Heteromultimeric delayed-rectifier K+ channels in schwann cells: developmental expression and role in cell proliferation

Schwann cells (SCs) are responsible for myelination of nerve fibers in the peripheral nervous system. Voltage-dependent K+ currents, including inactivating A-type (KA), delayed-rectifier (KD), and inward-rectifier (KIR) K+ channels, constitute the main conductances found in SCs. Physiological studies have shown that KD channels may play an important role in SC proliferation and that they are downregulated in the soma as proliferation ceases and myelination proceeds. Recent studies have begun to address the molecular identity of K+ channels in SCs. Here, we show that a large repertoire of K+ channel alpha subunits of the Shaker (Kv1.1, Kv1.2, Kv1.4, and Kv1.5), Shab (Kv2.1), and Shaw (Kv3.1b and Kv3.2) families is expressed in mouse SCs and sciatic nerve. We characterized heteromultimeric channel complexes that consist of either Kv1.5 and Kv1.2 or Kv1.5 and Kv1.4. In postnatal day 4 (P4) sciatic nerve, most of the Kv1.2 channel subunits are involved in heteromultimeric association with Kv1.5. Despite the presence of Kv1. 1 and Kv1.2 alpha subunits, the K+ currents were unaffected by dendrotoxin I (DTX), suggesting that DTX-sensitive channel complexes do not account substantially for SC KD currents. SC proliferation was found to be potently blocked by quinidine or 4-aminopyridine but not by DTX. Consistent with previous physiological studies, our data show that there is a marked downregulation of all KD channel alpha subunits from P1-P4 to P40 in the sciatic nerve. Our results suggest that KD currents are accounted for by a complex combinatorial activity of distinct K+ channel complexes and confirm that KD channels are involved in SC proliferation.     

Regional and cellular expression patterns of four K+ channel mRNAs in the adult rat brain

Potassium (K+) channels are involved in the modulation and fine tuning of the excitable properties of neurons and glia in the nervous system. In the present report, in situ hybridization histochemistry was used to determine the regional and cellular distribution patterns in the adult rat brain of four mRNAs encoding subunits of voltage-gated K+ channels. These are Kv1.1, Kv1.6, K13 and IK8. All K+ channels examined showed distinct yet overlapping expression patterns. Expression of Kv1.1 mRNA was high in cells of certain motor-related structures of the brainstem. Kv1.6 mRNA expression was observed in cerebellar Purkinje cells and in various olfactory and amygdaloid structures. K13 was the only mRNA expressed in both neuronal and non-neuronal cell populations, including the cells of choroid plexus and pia. IK8 expression was observed only in the forebrain structures. In many brain regions, mRNAs for Kv1.1 and Kv1.6, both encoding K+ channel subunits belonging to the Shaker subfamily, were co-expressed, a necessary condition for heteromultimer formation.     

Hypomyelination alters K+ channel expression in mouse mutants shiverer and Trembler

Voltage-gated K+ channels are localized to juxtaparanodal regions of myelinated axons. To begin to understand the role of normal compact myelin in this localization, we examined mKv1.1 and mKv1.2 expression in the dysmyelinating mouse mutants shiverer and Trembler. In neonatal wild-type and shiverer mice, the focal localization of both proteins in axon fiber tracts is similar, suggesting that cues other than mature myelin can direct initial K+ channel localization in shiverer mutants. In contrast, K+ channel localization is altered in hypomyelinated axonal fiber tracts of adult mutants, suggesting that abnormal myelination leads to channel redistribution. In shiverer adult, K+ channel expression is up-regulated in both axons and glia, as revealed by immunocytochemistry, RNase protection, and in situ hybridization studies. This up-regulation of K+ channels in hypomyelinated axon tracts may reflect a compensatory reorganization of ionic currents, allowing impulse conduction to occur in these dysmyelinating mouse mutants.     

Association and colocalization of the Kvbeta1 and Kvbeta2 beta-subunits with Kv1 alpha-subunits in mammalian brain K+ channel complexes

The differential expression and association of cytoplasmic beta-subunits with pore-forming alpha-subunits may contribute significantly to the complexity and heterogeneity of voltage-gated K+ channels in excitable cells. Here we examined the association and colocalization of two mammalian beta-subunits, Kvbeta1 and Kvbeta2, with the K+ channel alpha-subunits Kv1.1, Kv1.2, Kv1.4, Kv1.6, and Kv2.1 in adult rat brain. Reciprocal coimmunoprecipitation experiments using subunit-specific antibodies indicated that Kvbeta1 and Kvbeta2 associate with all the Kv1 alpha-subunits examined, and with each other, but not with Kv2.1. A much larger portion of the total brain pool of Kv1-containing channel complexes was found associated with Kvbeta2 than with Kvbeta1. Single- and multiple-label immunohistochemical staining indicated that Kvbeta1 codistributes extensively with Kv1.1 and Kv1.4 in cortical interneurons, in the hippocampal perforant path and mossy fiber pathways, and in the globus pallidus and substantia nigra. Kvbeta2 codistributes extensively with Kv1.1 and Kv1.2 in all brain regions examined and was strikingly colocalized with these alpha-subunits in the juxtaparanodal region of nodes of Ranvier as well as in the axons and terminals of cerebellar basket cells. Taken together, these data provide a direct demonstration that Kvbeta1 and Kvbeta2 associate and colocalize with Kv1 alpha-subunits in native tissues and provide a biochemical and neuroanatomical basis for the differential contribution of Kv1 alpha- and beta-subunits to electrophysiologically diverse neuronal K+ currents.     

Cyclic AMP regulates potassium channel expression in C6 glioma by destabilizing Kv1.1 mRNA

The tissue distributions and physiological properties of a variety of cloned voltage-gated potassium channel genes have been characterized extensively, yet relatively little is known about the mechanisms controlling expression of these genes. Here, we report studies on the regulation of Kv1.1 expressed endogenously in the C6 glioma cell line. We demonstrate that elevation of intracellular cAMP leads to the accelerated degradation of Kv1.1 RNA. The cAMP-induced decrease in Kv1.1 RNA is followed by a decrease in Kv1. 1 protein and a decrease in the whole cell sustained K+ current amplitude. Dendrotoxin-I, a relatively specific blocker of Kv1.1, blocks 96% of the sustained K+ current in glioma cells, causing a shift in the resting membrane potential from -40 mV to -7 mV. These data suggest that expression of Kv1.1 contributes to setting the resting membrane potential in undifferentiated glioma cells. We therefore suggest that receptor-mediated elevation of cAMP reduces outward K+ current density by acting at the translational level to destabilize Kv1.1 RNA, an additional mechanism for regulating potassium channel gene expression.     

Susceptibility of cloned K+ channels to reactive oxygen species

Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states.     

Activation of a metabotropic glutamate receptor and protein kinase C reduce the extent of inactivation of the K+ channel Kv1.1/Kvbeta1.1 via dephosphorylation of Kv1.1

Various brain K+ channels, which may normally exist as complexes of alpha (pore-forming) and beta (auxiliary) subunits, were subjected to regulation by metabotropic glutamate receptors. Kv1.1/Kvbeta1.1 is a voltage-dependent K+ channel composed of alpha and beta proteins that are widely expressed in the brain. Expression of this channel in Xenopus oocytes resulted in a current that had fast inactivating and noninactivating components. Previously we showed that basal and protein kinase A-induced phosphorylation of the alpha subunit at Ser-446 decreases the fraction of the noninactivating component. In this study we investigated the effect of protein kinase C (PKC) on the channel. We showed that a PKC-activating phorbol ester (phorbol 12-myristate 13-acetate (PMA)) increased the noninactivating fraction via activation of a PKC subtype that was inhibited by staurosporine and bisindolylmaleimide but not by calphostin C. However, it was not a PKC-induced phosphorylation but rather a dephosphorylation that mediated the effect. PMA reduced the basal phosphorylation of Ser-446 significantly in plasma membrane channels and failed to affect the inactivation of channels having an alpha subunit that was mutated at Ser-446. Also, the activation of coexpressed mGluR1a known to activate phospholipase C mimicked the effect of PMA on the inactivation via induction of dephosphorylation at Ser-446. Thus, this study identified a potential neuronal pathway initiated by activation of metabotropic glutamate receptor 1a coupled to a signaling cascade that possibly utilized PKC to induce dephosphorylation and thereby to decrease the extent of inactivation of a K+ channel.     

Toxin and subunit specificity of blocking affinity of three peptide toxins for heteromultimeric, voltage-gated potassium channels expressed in Xenopus oocytes

The ability of voltage-gated potassium channel alpha-subunits to form heteromultimers has complicated efforts to use toxins to characterize potassium channels in native cells. Here I investigate the effects of subunit composition on toxin blocking affinity, using three members of the Shaker subfamily of potassium channel alpha-subunits (Kv1.1, Kv1.2 and Kv1.4), which are known to form heteromultimers in vivo, in the Xenopus oocyte expression system. These subunits were coexpressed as pairs in which one member was toxin-sensitive and the other relatively insensitive. The blocking affinity of two dendrotoxins (DTX-I and delta-DTX) and a scorpion toxin (tityustoxin-Kalpha) on the resulting mixed population of channels was measured to evaluate three models of toxin block. The single subunit model, in which a single toxin-sensitive subunit renders the channel toxin sensitive, best described all of the data for the two dendrotoxins and the block of tityustoxin-Kalpha for a mixed population of channels composed of Kv1.1 and Kv1.2 subunits. However, with tityustoxin-Kalpha, the data for a mixed population of Kv1.2 and Kv1.4 subunits was fit best by a model in which the toxin interacts with all four subunits for high-affinity block. The data suggest that subunit composition of potassium channels can have a large effect on toxin block and that different toxins yield strikingly diverse results with the same pair of subunits, even when they are nearly identical in blocking affinity for the toxin-sensitive subunit.     

Specific block of cloned Herg channels by clofilium and its tertiary analog LY97241

The class III antiarrhythmic drug clofilium is known to block diverse delayed rectifier K+ channels at micromolar concentrations. In the present study we investigated the potency of clofilium and its tertiary analog LY97241 to inhibit K+ channels, encoded by the human ether-a-go-go related gene (HERG). Clofilium blocked HERG channels in a voltage-dependent fashion with an IC50 of 250 nM and 150 nM at 0 and +40 mV, respectively. LY97241 was almost 10-fold more potent (IC50 of 19 nM at +40 mV). Other cloned K+ channels which are also expressed in cardiac tissue, Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv4.2, Kir2.1, or I(Ks), were not affected by 100-fold higher concentrations. Block of HERG channels by LY97241 was voltage dependent and the rate of HERG inactivation was increased by LY97241. A rise of [K+]0 decreased both, rate of HERG inactivation and LY97241 affinity. The HERG S631A and S620T mutant channels which have a strongly reduced degree of inactivation were 7-fold and 33-fold less sensitive to LY97241 blockade, indicating that LY97241 binding is affected by HERG channel inactivation. In summary, the antiarrhythmic action of clofilium and its analog LY97241 appears to be caused by their potent, but distinct ability for blocking HERG channels.     

K+ channel modulation in arterial smooth muscle

Potassium channels play an essential role in the membrane potential of arterial smooth muscle, and also in regulating contractile tone. Four types of K+ channel have been described in vascular smooth muscle: Voltage-activated K+ channels (Kv) are encoded by the Kv gene family, Ca(2+)-activated K+ channels (BKCa) are encoded by the slo gene, inward rectifiers (KIR) by Kir2.0, and ATP-sensitive K+ channels (KATP) by Kir6.0 and sulphonylurea receptor genes. In smooth muscle, the channel subunit genes reported to be expressed are: Kv1.0, Kv1.2, Kv1.4-1.6, Kv2.1, Kv9.3, Kv beta 1-beta 4, slo alpha and beta, Kir2.1, Kir6.2, and SUR1 and SUR2. Arterial K+ channels are modulated by physiological vasodilators, which increase K+ channel activity, and vasoconstrictors, which decrease it. Several vasodilators acting at receptors linked to cAMP-dependent protein kinase activate KATP channels. These include adenosine, calcitonin gene-related peptide, and beta-adrenoceptor agonists. beta-adrenoceptors can also activate BKCa and Kv channels. Several vasoconstrictors that activate protein kinase C inhibit KATP channels, and inhibition of BKCa and Kv channels through PKC has also been described. Activators of cGMP-dependent protein kinase, in particular NO, activate BKCa channels, and possibly KATP channels. Hypoxia leads to activation of KATP channels, and activation of BKCa channels has also been reported. Hypoxic pulmonary vasoconstriction involves inhibition of Kv channels. Vasodilation to increased external K+ involves KIR channels. Endothelium-derived hyperpolarizing factor activates K+ channels that are not yet clearly defined. Such K+ channel modulations, through their effects on membrane potential and contractile tone, make important contributions to the regulation of blood flow.     

Radiation therapy of esophageal carcinoma: a report on 180 cases]

To determine whether the charybdotoxin-sensitive subtypes of voltage-gated K+ channels (Kv1.2 and Kv1.3) exist in inhibitory pre-synaptic terminals, effects of K+ channel blockers including TEA, charybdotoxin (ChTX), iberiotoxin (IbTX), kaliotoxin (KTX) and margatoxin (MgTX) on the inhibitory transmission were examined with cultured rat hippocampal neurons. Monosynaptic inhibitory postsynaptic currents (IPSCs) evoked by electrical stimulation of single presynaptic neurons were recorded from the whole-cell clamped postsynaptic neurons. In the presence of TEA, application of ChTX greatly increased the amplitude of IPSCs. A specific maxi-K+ channel blocker IbTX failed to augment IPSCs. KTX and MgTX, both of which block Kv1.3 but not Kv1.2, mimicked the facilitating effect of ChTX. In the absence of TEA, application of ChTX increased the IPSC amplitude significantly, while IbTX was without effect. These results indicate that the ChTX-sensitive subtypes of voltage-gated K+ channels, most likely Kv1.3, contribute to the repolarization of action potentials at presynaptic terminals of hippocampal inhibitory neurons, and that the ChTX-induced facilitation of the transmission can be explained by its effects on the Kv channels rather than maxi-K+ channels.     

Modifications of current properties by expression of a foreign potassium channel gene in Xenopus embryonic cells

The development of excitable cells is characterized by highly organized patterns of expression of ion channels. During the terminal differentiation of Xenopus muscle somites, potassium currents are expressed first just after Stage 15 (early-mid neurula), following a long period during which no voltage-dependent currents can be detected in any cell in the dorsal embryo. We have investigated whether early expression of a foreign delayed rectifier potassium channel may affect this endogenous pattern of electrical development. We injected the purified cRNA of the mammalian brain Shaker-like potassium channel, Kv1.1, into fertilized Xenopus eggs. The resulting currents were analyzed in blastomeres during a 12-hr period prior to Stage 15 and in differentiating muscle cells after Stage 15. In injected embryos, a high fraction of blastomeres expressed a delayed rectifier-type current. The Kv1.1 current could be distinguished from the endogenous muscle delayed potassium current (IK,X) by its very different voltage dependence. Separation of currents based on this difference indicated that, in injected embryos, IK,X appeared much earlier in development than in control embryos. Furthermore, even in cells which expressed solely Kv1.1-type current, the sensitivity of the current to dendrotoxin declined dramatically during development, approaching that of IK,X. These data suggest an interaction between Kv1.1 and endogenous channel subunits, and/or modification of the Kv1.1 protein by the embryonic cells in ways not seen in Xenopus oocytes or mammalian cell lines.     

A snake toxin inhibitor of inward rectifier potassium channel ROMK1

Mamba snake dendrotoxins have been used extensively in biochemical and physiological studies of K+ channels of the brain. Their known targets of inhibition have been limited to the family of voltage-gated K+ channels. We report the isolation of a dendrotoxin inhibitor of ROMK1, a channel belonging to the inward rectifier family of K+ channels. The inhibitory activity, fractionated to purity with FPLC and HPLC, is identical to a previously identified delta-dendrotoxin. To verify that delta-dendrotoxin blocks ROMK1 channels, a cDNA encoding the toxin was synthesized and recombinant toxin expressed in Escherichia coli. Electrophysiological recordings reveal that recombinant delta-dendrotoxin has a half-maximal inhibition constant (Kd) of 150 nM when applied to ROMK1 channels expressed in Xenopus laevis oocytes. That the delta-dendrotoxin binding site exists on separate K+ channel classes is shown by its high affinity for two of the voltage-gated family of channels, Kv1.1 (Kd < 0.1 nM) and Kv1.6 (Kd = 23 nM). Single amino acid substitutions in ROMK1 indicate that delta-dendrotoxin binds to the pore region of ROMK1 even though it does not completely block conduction through the pore. These results suggest that dendrotoxins inhibit K+ channels by recognizing the structurally conserved pore region of these channels.     

Differential K+ channel clustering activity of PSD-95 and SAP97, two related membrane-associated putative guanylate kinases

The molecular mechanisms underlying the clustering and localization of K+ channels in specific microdomains on the neuronal surface are largely unknown. The Shaker subclass of voltage-gated K+ channel alpha-subunits interact through their cytoplasmic C-terminus with a family of membrane-associated putative guanylate kinases, including PSD-95 and SAP97. We show here that heterologous coexpression of either sap97 or PSD-95 with various Shaker-type subunits results in the coclustering of these proteins with the K+ channels. Mutation of the C-terminal sequence (-ETDV) of the Shaker subunit Kv1.4 abolishes its binding to, and prevents its clustering with, SAP97 and PSD-95. Whereas PSD-95 induces plaque-like clusters of K+ channels at the cell surface; however, SAP97 coexpression results in the formation of large round intracellular aggregates into which both SAP97 and the K+ channel proteins are colocalized. The efficiency of surface clustering by PSD-95 varies with different Shaker subunits: striking Kv1.4 clustering occurs in > 60% of cotransfected cells, whereas Kv1.1 and Kv1.2 form convincing clusters with PSD-95 only in approximately 10% of cells.     

Structural and functional characterization of human potassium channel subunit beta 1 (KCNA1B)

Voltage-activated Shaker-related potassium channels (kv1) consist of alpha and beta subunits. We have analysed the structure of the human KCNA1B (hKv beta 1) gene. KCNA1B is > 250 kb in size and encodes at least three Kv beta 1 splice variants. The Kv beta 1 open reading frame is divided into 14 exons. In contrast, genes coding for family members of KCNA (Kv 1 alpha) subunits are markedly smaller and have intronless open reading frames. The expression of Kv 1 alpha and Kv beta mRNA was compared in Northern blots of poly(A+) RNA isolated from various human brain tissues. The results suggest an intricate and cell-specific regulation of Kv 1 alpha and Kv beta mRNA synthesis such that distinct combinations of alpha and beta subunits would occur in different nuclei of the brain. The splice variants hKv beta 1.1 and hKv beta 1.2 were functionally characterized in coexpression studies with hKv 1.5 alpha subunits in 293 cells. It is shown that the confer rapid inactivation on hKv 1.5 channels with different potencies. This may be due to differences in their amino terminal sequences and/or inactivating domains. It is also shown that the amino terminal Kv beta 1.1 and Kv 1.4 alpha inactivating domains compete with each other, probably for the binding to the same receptor site(s) on Kv 1 alpha-subunits.     

A 12R-lipoxygenase in human skin: mechanistic evidence, molecular cloning, and expression

Sea anemone venom is known to contain toxins that are active on voltage-sensitive Na+ channels, as well as on delayed rectifier K+ channels belonging to the Kv1 family. This report describes the properties of a new set of peptides from Anemonia sulcata that act as blockers of a specific member of the Kv3 potassium channel family. These toxins, blood depressing substance (BDS)-I and BDS-II, are 43 amino acids long and differ at only two positions. They share no sequence homologies with other K+ channel toxins from sea anemones, such as AsKS, AsKC, ShK, or BgK. In COS-transfected cells, the Kv3.4 current was inhibited in a reversible manner by BDS-I, with an IC50 value of 47 nM. This inhibition is specific because BDS-I failed to block other K+ channels in the Kv1, Kv2, Kv3, and Kv4 subfamilies. Inward rectifier K+ channels are also insensitive to BDS-I. BDS-I and BDS-II share the same binding site on brain synaptic membranes, with K0.5 values of 12 and 19 nM, respectively. We observed that BDS-I and BDS-II have some sequence homologies with other sea anemone Na+ channels toxins, such as AsI, AsII, and AxI. However, they had a weak effect on tetrodotoxin-sensitive Na+ channels in neuroblastoma cells and no effect on Na+ channels in cardiac and skeletal muscle cells. BDS-I and BDS-II are the first specific blockers identified so far for the rapidly inactivating Kv3.4 channel.     

Alkoxypsoralens, novel nonpeptide blockers of Shaker-type K+ channels: synthesis and photoreactivity

A series of psoralens and structurally related 5,7-disubstituted coumarins was synthesized and investigated for their K+ channel blocking activity as well as for their phototoxicity to Artemia salina and their ability to generate singlet oxygen and to photomodify DNA. After screening the compounds on Ranvier nodes of the toad Xenopus laevis, the affinities of the most promising compounds, which proved to be psoralens bearing alkoxy substituents in the 5-position or alkoxymethyl substituents in the neighboring 4- or 4'-position, to a number of homomeric K+ channels were characterized. All compounds exhibited the highest affinity to Kv1.2. 5,8-Diethoxypsoralen (10d) was found to be an equally potent inhibitor of Kv1.2 and Kv1.3, while lacking the phototoxicity normally inherent in psoralens. The reported compounds represent a novel series of nonpeptide blockers of Shaker-type K+ channels that could be further developed into selective inhibitors of Kv1.2 or Kv1. 3.     

Topical lomefloxacin twice daily compared with fucidic acid in acute bacterial conjunctivitis

Possible heteromultimer formation between Kv- and Kir-type K+ channels was investigated, in connection with the known functional diversity of K+ channels in vivo. Voltage-clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1-Kv1.1 mRNA, or co-injected with Kv1.1 and Kir2.1 mRNA. K+ currents could be approximated by the algebraic sum of the 2 K+ current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 alpha-subunit proteins fail to assemble and do not contribute functional diversity to K+ channels.     

NMR structure of inactivation gates from mammalian voltage-dependent potassium channels

The electrical signalling properties of neurons originate largely from the gating properties of their ion channels. N-type inactivation of voltage-gated potassium (Kv) channels is the best-understood gating transition in ion channels, and occurs by a 'ball-and-chain' type mechanism. In this mechanism an N-terminal domain (inactivation gate), which is tethered to the cytoplasmic side of the channel protein by a protease-cleavable chain, binds to its receptor at the inner vestibule of the channel, thereby physically blocking the pore. Even when synthesized as a peptide, ball domains restore inactivation in Kv channels whose inactivation domains have been deleted. Using high-resolution nuclear magnetic resonance (NMR) spectroscopy, we analysed the three-dimensional structure of the ball peptides from two rapidly inactivating mammalian K. channels (Raw3 (Kv3.4) and RCK4 (Kv1.4)). The inactivation peptide of Raw3 (Raw3-IP) has a compact structure that exposes two phosphorylation sites and allows the formation of an intramolecular disulphide bridge between two spatially close cysteine residues. Raw3-IP exhibits a characteristic surface charge pattern with a positively charged, a hydrophobic, and a negatively charged region. The RCK4 inactivation peptide (RCK4-IP) shows a similar spatial distribution of charged and uncharged regions, but is more flexible and less ordered in its amino-terminal part.