Channel cDNA Research Articles

Characterization of an ABA-Induced and K+ Channel Gene FaKAT1 that Regulates Strawberry Fruit Ripening

Potassium (K+) plays an important role in the regulation of plant growth and development. However, how potassium works in K+ channels in strawberry fruit development is yet unknown. Here, a 2205 bp potassium channel cDNA encoding 734 amino acids, named FaKAT1, was cloned from strawberry fruits. Bioinformatics analysis showed that the cis-element ABRE responsive to ABA and conserved domains characteristic to the KAT1 superfamily were found in the promoter and encoding protein of FaKAT1. Transcription analysis revealed that FaKAT1 was expressed higher in stems and leaves, followed by fruits, but was less in roots and flowers, and that coupled with the fruit red-coloring, its transcripts increased rapidly, suggesting that FaKAT1 might play a role in ripening. Using tobacco rattle virus-induced gene silencing, downregulation of the FaKAT1 transcripts resulted in significant unripening of fruits, which is consistent with changes of the ripening-related events and genes, such as fruit firmness, contents of titratable acid, sugar, anthocyanin, and abscisic acid (ABA), as well as transcripts of PYR1, ABI1, SnRK2, GAL6, PE5, EXP2, XYL2, CA4H, CHS, CHI, DFR, and 4CL. Furthermore, ABA and the ABA inhibitor, fluridone, promoted and inhibited the mRNA expression levels of FaKAT1, respectively. Importantly, a K+ channel blocker, tetraethylammonium, also inhibited FaKAT1 expression and fruit red coloring. In conclusion, FaKAT1 is an ABA-induced K+ channel, which regulates strawberry fruit ripening.

T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels.

The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling.

Allocryptopine and benzyltetrahydropalmatine block hERG potassium channels expressed in HEK293 cells

Allocryptopine (ALL) is an alkaloid extracted from Corydalis decumbens (Thunb) Pers. Papaveraceae, whereas benzyltetrahydropalmatine (BTHP) is a derivative of tetrahydropalmatine extracted from Corydalis ambigua (Pall) Cham et Schlecht. The aim of this study was to investigate the effects of ALL and BTHP on the human ether-a-go-go related gene (hERG) current expressed in HEK293 cells. Cultured HEK293 cells were transiently transfected with hERG channel cDNA plasmid pcDNA3.1 using Lipofectamine. The whole-cell current IHERG was evoked and recorded using Axon MultiClamp 700B amplifier. The drugs were applied via supserfusion. Both ALL and BTHP reversibly suppressed the amplitude and density of IHERG in concentration- and voltage-dependent manners (the respective IC50 value was 49.65 and 22.38 μmol/L). BTHP (30 μmol/L) caused a significant negative shift of the steady-state inactivation curve of IHERG, while ALL (30 μmol/L) did not affect the steady-state inactivation of IHERG. Furthermore, BTHP, but not ALL, shortened the time constants of fast inactivation and slow time constants of deactivation of IHERG. But both the drugs markedly lengthened the time constants for recovery of IHERG from inactivation. Using action potential waveform pulses, it was found that both the drugs at 30 μmol/L significantly suppressed the current densities in the late phase of action potential, but did not significantly affect the current densities in the early phase of action potential. Both ALL and BTHP derived from Chinese herbs potently block hERG current.

Molecular characterization of genes encoding inward rectifier potassium (Kir) channels in the bed bug (Cimex lectularius)

The molecular genetics of inward-rectifier potassium (Kir) channels in insects is poorly understood. To date, Kir channel genes have been characterized only from a few representative dipterans (i.e., fruit flies and mosquitoes). The goal of the present study was to characterize Kir channel cDNAs in a hemipteran, the bed bug (Cimex lectularius). Using our previously reported bed bug transcriptome (RNA-seq), we identified two cDNAs that encode putative Kir channels. One was a full-length cDNA that encodes a protein belonging to the insect 'Kir3' clade, which we designate as 'ClKir3'. The other was a partial cDNA that encodes a protein with similarity to both the insect 'Kir1' and 'Kir2' clades, which we designate as 'ClKir1/2'. Quantitative real-time PCR analysis revealed that ClKir1/2 and ClKir3 exhibited peak expression levels in late-instar nymphs and early-instar nymphs, respectively. Furthermore, ClKir3, but not ClKir1/2, showed tissue-specific expression in Malpighian tubules of adult bed bugs. Lastly, using an improved procedure for delivering double-stranded RNA (dsRNA) to male and female bed bugs (via the cervical membrane) we demonstrate rapid and systemic knockdown of ClKir3 transcripts. In conclusion, we demonstrate that the bed bug possesses at least two genes encoding Kir channels, and that RNAi is possible for at least Kir3, thereby offering a potential approach for elucidating the roles of Kir channel genes in bed bug physiology.

T-type Cav3.2 Ca2+ channel is predominantly expressed in Xenopus laevis testis and involved in the fertilization process

The acrosome reaction (AR), a calcium-dependent exocytosis, is required for the mammalian fertilization process. Various ion channels, including T-type calcium channels presenting in the plasma membrane of the sperm head, play an important role in AR. In order to investigate the expression pattern and function of T-type calcium channels in Xenopus fertilization, we cloned partial cDNA sequences of cytoplasmic loops connecting domains II and III of T-type channel isoforms from the Xenopus laevis testis. The translated amino acid sequences of cloned Cav3.1 and Cav3.2 T-type channel cDNAs shared 81 and 96 % identity with those of Xenopus tropicalis, respectively. In addition, in situ hybridization showed that Cav3.1 and Cav3.2 transcripts are expressed in the testis tissues of X. laevis, with Cav3.2 transcripts more greatly expressed in testis than Cav3.1 transcripts. Pharmacological studies showed that treatment with nickel or the drug mibefradil significantly reduced the rate of fertilization in a concentration-dependent manner. Collectively, our findings suggest that Cav3.2 T-type calcium channels may play essential roles in the fertilization process of the amphibian species.

HERG Activation Gating Is Rapid: Evidence from Gating-Current Recordings and MTSET Modification of the Voltage Sensor

Background The human ether-a-go-go related gene protein (hERG) K + channel (Kv11.1) plays a major role in cardiac action potential repolarization, and defects in its function/expression can lead to long QT syndrome. The channel's key physiologic function as the main repolarization current is attributable in part to its unconventional slow activation kinetics, which are an order of magnitude slower than other members of the voltage-gated potassium channel superfamily. Methods We recorded gating-currents of hERG channels in tsa201 cells in order to directly identify the rate-limiting step in the activation of hERG channels. Expression of hERG channels was optimized by subcloning channel cDNA into a pGW1H expression vector and transfection into tsa201 cells. Gating currents were recorded in NMG + -based potassium-free internal and external solutions. As well, we used MTS modification of an externally facing residue in S4, I521C, to track the time course of movement of the S4 voltage sensor. I521 was mutated to a cysteine residue expressed in Xenopus or tsa201 cells, and various thiol-modifying reagents were used to modulate hERG activation and deactivation kinetics. Results hERG gating currents recorded from tsa201 cells were observed to activate on a millisecond time scale with time constants of on-gating current movement at 0 mV of 3.7 and 11.3 ms, much faster than ionic current activation, with bimodal voltage dependence (V 1/2-1 = –36.6 mV, k 1 = 8.3 mV, V 1/2-2 = 42.8 mV, k 2 = 15.5 mV, r² = 0.9912; A1=26%). Direct outward movement of the S4 domain as tracked by the modification of hERG channel I521C by MTSET was complete at 50 ms for pulses to 0 mV and within 25 ms for pulses to +60 mV. Conclusions This study represents the first report of hERG gating currents observed from mammalian cells and the first report of rapid voltage sensor modification associated with activation in hERG channels. Gating current recordings from tsa201 cells and the rate of MTSET modification of hERG voltage sensors suggest that the delayed activation kinetics of hERG cannot be attributed to slow voltage sensor movement but may be rate limited by a slow coupling process to the opening pore.

Evolutionary Adaptation of the Amino Acid and Codon Usage of the Mosquito Sodium Channel following Insecticide Selection in the Field Mosquitoes

Target site insensitivity resulting from point mutations within the voltage-gated sodium channel of the insect nervous system is known to be of primary importance in the development of resistance to pyrethroid insecticides. This study shifts current research paradigms by conducting, for the first time, a global analysis of all the naturally occurring mutations, both nonsynonymous and synonymous mutations, as well as mutation combinations in the entire mosquito sodium channel of Culex quinquefasciatus and analyzing their evolutionary and heritable feature and roles in insecticide resistance. Through a systematic analysis of comparing nucleotide polymorphisms in the entire sodium channel cDNAs of individuals between susceptible and resistant mosquito strains, between field parental mosquitoes and their permethrin selected offspring, and among different mosquito groups categorized by their levels of tolerance to specific permethrin concentrations within and among the mosquito strains of the field parental strains and their permethrin selected offspring, 3 nonsynonymous (A109S, L982F, and W1573R) and 6 synonymous (L852, G891, A1241, D1245, P1249, and G1733) mutations were identified. The co-existence of all 9 mutations, both nonsynonymous and synonymous, and their homozygousity were found to be important factors for high levels of resistance. Our study, for the first time, provide a strong case demonstrating the co-existence of both nonsynonymous and synonymous mutations in the sodium channel of resistant mosquitoes in response to insecticide resistance and the inheritance of these mutations in the offspring of field mosquito strains following insecticide selection.

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

Intracellular Proton-mediated Activation of TRPV3 Channels Accounts for the Exfoliation Effect of α-Hydroxyl Acids on Keratinocytes

α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.

Analysis of Voltage-Gated Sodium Channel Membrane Dynamics in Hippocampal Neurons via a Fluorescent Protein and Biotin Tagged Nav1.6 Channel

Voltage-gated sodium channels (Nav) are densely accumulated at the axon initial segment (AIS) of neurons where they are responsible for action potential initiation. The dense clustering of channels at the AIS involves ankyrinG binding, however the details of trafficking these channels to the AIS remains elusive. Furthermore, it is unclear what percentage of AIS channels is actually conducting. Since the large sodium channel cDNAs are difficult to manipulate and suffer from rearrangements in E. coli, the most elegant trafficking work to date has utilized chimeric proteins containing the sodium channel ankyrin-binding motif fused to other membrane proteins. To fully address trafficking in real time, an appropriately tagged full-length and functional channel is required. Therefore, we developed a Nav1.6 tagged with either GFP or Dendra2 fluorescent-proteins on the C-terminus and an extracellular biotin-acceptor-domain (BAD). The BAD allows for visualization and single molecule tracking of quantum-dot-bound sodium channels on the neuronal surface. This modified Nav1.6 demonstrated wild-type activity when expressed in hippocampal neurons. The tagged channel efficiently trafficked to the cell surface and was localized at the AIS as indicated by both confocal and TIRF microscopy. Alexafluor 594-conjugated-streptavidin binding indicated the surface-density of channels at the AIS was approximately 60 times greater than on the soma, comparable to endogenous Nav1.6 channels. Fluorescence recovery after photobleaching (FRAP) and single particle tracking showed that channels at the AIS had recovery time constants of greater than 2 hours and were confined to 60nm +/- 20nm compartments. In summary, we constructed a sodium channel with fluorescent protein and extracellular biotin reporters that has both wild-type trafficking and biophysical properties. This construct will permit the examination of sodium channel turnover, trafficking, diffusion and location-dependent function in neuronal cells.

Abstract P222: T-Type Calcium Channels Regulate NFAT Signaling in Cardiomyocytes and Are Expressed in Chronic Hypoxic Right Ventricle

Alterations in intracellular calcium (Ca 2+ ) have a significant impact on Ca 2+ signaling, an integral part of the cardiac response to pathological conditions. The calcineurin/NFAT signaling pathway is a well established Ca 2+ -dependent mechanism for transcriptional regulation of ventricular remodeling target genes. Although low voltage-activated T-type Ca 2+ channels are not normally present in the adult myocardium, they reappear in conditions of hypertrophy and heart failure, where their functions remain poorly understood. To further investigate T-type Ca 2+ channels, we have employed cultured neonatal rat ventricular myocytes (NRVM) and rat models of pulmonary hypertension that lead to right ventricular (RV) hypertrophy and heart failure. To assay for NFAT activation in cardiomyocytes, NRVM were infected with NFATc1-GFP and nuclear localization of GFP was measured. NFAT activation was markedly stimulated by over expressing the human Ca v 3.2 T-type Ca 2+ channel cDNA in NRVM, and this activation was inhibited by cyclosporin A (a calcineurin inhibitor), by T-type selective concentrations of mibefradil and NiCl 2 , and in the absence of extracellular Ca 2+ . To investigate the up regulation of cardiac T-type Ca 2+ channels in vivo , rats were maintained in chronic hypoxia, where they developed significantly elevated RV systolic pressure, RV hypertrophy and reduced cardiac output. Electrophysiological measurements revealed elevated membrane capacitance in RV myocytes indicative of RV hypertrophy, and T-type current density was also increased. Myocytes isolated from the RV of pulmonary hypertensive rats that displayed elevated T-type currents and RV dysfunction, also showed impaired contractility with decreased sensitivity to Ca 2+ . The results in NRVM suggest that T-type channels contribute to hypertrophic signaling via their interaction with calcineurin/NFAT signaling intermediaries. Furthermore, T-type channels are expressed in the RV during pulmonary hypertension where they influence RV myocyte contractility. Thus, when expressed in pathological conditions, T-type Ca 2+ channels may play a dual role as a signaling intermediary, as well as a modifier of ventricular contractile function.

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28°C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28°C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37°C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28°C for further incubation. We find that the initial incubation at 28°C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

The Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) voltage-gated sodium channel and mutations associated with pyrethroid resistance in field-collected adult males

Helicoverpa zea is one of the most costly insect pests of food and fiber crops throughout the Americas. Pyrethroid insecticides are widely applied for its control as they are effective and relatively inexpensive; however, resistance to pyrethroids threatens agricultural systems sustainability because alternative insecticides are often more expensive or less effective. Although pyrethroid resistance has been identified in this pest since 1990, the mechanisms of resistance have not yet been elucidated at the molecular level. Pyrethroids exert their toxicity by prolonging the open state of the voltage-gated sodium channel. Here we report the cDNA sequence of the H. zea sodium channel α-subunit homologous to the para gene from Drosophila melanogaster. In field-collected males which were resistant to cypermethrin as determined by the adult vial test, we identify known resistance-conferring mutations L1029H and V421M, along with two novel mutations at the V421 residue, V421A and V421G. An additional mutation, I951V, may be the first example of a pyrethroid resistance mutation caused by RNA editing. Identification of the sodium channel cDNA sequence will allow for testing hypotheses on target-site resistance for insecticides acting on this channel through modeling and expression studies. Understanding the mechanisms responsible for resistance will greatly improve our ability to identify and predict resistance, as well as preserve susceptibility to pyrethroid insecticides.

Synthetic Ciguatoxins Selectively Activate Nav1.8-derived Chimeric Sodium Channels Expressed in HEK293 Cells

The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Na(v)1.2, Na(v)1.4, and Na(v)1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879-889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Na(v)1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262). Thus, we hypothesized that ciguatoxin-induced activation of the Na(v)1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 mum CTX3C preferentially affected the activation process of the Na(v)1.8 channel compared with those of the Na(v)1.2 and Na(v)1.4 channels. Importantly, without stimulation, 0.1 mum CTX3C induced a large leakage current (I (L)). The conductance of the I (L) calculated relative to the maximum conductance (G (max)) was 10 times larger than that of Na(v)1.2 or Na(v)1.4. To determine the molecular domain of Na(v)1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Na(v)1.4 and Na(v)1.8. Chimeras containing the N-terminal half of Na(v)1.8 exhibited a large response similar to wild-type Na(v)1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.

D1 Receptors Physically Interact with N-Type Calcium Channels to Regulate Channel Distribution and Dendritic Calcium Entry

Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.

HCN2 and HCN4 Isoforms Self-assemble and Co-assemble with Equal Preference to Form Functional Pacemaker Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET(2)), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET(2) data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.

A comparative study of voltage‐gated sodium channels in the Insecta: implications for pyrethroid resistance in Anopheline and other Neopteran species

We report the complete cDNA sequence of the Anopheles gambiae voltage-gated sodium channel (VGSC) alpha-subunit isolated from mature adult mosquitoes. The genomic DNA contains 35 deduced exons with a predicted translation of <or= 2139 amino acid cDNAs. The transcription of the gene is, however, complex, alternate splicing being evident for at least five optional exons (or exon segments) and two sets of mutually exclusive exons. Overall gene organization was also compared with that of other VGSCs within the Insecta. Several insecticides used in mosquito control (including DDT and synthetic pyrethroids) target the VGSC. Isolation of the sodium channel cDNA for An. gambiae: (1) allows prediction of likely single nucleotide polymorphisms that may arise at residue L1014 to cause resistance to insecticides; (2) defines An. gambiae exon usage in key areas of the VGSC protein that are known (from previous studies in a range of different pest species) to have roles in altering insecticide susceptibility and in generating resistance; and (3) is a critical first step towards development of refined malarial control strategies and of new diagnostics for resistance monitoring.

Loss of CNGB1 Protein Leads to Olfactory Dysfunction and Subciliary Cyclic Nucleotide-gated Channel Trapping

Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1-/-) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1-/- mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1-/- mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca2+-calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1-/- mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function in vivo. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel.

A method for the simultaneous analysis of mRNA levels of multiple cardiac ion channels with a multi-probe RNase protection assay

Various pathological conditions can alter cardiac electrophysiological properties not only by physiological responses but also by modifying the gene expression of ion channels (electrical remodelling). To investigate the underlying mechanisms of the latter, electrophysiological alterations would require a simultaneous and comprehensive analysis of the mRNA level of the ion channel genes. We designed 19 cardiac ion channel cDNA templates to analyse the corresponding mRNAs and classified them into three template sets. Those sets were a voltage-dependent K(+) channel series (rat erg, KvLQT1, Kv4.3, Kv4.2, Kv2.1, Kv1.5, Kv1.4, Kv1.2), an inwardly rectifying K(+) channel series (rat Kir6.2, SUR2A/B, Kir3.4, Kir3.1, Kir2.2, Kir2.1), and an inward cationic ion channel series (rat SCN5A, alpha1C, beta2, alpha2delta2 of cardiac L-type Ca(2+) channel and alpha1G). These cDNA templates were used to synthesize antisense digoxigenin-labelled RNA probes. An amount of the total RNA of 25 microg was adequate to analyse simultaneously the mRNA levels of the ion channel genes with the use of multi-probe RPA, and these three multi-probe template sets enabled us to evaluate the profile of the spatial and temporal transcripts of the cardiac ion channels. The newly developed ion channel multi-probe RPA templates provide an aid in the comprehensive analysis of the electrical remodelling of the heart.

Comparative pharmacology and cloning of two novel arachnid sodium channels: Exploring the adaptive insensitivity of scorpion to its toxins

Scorpion toxins have been found lacking effect on Na + current of its own sodium channel, whereas the molecular mechanism remains mystery. In this study, the binding affinity of pharmacologically distinct scorpion toxins was found much weaker to scorpion ( Buthus martensii) nerve synaptosomes than to spider ( Ornithoctonus huwena) ones. The sodium channel cDNA from these two species were further cloned. The deduced proteins contain 1871 and 1987 amino acids respectively. Several key amino acid substitutions, i.e., A1610V, I1611L and S1617K, are found in IVS3–S4 constituting receptor site-3, and for receptor site-4, two residues (Leu-Pro) are inserted near IIS4 of scorpion sodium channel.