Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons.

Small-conductance Ca(2+)-activated K(+) channel activation is an emerging therapeutic approach for treatment of neurological diseases, including stroke, amyotrophic lateral sclerosis and schizophrenia. Our previous studies showed that activation of SK channels exerted neuroprotective effects through inhibition of NMDAR-mediated excitotoxicity. In this study, we tested the therapeutic potential of SK channel activation of NS309 (25 µM) in cultured human postmitotic dopaminergic neurons in vitro conditionally immortalized and differentiated from human fetal mesencephalic cells. Quantitative RT-PCR and western blotting analysis showed that differentiated dopaminergic neurons expressed low levels of SK2 channels and high levels of SK1 and SK3 channels. Further, protein analysis of subcellular fractions revealed expression of SK2 channel subtype in mitochondrial-enriched fraction. Mitochondrial complex I inhibitor rotenone (0.5 µM) disrupted the dendritic network of human dopaminergic neurons and induced neuronal death. SK channel activation reduced mitochondrial membrane potential, while it preserved the dendritic network, cell viability and ATP levels after rotenone challenge. Mitochondrial dysfunction and delayed dopaminergic cell death were prevented by increasing and/or stabilizing SK channel activity. Overall, our findings show that activation of SK channels provides protective effects in human dopaminergic neurons, likely via activation of both membrane and mitochondrial SK channels. Thus, SK channels are promising therapeutic targets for neurodegenerative disorders such as Parkinson's disease, where dopaminergic cell loss is associated with progression of the disease.

rKv1.2 overexpression in the central medial thalamic area decreases caffeine-induced arousal.

The voltage-gated potassium channel Kv1.2 belongs to the shaker-related family and has recently been implicated in the control of sleep profile on the basis of clinical and experimental evidence in rodents. To further investigate whether increasing Kv1.2 activity would promote sleep occurrence in rats, we developed an adeno-associated viral vector that induces overexpression of rat Kv1.2 protein. The viral vector was first evaluated in vitro for its ability to overexpress rat Kv1.2 protein and to produce functional currents in infected U2OS cells. Next, the adeno-associated Kv1.2 vector was injected stereotaxically into the central medial thalamic area of rats and overexpression of Kv1.2 was showed by in situ hybridization, ex vivo electrophysiology and immunohistochemistry. Finally, the functional effect of Kv1.2 overexpression on sleep facilitation was investigated using telemetry system under normal conditions and following administration of the arousing agent caffeine, during the light phase. While no differences in sleep profile were observed between the control and the treated animals under normal conditions, a decrease in the pro-arousal effect of caffeine was seen only in the animals injected with the adeno-associated virus-Kv1.2 vector. Overall, our data further support a role of the Kv1.2 channel in the control of sleep profile, particularly under conditions of sleep disturbance.

KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia.

Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca2+]i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca2+]i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (KCa2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of KCa2.2 channels within 3 h after the onset of glutamate exposure. Activation of KCa2 channels preserved KCa2 expression and significantly reduced pathological increases in [Ca2+]i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for KCa2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.

Endocytic trafficking signals in KCNMB2 regulate surface expression of a large conductance voltage and Ca(2+)-activated K+ channel.

Large conductance voltage and calcium-activated K(+) channels play critical roles in neuronal excitability and vascular tone. Previously, we showed that coexpression of the transmembrane beta2 subunit, KCNMB2, with the human pore-forming alpha subunit of the large conductance voltage and Ca(2+)-activated K(+) channel (hSlo) yields inactivating currents similar to those observed in hippocampal neurons [Hicks GA, Marrion NV (1998) Ca(2+)-dependent inactivation of large conductance Ca(2+)-activated K(+) (BK) channels in rat hippocampal neurones produced by pore block from an associated particle. J Physiol (Lond) 508 (Pt 3):721-734; Wallner M, Meera P, Toro L (1999b) Molecular basis of fast inactivation in voltage and Ca(2+)-activated K(+) channels: A transmembrane beta-subunit homolog. Proc Natl Acad Sci U S A 96:4137-4142]. Herein, we report that coexpression of beta2 subunit with hSlo can also modulate hSlo surface expression levels in HEK293T cells. We found that, when expressed alone, beta2 subunit appears to reach the plasma membrane but also displays a distinct intracellular punctuated pattern that resembles endosomal compartments. beta2 Subunit coexpression with hSlo causes two biological effects: i) a shift of hSlo's intracellular expression pattern from a relatively diffuse to a distinct punctated cytoplasmic distribution overlapping beta2 expression; and ii) a decrease of hSlo surface expression that surpassed an observed small decrease in total hSlo expression levels. beta2 Site-directed mutagenesis studies revealed two putative endocytic signals at the C-terminus of beta2 that can control expression levels of hSlo. In contrast, a beta2 N-terminal consensus endocytic signal had no effect on hSlo expression levels. Thus, beta2 subunit not only can influence hSlo currents but also has the ability to limit hSlo surface expression levels via an endocytic mechanism. This new mode of beta2 modulation of hSlo may depend on particular coregulatory mechanisms in different cell types.

An endoplasmic reticulum trafficking signal prevents surface expression of a voltage- and Ca2+-activated K+ channel splice variant.

Protein delivery to restricted plasma membrane domains is exquisitely regulated at different stages of the cell trafficking machinery. Traffic control involves the recognition of export/retention/retrieval signals in the endoplasmic reticulum (ER)/Golgi complex that will determine protein fate. A splice variant (SV), SV1, of the voltage- and Ca(2+)-activated K(+) channel alpha-subunit accumulates the channel in the ER, preventing its surface expression. We show that SV1 insert contains a nonbasic, hydrophobic retention/retrieval motif, CVLF, that does not interfere with proper folding and tetramerization of SV1. Localization of proteins in the ER by CVLF is independent of its position; originally, on the first internal loop, SV1 insert or CVLF perform equally well if placed at the middle or end of the alpha-subunit intracellular carboxyl terminus. Also, CVLF is able to restrict the traffic of an independently expressed transmembrane protein, beta 1-subunit. CVLF is present in proteins across species and in lower organisms. Thus, CVLF may have evolved to serve as a regulator of cellular traffic.

Cerebellar ataxia and Purkinje cell dysfunction caused by Ca2+-activated K+ channel deficiency.

Malfunctions of potassium channels are increasingly implicated as causes of neurological disorders. However, the functional roles of the large-conductance voltage- and Ca(2+)-activated K(+) channel (BK channel), a unique calcium, and voltage-activated potassium channel type have remained elusive. Here we report that mice lacking BK channels (BK(-/-)) show cerebellar dysfunction in the form of abnormal conditioned eye-blink reflex, abnormal locomotion and pronounced deficiency in motor coordination, which are likely consequences of cerebellar learning deficiency. At the cellular level, the BK(-/-) mice showed a dramatic reduction in spontaneous activity of the BK(-/-) cerebellar Purkinje neurons, which generate the sole output of the cerebellar cortex and, in addition, enhanced short-term depression at the only output synapses of the cerebellar cortex, in the deep cerebellar nuclei. The impairing cellular effects caused by the lack of postsynaptic BK channels were found to be due to depolarization-induced inactivation of the action potential mechanism. These results identify previously unknown roles of potassium channels in mammalian cerebellar function and motor control. In addition, they provide a previously undescribed animal model of cerebellar ataxia.

Presynaptic Ca2+-activated K+ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release.

Large-conductance Ca(2+)-activated K(+) channels (BK, also called Maxi-K or Slo channels) are widespread in the vertebrate nervous system, but their functional roles in synaptic transmission in the mammalian brain are largely unknown. By combining electrophysiology and immunogold cytochemistry, we demonstrate the existence of functional BK channels in presynaptic terminals in the hippocampus and compare their functional roles in somata and terminals of CA3 pyramidal cells. Double-labeling immunogold analysis with BK channel and glutamate receptor antibodies indicated that BK channels are targeted to the presynaptic membrane facing the synaptic cleft in terminals of Schaffer collaterals in stratum radiatum. Whole-cell, intracellular, and field-potential recordings from CA1 pyramidal cells showed that the presynaptic BK channels are activated by calcium influx and can contribute to repolarization of the presynaptic action potential (AP) and negative feedback control of Ca(2+) influx and transmitter release. This was observed in the presence of 4-aminopyridine (4-AP, 40-100 microm), which broadened the presynaptic compound action potential. In contrast, the presynaptic BK channels did not contribute significantly to regulation of action potentials or transmitter release under basal experimental conditions, i.e., without 4-AP, even at high stimulation frequencies. This is unlike the situation in the parent cell bodies (CA3 pyramidal cells), where BK channels contribute strongly to action potential repolarization. These results indicate that the functional role of BK channels depends on their subcellular localization.

Potassium channels regulate tone in rat pulmonary veins.

Intrapulmonary veins (PVs) contribute to pulmonary vascular resistance, but the mechanisms controlling PV tone are poorly understood. Although smooth muscle cell (SMC) K(+) channels regulate tone in most vascular beds, their role in PV tone is unknown. We show that voltage-gated (K(V)) and inward rectifier (K(ir)) K(+) channels control resting PV tone in the rat. PVs have a coaxial structure, with layers of cardiomyocytes (CMs) arrayed externally around a subendothelial layer of typical SMCs, thus forming spinchterlike structures. PVCMs have both an inward current, inhibited by low-dose Ba(2+), and an outward current, inhibited by 4-aminopyridine. In contrast, PVSMCs lack inward currents, and their outward current is inhibited by tetraethylammonium (5 mM) and 4-aminopyridine. Several K(V), K(ir), and large-conductance Ca(2+)-sensitive K(+) channels are present in PVs. Immunohistochemistry showed that K(ir) channels are present in PVCMs and PV endothelial cells but not in PVSMCs. We conclude that K(+) channels are present and functionally important in rat PVs. PVCMs form sphincters rich in K(ir) channels, which may modulate venous return both physiologically and in disease states including pulmonary edema.

Developmental expression of the small-conductance Ca(2+)-activated potassium channel SK2 in the rat retina.

Small-conductance Ca(2+)-activated potassium (SK) channels are present in most central neurons, where they mediate the afterhyperpolarizations (AHPs) following action potentials. SK channels integrate changes in intracellular Ca(2+) concentration with membrane potential and thus play an important role in controlling firing pattern and excitability. Here, we characterize the expression pattern of the apamin-sensitive SK subunits, SK2 and SK3, in the developing and adult rat retina using in situ hybridization and immunohistochemistry. The SK2 subunit showed a distinct and developmentally regulated pattern of expression. It appeared during the first postnatal week and located to retinal ganglion cells and to subpopulations of neurons in the inner nuclear layer. These neurons were identified as horizontal cells and dopaminergic amacrine cells by specific markers. In contrast to SK2, the SK3 subunit was detected neither in the developing nor in the adult retina. These results show cell-specific expression of the SK2 subunit in the retina and suggest that this channel underlies the apamin-sensitive AHP currents described in retinal ganglion cells.

Respiration and parturition affected by conditional overexpression of the Ca2+-activated K+ channel subunit, SK3.

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.

Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin.

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0

High affinity interaction of mibefradil with voltage-gated calcium and sodium channels.

Mibefradil is a novel Ca(2+) antagonist which blocks both high-voltage activated and low voltage-activated Ca(2+) channels. Although L-type Ca(2+) channel block was demonstrated in functional experiments its molecular interaction with the channel has not yet been studied. We therefore investigated the binding of [(3)H]-mibefradil and a series of mibefradil analogues to L-type Ca(2+) channels in different tissues. [(3)H]-Mibefradil labelled a single class of high affinity sites on skeletal muscle L-type Ca(2+) channels (K(D) of 2.5+/-0.4 nM, B(max)=56.4+/-2.3 pmol mg(-1) of protein). Mibefradil (and a series of analogues) partially inhibited (+)-[(3)H]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca(2+) channels and alpha1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. [(3)H]-Mibefradil also labelled a heterogenous population of high affinity sites in rabbit brain which was inhibited by a series of nonspecific Ca(2+) and Na(+)-channel blockers. Mibefradil and its analogue RO40-6040 had high affinity for neuronal voltage-gated Na(+)-channels as confirmed in binding (apparent K(i) values of 17 and 1.0 nM, respectively) and functional experiments (40% use-dependent inhibition of Na(+)-channel current by 1 microM mibefradil in GH3 cells). Our data demonstrate that mibefradil binds to voltage-gated L-type Ca(2+) channels with very high affinity and is also a potent blocker of voltage-gated neuronal Na(+)-channels. More lipophilic mibefradil analogues may possess neuroprotective properties like other nonselective Ca(2+)-/Na(+)-channel blockers.

Generating a high affinity scorpion toxin receptor in KcsA-Kv1.3 chimeric potassium channels.

The crystal structure of the bacterial K(+) channel, KcsA (Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77), and subsequent mutagenesis have revealed a high structural conservation from bacteria to human (MacKinnon, R., Cohen, S. L., Kuo, A., Lee, A., and Chait, B. T. (1998) Science 280, 106-109). We have explored this conservation by swapping subregions of the M1-M2 linker of KcsA with those of the S5-S6 linker of the human Kv-channel Kv1.3. The chimeric K(+) channel constructs were expressed in Escherichia coli, and their multimeric state was analyzed after purification. We used two scorpion toxins, kaliotoxin and hongotoxin 1, which bind specifically to Kv1.3, to analyze the pharmacological properties of the KcsA-Kv1.3 chimeras. The results demonstrate that the high affinity scorpion toxin receptor of Kv1.3 could be transferred to KcsA. Our biochemical studies with purified KcsA-Kv1.3 chimeras provide direct chemical evidence that a tetrameric channel structure is necessary for forming a functional scorpion toxin receptor. We have obtained KcsA-Kv1.3 chimeras with kaliotoxin affinities (IC(50) values of approximately 4 pm) like native Kv1.3 channels. Furthermore, we show that a subregion of the S5-S6 linker may be an important determinant of the pharmacological profile of K(+) channels. Using available structural information on KcsA and kaliotoxin, we have developed a structural model for the complex between KcsA-Kv1.3 chimeras and kaliotoxin to aid future pharmacological studies of K(+) channels.

Preparation of thiol-reactive Cy5 derivatives from commercial Cy5 succinimidyl ester.

The present study offers reliable protocols for the preparation of new thiol-reactive Cy5 derivatives which are urgently needed for single molecule fluorescence microscopy. In a systematic approach, two alternate strategies were found for the extension of commercial amine-reactive Cy5 with thiol-reactive end groups. In the two-step method, Cy5 succinimidyl ester was first reacted with ethylenediamine under conditions which gave approximately 99% asymmetric "Cy5-amine" and only approximately 1% symmetric product with two Cy5 residues. Subsequently, "Cy5-amine" was derivatized with commercial heterobifunctional cross-linkers to introduce thiol-reactive end groups (maleimide or pyridyldithio). Alternatively, commercial Cy5 succinimidyl ester was reacted with a primary amine (MTSEA, methanethiosulfonylethylamine, or PDEA, pyridyldithioethylamine) or a secondary amine (PEM, piperazinylethylmaleimide) to give the corresponding thiol-reactive derivatives in a single step. Results were good for MTSEA, moderate for PEM, and poor for PDEA. An additional drawback of the one-step method was the need for rigorous removal of unreacted Cy5 succinimidyl ester, which would label lysine residues on probe molecules. It is concluded that, except for the Cy5-MTSEA conjugate, the two-step method is much more general, reliable, and easier to follow by the typical biophysicist, biologist, etc., for whose benefit, these procedures are being published. All thiol-reactive Cy5 derivatives showed similar absorption and fluorescence properties as Cy5 succinimidyl ester, and fluorescence was fully retained after binding to thiols on proteins. The kinetics of protein labeling was also examined in order to get an idea of proper labeling conditions.

WIN 17317-3, a new high-affinity probe for voltage-gated sodium channels.

The iminodihydroquinoline WIN 17317-3 was previously shown to inhibit selectively the voltage-gated potassium channels, K(v)1.3 and K(v)1.4 [Hill, R. J., et al. (1995) Mol. Pharmacol. 48, 98-104; Nguyen, A., et al. (1996) Mol. Pharmacol. 50, 1672-1679]. Since these channels are found in brain, radiolabeled WIN 17317-3 was synthesized to probe neuronal K(v)1 channels. In rat brain synaptic membranes, [(3)H]WIN 17317-3 binds reversibly and saturably to a single class of high-affinity sites (K(d) 2.2 +/- 0.3 nM; B(max) 5.4 +/- 0.2 pmol/mg of protein). However, the interaction of [(3)H]WIN 17317-3 with brain membranes is not sensitive to any of several well-characterized potassium channel ligands. Rather, binding is modulated by numerous structurally unrelated sodium channel effectors (e.g., channel toxins, local anesthetics, antiarrhythmics, and cardiotonics). The potency and rank order of effectiveness of these agents in affecting [(3)H]WIN 17317-3 binding is consistent with their known abilities to modify sodium channel activity. Autoradiograms of rat brain sections indicate that the distribution of [(3)H]WIN 17317-3 binding sites is in excellent agreement with that of sodium channels. Furthermore, WIN 17317-3 inhibits sodium currents in CHO cells stably transfected with the rat brain IIA sodium channel with high affinity (K(i) 9 nM), as well as agonist-stimulated (22)Na uptake in this cell line. WIN 17317-3 interacts similarly with skeletal muscle sodium channels but is a weaker inhibitor of the cardiac sodium channel. Together, these results demonstrate that WIN 17317-3 is a new, high-affinity, subtype-selective ligand for sodium channels and is a potent blocker of brain IIA sodium channels.

Quantification and distribution of Ca(2+)-activated maxi K(+) channels in rabbit distal colon.

The Ca(2+)-activated maxi K(+) channel is an abundant channel type in the distal colon epithelium, but nothing is known regarding the actual number and precise localization of these channels. The aim of this study has therefore been to quantify the maxi K(+) channels in colon epithelium by binding of iberiotoxin (IbTX), a selective peptidyl ligand for maxi K(+) channels. In isotope flux measurements 75% of the total K(+) channel activity in plasma membranes from distal colon epithelium is inhibited by IbTX (K(0.5) = 4.5 pM), indicating that the maxi K(+) channel is the predominant channel type in this epithelium. Consistent with the functional studies, the radiolabeled double mutant (125)I-IbTX-D19Y/Y36F binds to the colon epithelium membranes with an equilibrium dissociation constant of approximately 10 pM. The maximum receptor concentration values (in fmol/mg protein) for (125)I-IbTX-D19Y/Y36F binding to colon epithelium are 78 for surface membranes and 8 for crypt membranes, suggesting that the maxi K(+) channels are predominantly expressed in the Na(+)-absorbing surface cells, as compared with the Cl(-)-secreting crypt cells. However, aldosterone stimulation of this tissue induced by a low-Na(+) diet does not change the total number of maxi K(+) channels.

High-conductance calcium-activated potassium channels in rat brain: pharmacology, distribution, and subunit composition.

In rat brain, high-conductance Ca2+-activated K+ (BK) channels are targeted to axons and nerve terminals [Knaus, H. G., et al. (1996) J. Neurosci. 16, 955-963], but absolute levels of their regional expression and subunit composition have not yet been fully established. To investigate these issues, an IbTX analogue ([125I]IbTX-D19Y/Y36F) was employed that selectively binds to neuronal BK channels with high affinity (Kd = 21 pM). Cross-linking experiments with [125I]IbTX-D19Y/Y36F in the presence of a bifunctional reagent led to covalent incorporation of radioactivity into a protein with an apparent molecular mass of 25 kDa. Deglycosylation and immunoprecipitation studies with antibodies raised against alpha- and smooth muscle beta-subunits of the BK channel suggest that the beta-subunit that is associated with the neuronal BK channel is a novel protein. Quantitative receptor autoradiography reveals the highest levels of BK channel expression in the outer layers of the neocortex, hippocampal perforant path projections, and the interpeduncular nucleus. This distribution pattern has also been confirmed in immunocytochemical experiments with a BK channel-selective antibody. Taken together, these findings imply that neuronal BK channels exhibit a restricted distribution in brain and have a subunit composition different from those of their smooth muscle congeners.

Scorpion toxins as tools for studying potassium channels.

The search for peptidyl inhibitors of K+ channels is a very active area of investigation. In addition to scorpion venoms, other venom sources have been investigated; all of these sources have yielded novel peptides with interesting properties. For instance, spider venoms have provided peptides that block other families of K+ channels (e.g., Kv2 and Kv4) that act via mechanisms which modify the gating properties of these channels. Such inhibitors bind to a receptor on the channel that is different from the pore region in which the peptides discussed in this chapter bind. In fact, it is possible to have a channel occupied simultaneously by both inhibitor types. It is expected that many of the methodologies concerning peptidyl inhibitors from scorpion venom, which have been developed in the past and outlined above, will be extended to the new families of K+ channel blockers currently under development.

Glucocorticoid regulation of calcium-activated potassium channels mediated by serine/threonine protein phosphatase.

Adrenal glucocorticoids exert powerful effects on cellular excitability in neuroendocrine cells and neurons, although the underlying mechanisms are poorly understood. In metabolically intact mouse anterior pituitary corticotrope (AtT20) cells glucocorticoid-induced proteins render large conductance calcium-activated potassium (BK) channels insensitive to inhibition by protein kinase A (PKA). In this study we have addressed whether this action of glucocorticoids is mediated via protein phosphatase activity at the level of single BK channels. In isolated inside-out patches from control AtT20 cells BK channels (125 pS) were inhibited by activation of closely associated PKA. Pretreatment (2 h) of cells with 1 microM dexamethasone before patch excision did not modify the intrinsic properties or expression levels of BK channel alpha-subunits in AtT20 cells. However, PKA-mediated inhibition of BK channel activity in isolated patches from steroid-treated cells was severely blunted. This effect of steroid was not observed using adenosine 5'-O-(3-thiotriphosphate) as phosphate donor or on exposure of the intracellular face of the patch with 10 nM of the protein phosphatase inhibitors okadaic acid or calyculin A but was mimicked by application of protein phosphatase 2A (PP2A) to the intracellular face of patches from control cells. Glucocorticoids did not modify total PP2A activity in AtT20 cells, suggesting that modified PP2A-like phosphatase activity closely associated with BK channels is required for glucocorticoid action.