies focusing on how apo (calcium-depleted) and calcium-saturated 13C-15N-CaM recognize an IQ-motif (IQxxxBGxxxB, B=K,R) located in the tail of NaV1.2 will be presented. These will be compared to studies of how CaM recognizes other sodium channels, unconventional myosin V (2IX7.pdb), and the small conductance potassium channel (1G4Y.pdb). NIH R01 GM57001.

Antecedent hydrogen sulfide elicits an anti-inflammatory phenotype in postischemic murine small intestine: role of BK channels

The objectives of this study were to determine the role of calcium-activated, small (SK), intermediate (IK), and large (BK) conductance potassium channels in initiating the development of an anti-inflammatory phenotype elicited by preconditioning with an exogenous hydrogen sulfide (H(2)S) donor, sodium hydrosulfide (NaHS). Intravital microscopy was used to visualize rolling and firmly adherent leukocytes in vessels of the small intestine of mice preconditioned with NaHS (in the absence and presence of SK, IK, and BK channel inhibitors, apamin, TRAM-34, and paxilline, respectively) or SK/IK (NS-309) or BK channel activators (NS-1619) 24 h before ischemia-reperfusion (I/R). I/R induced marked increases in leukocyte rolling and adhesion, effects that were largely abolished by preconditioning with NaHS, NS-309, or NS-1619. The postischemic anti-inflammatory effects of NaHS-induced preconditioning were mitigated by BKB channel inhibitor treatment coincident with NaHS, but not by apamin or TRAM-34, 24 h before I/R. Confocal imaging and immunohistochemistry were used to demonstrate the presence of BKα subunit staining in both endothelial and vascular smooth muscle cells of isolated, pressurized mesenteric venules. Using patch-clamp techniques, we found that BK channels in cultured endothelial cells were activated after exposure to NaHS. Bath application of the same concentration of NaHS used in preconditioning protocols led to a rapid increase in a whole cell K(+) current; specifically, the component of K(+) current blocked by the selective BK channel antagonist iberiotoxin. The activation of BK current by NaHS could also be demonstrated in single channel recording mode where it was independent of a change in intracellular Ca(+) concentration. Our data are consistent with the concept that H(2)S induces the development of an anti-adhesive state in I/R in part mediated by a BK channel-dependent mechanism.

Differential Involvement of Potassium Channel Subtypes in Early and Late Sepsis-induced Hyporesponsiveness to Vasoconstrictors

This study investigated the involvement of potassium channel subtypes in the hyporesponsiveness to vasoconstrictors of an experimental model of sepsis [cecal ligation and puncture (CLP)], at 2 time points, namely, 6 and 24 hours after sepsis onset. Wistar rats were submitted to CLP or sham surgery, and 6 and 24 hours later, responses to phenylephrine were obtained before and 30 minutes after injection of potassium channel blockers. The potassium channel blockers used were tetraethylammonium (TEA; a nonselective channel blocker), glibenclamide (GLB; an adenosine triphosphate -dependent channel blocker), 4-aminopyridine (4-AP; a voltage-dependent channel blocker), apamin (APA; a small-conductance calcium-dependent channel blocker), and iberiotoxin (IBTX; a large-conductance calcium-dependent channel blocker). It was found that (1) sepsis caused a severe vascular hyporesponsiveness to phenylephrine both 6 and 24 hours after CLP, (2) TEA partially reversed the hyporesponsiveness 6 hours after CLP and completely restored vascular response to phenylephrine 24 hours after CLP, (3) apamin reversed hyporesponsiveness 6 but not 24 hours after CLP, (4) GLB restored phenylephrine response only 24 hours after CLP, and (5) IBTX and 4-AP were ineffective in all periods studied. Our results suggest that potassium channels are important effectors of sepsis-induced vascular dysfunction in vivo and that different subtypes of potassium channels are involved in early (small-conductance calcium-dependent potassium channels) and late (adenosine triphosphate -dependent potassium channels) hyporesponsiveness to vasoconstrictors caused by sepsis.

Constitutive Expression of the α10 Nicotinic Acetylcholine Receptor Subunit Fails to Maintain Cholinergic Responses in Inner Hair Cells After the Onset of Hearing

Efferent inhibition of cochlear hair cells is mediated by alpha9alpha10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express alpha10 into adulthood by expressing the alpha10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the alpha10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 alpha10 transgenic mice backcrossed to a Chrna10(-/-) background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the alpha10 transgene restored nAChR function. However, in the alpha10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.

Expression of the SK2 calcium‐activated potassium channel is required for cholinergic function in mouse cochlear hair cells

Efferent inhibition of cochlear hair cells is mediated by 'nicotinic' cholinergic receptors functionally coupled to calcium-activated, small conductance (SK2) potassium channels. We recorded from cochlear hair cells in SK2 knockout mice to evaluate further the role of this channel in efferent function. Since cholinergic inhibitory synapses can be found on inner or outer hair cells, depending on developmental age, both cell types were studied. To determine if SK channel activity was indeed eliminated, seconds-long voltage-gated calcium influx was used to activate slowly rising and falling calcium-dependent potassium currents. These were identified as SK currents by their time course, calcium dependence and sensitivity to block by apamin in wild-type IHCs. IHCs from knockout mice had no SK current by these same criteria. Thus, the SK2 gene is solely responsible for encoding the SK channels of inner hair cells. Other aspects of hair cell excitability remained relatively unaffected. Unexpectedly, cholinergic synaptic currents were entirely absent from both inner and outer SK2-knockout hair cells. Further, direct application of ACh caused no change in membrane current, implying absent or otherwise dysfunctional ACh receptors. Immunohistology of whole-mounts using the antibody to the synaptic vesicle protein 2 (SV2) revealed a pronounced reduction of efferent innervation to outer hair cells (OHCs) in the knockout cochleas. Quantitative RT-PCR analysis, however, showed no change in the mRNA levels of alpha9 and alpha10 nicotinic ACh receptor (nAChR) genes. Thus, some aspect of translation or subsequent protein processing leads to non-functional or absent ACh receptors. These results indicate that SK2 channels are required both for expression of functional nAChRs, and for establishment and/or maintenance of efferent terminals in the cochlea.

A simplified model of dopaminergic neuron

A minimal model of a dopamine cell in vitro in the pres-ence of tetrodoxin (TTX) and tetraethyl ammonium (TEA)was developed to simulate two types of subthresholdoscillations: the slow oscillatory potential (SOP – see Fig-ure 1A) and the square wave (Figure 1B) that can be pro-duced by the abolition of the SOP by apamin. The L-typecalcium current drives both types of oscillation. The smallconductance potassium channel repolarizes the SOP, andwe propose that the ether-a-go-go related potassium chan-nel repolarizes the square wave. We further suggest thatthe role of this current in vivo is to relieve depolarizationblock. The model predicts that blocking the ether-a-go-gocurrent will elongate the plateau of the square wave andblocking the hyperpolarization-activated potassium cur-rent shortens the period of oscillations. In addition, themodel predicts that blocking the ether-a-go-go currentwill speed up the SOP. The model also predicts that cal-cium chelation converts SOPs into square waves by block-ing the increase in the small conductance (SK) current.Multiple-parameter bifurcation diagrams were used toinvestigate the properties of the firing patterns generatedby our model (Figure 2).

T1386 Effect of Small and Intermediate Conductance Potassium Channel Modulators On Visceral Hypersensitivity and Function

T1386 Effect of Small and Intermediate Conductance Potassium Channel Modulators On Visceral Hypersensitivity and Function