Cloning and expression of an Aplysia K+ channel and comparison with native Aplysia K+ currents

We describe here the cloning of the Aplysia K+ channel AK01a.AK01a codes for a protein of 515 amino acids, shows considerable homology to other cloned potassium channels, and can be classified as a member of the ShakerK+ channel family. Expression of the AK01a channel in Xenopus oocytes produces a rapidly inactivating outward potassium current (IAK01a) resembling the A-type currents of Drosophila Shaker. Gating for this current is shifted to potentials considerably more positive than the traditional A-currents of Aplysia; we have, however, identified a novel transient potassium current (IAdepoI) in a subset of Aplysia neurons that has similar gating and pharmacological properties to IAK01a.     

Molecular physiology and modulation of somatodendritic A-type potassium channels

The somatodendritic subthreshold A-type K+ current (ISA) in nerve cells is a critical component of the ensemble of voltage-gated ionic currents that determine somatodendritic signal integration. The underlying K+ channel belongs to the Shal subfamily of voltage-gated K+ channels. Most Shal channels across the animal kingdom share a high degree of structural conservation, operate in the subthreshold range of membrane potentials, and exhibit relatively fast inactivation and recovery from inactivation. Mammalian Shal K+ channels (Kv4) undergo preferential closed-state inactivation with features that are generally inconsistent with the classical mechanisms of inactivation typical of Shaker K+ channels. Here, we review (1) the physiological and genetic properties of ISA, 2 the molecular mechanisms of Kv4 inactivation and its remodeling by a family of soluble calcium-binding proteins (KChIPs) and a membrane-bound dipeptidase-like protein (DPPX), and (3) the modulation of Kv4 channels by protein phosphorylation.     

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.     

An essential 'set' of K+ channels conserved in flies, mice and humans.

The molecular genetic approach to studying K+ channels has revealed that at least four subfamilies of voltage-gated K+ channels originally discovered in Drosophila are conserved in mice and humans. This conservation of the K+ channel subfamilies Shaker, Shal, Shab, and Shaw suggests that not only the broad outlines of membrane electrical properties but also many molecular details as well evolved in the parent species ancestral to both invertebrate and vertebrate life. Shaker, Shal, Shab, and Shaw K+ channels have similar structures, but appear to be independent channel systems: when co-expressed in Xenopus oocytes, all four function independently. These four K+ channel subfamilies may be part of an essential 'set' of excitable channels required by most nervous systems. The task now remaining is to understand the functions of each member of the set.     

A novel leg-shaking Drosophila mutant defective in a voltage-gated K(+)current and hypersensitive to reactive oxygen species.

1,1'-Dimethyl-4,4'-bipyridinium dichloride (methyl viologen; paraquat), an herbicide that causes depletion of NADPH and generates excessive reactive oxygen species (ROS) in vivo, has been used to screen for ROS-sensitive Drosophila mutants. One mutant so isolated, named quiver(1) (qvr(1)), has a leg-shaking phenotype. Mutants of the Shaker (Sh), Hyperkinetic (Hk), and ether a go-go (eag) genes, which encode different K(+) channel subunits that regulate the A-type K(+) current (I(A)) in different ways, exhibit leg shaking under ether anesthesia and have heightened metabolic rates and shortened life spans. We found that Sh, Hk, and eag mutant flies were all hypersensitive to paraquat. Double-mutant combinations among the three channel mutations and qvr(1) had drastically enhanced sensitivity to paraquat. Synaptic transmission at the larval neuromuscular junction was increased in the qvr(1) mutant to the level of Sh mutants. Similar to eag Sh double mutants, double mutants of eag and qvr(1) showed striking enhancement in synaptic transmission and a wings-down phenotype, the hallmarks of extreme hyperexcitability. Voltage-clamp experiments demonstrated that the qvr(1) mutation specifically disrupted the Sh-dependent I(A) current without altering the other currents [I(K), Ca(2+)-activated fast (I(CF)) and slow (I(CS)) currents, and I(Ca)] in larval muscles. Several deficiency strains of the qvr locus failed to complement qvr(1) and confirmed that ether-induced leg shaking, reduced I(A) current, and paraquat hypersensitivity map to the same locus. Our results suggest that the qvr gene may encode a novel K(+) channel-related polypeptide and indicate a strong link between a voltage-activated K(+) current and vulnerability to ROS.     

Shaker Mutants Lack Post-tetanic Potentiation at Motor End-plates

The two-electrode voltage clamp technique was employed to measure end-plate currents in larval neuromuscular junctions of wild-type (Canton-S) and of three different Drosophila Shaker mutants: Shaker^KS133, Shaker¹⁰² and f⁵Shaker⁵. In the Shaker mutants, nerve-evoked end-plate currents (neepc) were 4–5-fold larger than those measured in Canton-S. Shaker motor end-plates were found to lack post-tetanic potentiation (PTP), but could undergo facilitation. Moreover, PTP but not facilitation was lost in wild-type larvae if the neuromuscular junction was exposed to 4-aminopyridine (4-AP), a blocker of Shaker A-type K⁺ currents. End-plate currents were depressed by Ca²⁺ channel blockers like Mg²⁺, at millimolar concentrations, and Co²⁺ and Cd²⁺, at micromolar concentrations, but not by nifedipine (100 nM) and verapamil (100 nM). After exposure to Ca²⁺ channel blockers, Shaker end-plates exhibited PTP. In particular, Cd²⁺ was most effective in depressing neepes and in restoring PTP in all Shaker mutants. The results obtained indicate the abnormal function of Shaker K⁺ channels at motor nerves specifically abolishes PTP in Drosophila larval neuromuscular junctions.     

Identification of the Kv2.1 K+ channel as a major component of the delayed rectifier K+ current in rat hippocampal neurons.

Molecular cloning studies have revealed the existence of a large family of voltage-gated K+ channel genes expressed in mammalian brain. This molecular diversity underlies the vast repertoire of neuronal K+ channels that regulate action potential conduction and neurotransmitter release and that are essential to the control of neuronal excitability. However, the specific contribution of individual K+ channel gene products to these neuronal K+ currents is poorly understood. We have shown previously, using an antibody, "KC, " specific for the Kv2.1 K+ channel alpha-subunit, the high-level expression of Kv2.1 protein in hippocampal neurons in situ and in culture. Here we show that KC is a potent blocker of K+ currents expressed in cells transfected with the Kv2.1 cDNA, but not of currents expressed in cells transfected with other highly related K+ channel alpha-subunit cDNAs. KC also blocks the majority of the slowly inactivating outward current in cultured hippocampal neurons, although antibodies to two other K+ channel alpha-subunits known to be expressed in these cells did not exhibit blocking effects. In all cases the blocking effects of KC were eliminated by previous incubation with a recombinant fusion protein containing the KC antigenic sequence. Together these studies show that Kv2.1, which is expressed at high levels in most mammalian central neurons, is a major contributor to the delayed rectifier K+ current in hippocampal neurons and that the KC antibody is a powerful tool for the elucidation of the role of the Kv2.1 K+ channel in regulating neuronal excitability.     

Antibodies against Drosophila potassium channels identify membrane proteins across species

Shaker is a complex locus (ShC) in Drosophila that encodes components of the K+ channel responsible for the IA current. We have raised antibodies against synthetic peptides of selected sequences from the Sh products. One of the antisera identifies a 71 kDa protein band in immunoblots from Drosophila neural membrane proteins. We demonstrate that this protein is encoded within the viable (V) region of the ShC since deletions and breakpoints in this part of the complex eliminate this band from the immunoblots. Certain Sh mutations abolish the production of this product while other do not seem to interfere with it. The same antiserum identifies bands of different apparent molecular weight (Mr) in membrane extracts of nervous systems of a variety of organisms including vertebrates.     

Functional identification of a cloned squid presynaptic voltage-dependent calcium channel

We previously cloned a voltage-dependent Ca2+ channel alpha1 subunit LoCa(v)2 cDNA from the squid optic lobe. LoCa(v)2 is designated as a non-L-type voltage-dependent Ca2+ channel based on its amino acid sequence. We performed functional expression experiments of LoCa(v)2 in oocytes and characterized the expressed currents electrophysiologically and pharmacologically. The LoCa(v)2 current was high voltage-activated and the peak current was maximal at +20 mV and lasted for long during activation. The LoCa(v)2 current was not inhibited by the drugs and toxins examined except for omega-agatoxin IVA and PLTX-II. Omega-agatoxin IVA, which is a P-type channel blocker, moderately inhibited the LoCa(v)2 current at higher concentration. PLTX-II, which blocks insect presynaptic Ca2+ channel, inhibited the LoCa(v)2 current at lower concentration. Immunohistochemical investigation showed that the LoCa(v)2 protein may exist at presynaptic terminals in the squid optic lobe. These results suggest that LoCa(v)2 is an omega-agatoxin IVA and PLTX-II-sensitive presynaptic Ca2+ channel in the squid nervous system.     

Voltage clamp analysis of membrane currents in larval muscle fibers of Drosophila: alteration of potassium currents in Shaker mutants

The body wall muscles in Drosophila larvae are suitable for voltage clamp analysis of changes in membrane excitability caused by mutations. Both inward and outward ionic currents are present in these muscle fibers. The inward current is mediated by voltage-dependent Ca2+ channels. In Ca2+-free saline, the inward current is eliminated. The remaining outward K+ currents consist of two distinct components, an early transient IA and a delayed steady IK, which are separable by differences in the rate and voltage dependence of activation and inactivation. The steady-state and kinetic properties of the activation and inactivation processes of these two currents are analyzed. The results provide a basis for quantitative analysis of altered membrane currents in behavioral mutants of Drosophila. Previous studies indicate that mutations in the Shaker (Sh) locus alter excitability in both nerve and muscle in Drosophila. Our results support the idea that the channels mediating IA are molecularly distinct from those mediating IK. All Sh mutations studied specifically affect IA without changing the properties of the calcium current and IK. In certain alleles (ShKS133, Sh102, and ShM) IA is eliminated, permitting detailed studies of IK in isolation of IA. Studies of the alleles that do not eliminate IA provide additional information of the channels. In one such allele, Sh5, voltage dependence of IA activation is shifted to more positive potentials. This is accompanied by a less pronounced shift in the voltage dependence of inactivation. These results suggest that Sh5 mutation affects the voltage-sensitive mechanism of both activation and inactivation processes and that these two processes are not controlled by independent parts of the channel. Furthermore, the differential effects of these alleles on different excitable membranes imply that other genes take part in the control of IA. The effects of Sh5 on muscle depend on developmental stage. In larval muscle, Sh5 reduces the amplitude of IA because of the shift in the current-voltage (I-V) relation. In contrast, in adult Sh5 muscles, IA is reported to be normal in amplitude but shows abnormally rapid inactivation (Salkoff, L., and R. Wyman (1981) Nature 293: 228-230). A different allele, ShrK0120, causes a clear defect in nerve excitability, but analysis of IA in ShrK0120 larval muscle reveals I-V relations, inactivation, and recovery from inactivation similar to those seen in normal fibers. We suggest a possible mechanism of combinations of multiple interacting genes participating in the control of potassium channels to account for the presence of a variety of potassium channels in different excitable membranes.     

Identification and localization of Ca(2+)-activated K+ channels in rat sciatic nerve.

To understand the physiology of Schwann cells and myelinated nerve, we have been engaged in identifying K+ channels in sciatic nerve and determining their subcellular localization. In the present study, we examined the slo family of Ca(2+)-activated K+ channels, a class of channel that had not previously been identified in myelinated nerve. We have determined that these channels are indeed expressed in peripheral nerve, and have cloned rat homologues of slo that are more than 95% identical to the murine slo. We found that sciatic nerve RNA contained numerous alternatively spliced variants of the slo homologue, as has been seen in other tissues. We raised a polyclonal antibody against a peptide from the carboxyl terminal of the channels. Immunocytochemistry revealed that the channel proteins are in Schwann cells and are associated with canaliculi that run along the outer surface of the cells. They are also relatively concentrated near the node of Ranvier in the Schwann cell outer membrane. This staining pattern is quite similar to what we previously reported for the voltage-dependent K+ channel Kv 1.5. We did not observe staining of axons or connective tissue in the nerve and so it seems likely that most or all of the splicing variants are located in the Schwann cells. The localization of these channels also suggests that they may participate in maintaining the resting potential of the Schwann cells during K+ buffering.     

Gene dosage and complementation analysis of the Shaker locus in Drosophila

Mutations of the Shaker (Sh) locus alter or eliminate a transient, voltage-sensitive potassium current, the "A" current, in flight muscle of Drosophila. We show that the amplitude of the A current is reduced when the dosage of Sh+ is lower than normal, but that A current amplitude does not increase as extra copies of Sh+ are added. We have also examined 14 Shaker mutants by voltage clamp and by intracellular recording at the larval neuromuscular junction. In 10 of these mutants there is no detectable fast component of the transient outward current. In each of these 10, however, a small, slowly inactivating, outward current is present. The 10 mutations null for the fast component of the transient, outward current are all partially dominant, giving 50-80% of the normal A current in Shnull/Sh+ heterozygotes. Because as little as 5% of the normal A current can be detected, complementation tests are feasible. The 10 null mutations are members of a single complementation group. The remaining 4 mutations have reduced A currents in pupal flight muscle. In all cases, crosses between these leaky mutants and null mutants give progeny with less A current than found in the leaky parental lines, as would be expected if the leaky and null mutations are in the same complementation group. For 1 of the mutations, ShrKO120, the mutant phenotype is much more severe in nerve than in muscle. That part of the Shaker locus required for the production of the A channel lies between the B55 and the V7 translocation breakpoints, in region 16F of the X chromosome.     

Distinct spatial and temporal expression patterns of K+ channel mRNAs from different subfamilies.

Different types of K+ channels play important roles in many aspects of excitability. The isolation of cDNA clones from Drosophila, Aplysia, Xenopus, and mammals points to a large multigene family with several distinct members encoding K+ channels with unique electrophysiological and pharmacological properties. Given the pivotal role K+ channels play in the fine tuning of electrical properties of excitable tissues, we studied the spatial and temporal basis of K+ channel diversity. We report the isolation of two putative K+ channels that define two new subfamilies based upon amino acid sequence similarities with other known K+ channels. Northern blot and in situ hybridization studies revealed differences in the spatial and temporal expression patterns for these two new clones along with mRNAs from other K+ channel subfamilies. Two of the K+ channels studied are predominantly expressed in the brain. One of the "brain-specific" K+ channels is first expressed after about 2 weeks of postnatal cerebellar development and remains at levels about 10-fold higher in the cerebellum than in the rest of the brain.     

Voltage-gated potassium channels in larval CNS neurons of Drosophila

The availability of genetic, molecular, and biophysical techniques makes Drosophila an ideal system for the study of ion channel function. We have used the patch-clamp technique to characterize voltage-gated K+ channels in cultured larval Drosophila CNS neurons. Whole-cell currents from different cells vary in current kinetics and magnitude. Most of the cells contain a transient A-type 4-AP-sensitive current. In addition, many cells also have a more slowly inactivating TEA-sensitive component and/or a sustained component. No clear correlation between cell morphology and whole-cell current kinetics was observed. Single-channel analysis in cell-free patches revealed that 3 types of channels, named A2, KD, and K1 can account for the whole-cell currents. None of these channels requires elevated intracellular calcium concentration for activation. The A2 channels have a conductance of 6-8 pS and underlie the whole-cell A current. They turn on rapidly, inactivate in response to depolarizing voltage steps, and are completely inactivated by prepulses to -50 mV. The KD (delayed) channels have a conductance of 10-16 pS and can account, in part, for the more slowly inactivating component of whole-cell current. They have longer open times and activate and inactivate more slowly than the A2 channels. The K1 channels have a slope conductance, measured between 0 and +40 mV, of 20-40 pS. These channels do not inactivate during 500 msec voltage steps and thus can contribute to the sustained component of current. They exhibit complex gating behavior with increased probability of being open at higher voltages. Although the K1 channels are sufficient to account for the noninactivating component of whole-cell current, we have observed several other channel types that have a similar voltage dependence and average kinetics.     

From worm to man: three subfamilies of TRP channels

A steadily increasing number of cDNAs for proteins that are structurally related to the TRP ion channels have been cloned in recent years. All these proteins display a topology of six transmembrane segments that is shared with some voltage-gated channels and the cyclic-nucleotide-gated channels. The TRP channels can be divided, on the basis of their homology, into three TRP channel (TRPC) subfamilies: short (S), long (L) and osm (O). From the evidence available to date, this subdivision can also be made according to channel function. Thus, the STRPC family, which includes Drosophila TRP and TRPL and the mammalian homologues, TRPC1-7, is a family of Ca2+-permeable cation channels that are activated subsequent to receptor-mediated stimulation of different isoforms of phospholipase C. Members of the OTRPC family are Ca2+-permeable channels involved in pain transduction (vanilloid and vanilloid-like receptors), epithelial Ca2+ transport and, at least in Caenorhabditis elegans, in chemo-, mechano- and osmoregulation. The LTRPC family is less well characterized.     

Subthreshold changes of voltage-dependent activation of the K(V)7.2 channel in neonatal epilepsy

Benign familial neonatal convulsions (BFNC) is an epileptic disorder caused by dominant mutations in the genes KCNQ2 and KCNQ3 encoding the K+ channels K(V)7.2 and K(V)7.3. We identified two novel KCNQ2 mutations in two BFNC families. One mutation predicted a truncated protein (S247X) that lacks the channel's pore region, the other resulted in the amino acid substitution S122L in the S2 segment of K(V)7.2. In comparison to wild-type (WT) K(V)7.2, functional analysis of S122L mutant channels in Xenopus oocytes revealed a significant positive shift and increased slope of the activation curve leading to significant current reduction in the subthreshold range of an action potential (75% reduction at -50 mV). Our results establish an important role of the K(V)7.2 S2 segment in voltage-dependent channel gating and demonstrate in a human disease that subthreshold voltages are likely to represent the physiologically relevant range for this K+ channel to regulate neuronal firing.     

Inhibitory effect of lamotrigine on A-type potassium current in hippocampal neuron-derived H19-7 cells

PURPOSE: We investigated the effects of lamotrigine (LTG) on the rapidly inactivating A-type K+ current (IA) in embryonal hippocampal neurons. METHODS: The whole-cell configuration of the patch-clamp technique was applied to investigate the ion currents in cultured hippocampal neuron-derived H19-7 cells in the presence of LTG. Effects of various related compounds on IA in H19-7 cells were compared. RESULTS: LTG (30 microM-3 mM) caused a reversible reduction in the amplitude of IA. The median inhibitory concentration (IC50) value required for the inhibition of IA by LTG was 160 microM. 4-Aminopyridine (1 mM), quinidine (30 microM), and capsaicin (30 microM) were effective in suppressing the amplitude of IA, whereas tetraethylammonium chloride (1 mM) and gabapentin (100 microM) had no effect on it. The time course for the inactivation of IA was changed to the biexponential process during cell exposure to LTG (100 microM). LTG (300 microM) could shift the steady-state inactivation of IA to a more negative membrane potential by approximately -10 mV, although it had no effect on the slope of the inactivation curve. Moreover, LTG (100 microM) produced a significant prolongation in the recovery of IA inactivation. Therefore in addition to the inhibition of voltage-dependent Na+ channels, LTG could interact with the A-type K+ channels to suppress the amplitude of IA. The blockade of IA by LTG does not simply reduce current magnitude, but alters current kinetics, suggesting a state-dependent blockade. LTG might have a higher affinity to the inactivated state than to the resting state of the IA channel. CONCLUSIONS: This study suggests that in hippocampal neurons, during exposure to LTG, the LTG-mediated inhibition of these K+ channels could be one of the ionic mechanisms underlying the increased neuronal excitability.