ABSTRACT
Nociceptor sensitization following nerve injury or inflammation leads to chronic pain. An increase in the nociceptor hyperpolarization-activated current, Ih, is observed in many models of pathological pain. Pharmacological blockade of Ih prevents the mechanical and thermal hypersensitivity that occurs during pathological pain. Alterations in the Hyperpolarization-activated Cyclic Nucleotide-gated ion channel 2 (HCN2) mediate Ih-dependent thermal and mechanical hyperalgesia. Limited knowledge exists regarding the nature of these changes during chronic inflammatory pain. Modifications in HCN2 expression and post-translational SUMOylation have been observed in the Complete Freund's Adjuvant (CFA) model of chronic inflammatory pain. Intra-plantar injection of CFA into the rat hindpaw induces unilateral hyperalgesia that is sustained for up to 14 days following injection. The hindpaw is innervated by primary afferents in lumbar DRG, L4-6. Adjustments in HCN2 expression and SUMOylation have been well-documented for L5 DRG during the first 7 days of CFA-induced inflammation. Here, we examine bilateral L4 and L6 DRG at day 1 and day 3 post-CFA. Using L4 and L6 DRG cryosections, HCN2 expression and SUMOylation were measured with immunohistochemistry and proximity ligation assays, respectively. Our findings indicate that intra-plantar injection of CFA elicited a bilateral increase in HCN2 expression in L4 and L6 DRG at day 1, but not day 3, and enhanced HCN2 SUMOylation in ipsilateral L6 DRG at day 1 and day 3. Changes in HCN2 expression and SUMOylation were transient over this time course. Our study suggests that HCN2 is regulated by multiple mechanisms during CFA-induced inflammation.
Subject(s)
Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Animals , Male , RatsABSTRACT
Norepinephrine (NE) released from the sympathetic nerves innervating white adipose tissue (WAT) is the principal initiator of lipolysis in mammals. Central WAT sympathetic outflow neurons express melanocortin 4-receptor (MC4-R) mRNA. Single central injection of melanotan II (MTII; MC3/4-R agonist) nonuniformly increases WAT NE turnover (NETO), increases interscapular brown adipose tissue (IBAT) NETO, and increases the circulating lipolytic products glycerol and free fatty acid. The WAT pads that contributed to this lipolysis were inferred from the increases in NETO. Because phosphorylation of perilipin A (p-perilipin A) and hormone-sensitive lipase are necessary for NE-triggered lipolysis, we tested whether MTII would increase these intracellular markers of lipolysis. Male Siberian hamsters received a single 3rd ventricular injection of MTII or saline. Trunk blood was collected at 0.5, 1.0, and 2.0 h postinjection from excised inguinal, retroperitoneal, and epididymal WAT (IWAT, RWAT, and EWAT, respectively) and IBAT pads. MTII increased circulating glycerol concentrations at 0.5 and 1.0 h, whereas free fatty acid concentrations were increased at 1.0 and 2.0 h. Western blot analysis showed that MTII specifically increased p-perilipin A and hormone-sensitive lipase only in fat pads that previously had MTII-induced increases in NETO. Phosphorylation increased in IWAT at all time points and IBAT at 0.5 h, but not RWAT or EWAT at any time point. These results show for the first time in rodents that p-perilipin A can serve as an in vivo, fat pad-specific indictor of lipolysis and extend our previous findings showing that central melanocortin stimulation increases WAT lipolysis.
Subject(s)
Adipose Tissue/metabolism , Lipolysis/physiology , Adipose Tissue/innervation , Adipose Tissue/physiology , Adipose Tissue, Brown/metabolism , Adipose Tissue, White/metabolism , Animals , Carrier Proteins , Central Nervous System Stimulants/metabolism , Cricetinae , Epididymis/metabolism , Fatty Acids, Nonesterified/blood , Fatty Acids, Nonesterified/metabolism , Glycerol/blood , Glycerol/metabolism , Male , Melanocortins/metabolism , Norepinephrine/blood , Norepinephrine/metabolism , Peptides, Cyclic , Perilipin-1 , Phodopus , Phosphoproteins , Phosphorylation , Receptor, Melanocortin, Type 4/genetics , Receptor, Melanocortin, Type 4/metabolism , Sterol Esterase/metabolism , Sympathetic Nervous System/metabolism , Sympathetic Nervous System/physiology , alpha-MSH/analogs & derivativesABSTRACT
Organotypic cultures of the lobster (Homarus gammarus) stomatogastric nervous system (STNS) were used to assess changes in membrane properties of neurons of the pyloric motor pattern-generating network in the long-term absence of neuromodulatory inputs to the stomatogastric ganglion (STG). Specifically, we investigated decentralization-induced changes in the distribution and density of the transient outward current, I(A), which is encoded within the STG by the shal gene and plays an important role in shaping rhythmic bursting of pyloric neurons. Using an antibody against lobster shal K(+) channels, we found shal immunoreactivity in the membranes of neuritic processes, but not somata, of STG neurons in 5 d cultured STNS with intact modulatory inputs. However, in 5 d decentralized STG, shal immunoreactivity was still seen in primary neurites but was likewise present in a subset of STG somata. Among the neurons displaying this altered shal localization was the pyloric dilator (PD) neuron, which remained rhythmically active in 5 d decentralized STG. Two-electrode voltage clamp was used to compare I(A) in synaptically isolated PD neurons in long-term decentralized STG and nondecentralized controls. Although the voltage dependence and kinetics of I(A) changed little with decentralization, the maximal conductance of I(A) in PD neurons increased by 43.4%. This increase was consistent with the decentralization-induced increase in shal protein expression, indicating an alteration in the density and distribution of functional A-channels. Our results suggest that, in addition to the short-term regulation of network function, modulatory inputs may also play a role, either directly or indirectly, in controlling channel number and distribution, thereby maintaining the biophysical character of neuronal targets on a long-term basis.
Subject(s)
Biological Clocks/physiology , Ion Channels/metabolism , Motor Neurons/metabolism , Neurotransmitter Agents/metabolism , Potassium Channels, Voltage-Gated , Animals , Antibodies/pharmacology , Biological Clocks/drug effects , Cell Membrane/metabolism , Digestive System/innervation , Ganglia, Invertebrate/cytology , Ganglia, Invertebrate/drug effects , Ganglia, Invertebrate/metabolism , In Vitro Techniques , Ion Channels/antagonists & inhibitors , Motor Neurons/drug effects , Nephropidae , Nerve Net/cytology , Nerve Net/drug effects , Nerve Net/metabolism , Neurites/metabolism , Patch-Clamp Techniques , Periodicity , Potassium/metabolism , Potassium Channel Blockers , Potassium Channels/metabolism , Shal Potassium ChannelsABSTRACT
The pyloric motor system, in the crustacean stomatogastric ganglion, produces a continuously adaptive behavior. Each cell type in the neural circuit possesses a distinct yet dynamic electrical phenotype that is essential for normal network function. We previously demonstrated that the transient potassium current (I(A)) in the different component neurons is unique and modulatable, despite the fact that the shal gene encodes the alpha-subunits that mediate I(A) in every cell. We now examine the hypothesis that alternate splicing of shal is responsible for pyloric I(A) diversity. We found that alternate splicing generates at least 14 isoforms. Nine of the isoforms were expressed in Xenopus oocytes and each produced a transient potassium current with highly variable properties. While the voltage dependence and inactivation kinetics of I(A) vary significantly between pyloric cell types, there are few significant differences between different shal isoforms expressed in oocytes. Pyloric I(A) diversity cannot be reproduced in oocytes by any combination of shal splice variants. While the function of alternate splicing of shal is not yet understood, our studies show that it does not by itself explain the biophysical diversity of I(A) seen in pyloric neurons.
Subject(s)
Alternative Splicing/genetics , Ganglia, Invertebrate/metabolism , Movement/physiology , Nerve Net/metabolism , Neurons/metabolism , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Animals , DNA, Complementary/analysis , Female , Ganglia, Invertebrate/cytology , Membrane Potentials/genetics , Molecular Sequence Data , Nephropidae/cytology , Nephropidae/genetics , Nephropidae/metabolism , Nerve Net/cytology , Neurons/cytology , Oocytes/cytology , Oocytes/metabolism , Open Reading Frames/genetics , Potassium Channels/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Pylorus/cytology , Pylorus/innervation , Pylorus/physiology , RNA, Messenger/isolation & purification , Sequence Homology, Amino Acid , Shal Potassium Channels , Xenopus/genetics , Xenopus/metabolismABSTRACT
We investigated the contribution of sialic acid residues to the K(+) currents involved in the repolarization of mouse ventricular myocytes. Ventricular K(+) currents had a rapidly inactivating component followed by slowly decaying and sustained components. This current was produced by the summation of three distinct currents: I(to), which contributed to the transient component; I(ss), which contributed to the sustained component; and I(K,slow), which contributed to both components. Incubation of ventricular myocytes with the sialidase neuraminidase reduced the amplitude of I(to) without altering I(K,slow) and I(ss). We found that the reduction in I(to) amplitude resulted from a depolarizing shift in the voltage of activation and a reduction in the conductance of I(to). Expression of Kv4.3 channels, a major contributor to I(to) in the ventricle, in a sialylation-deficient Chinese hamster ovary cell line (lec2) mimicked the effects of neuraminidase on the ventricular I(to). Furthermore, we showed that sialylated glycolipids have little effect on the voltage dependence of I(to). Finally, consistent with its actions on I(to), neuraminidase produced an increase in the duration of the action potential of ventricular myocytes and the frequency of early afterdepolarizations. We conclude that sialylation of the proteins forming Kv4 channels is important in determining the voltage dependence and conductance of I(to) and that incomplete glycosylation of these channels could lead to arrhythmias.
Subject(s)
N-Acetylneuraminic Acid/physiology , Potassium Channels, Voltage-Gated , Potassium Channels/physiology , Ventricular Function , Action Potentials/drug effects , Animals , CHO Cells , Calcium/pharmacology , Cricetinae , Electric Conductivity , Electrophysiology , Glycolipids/metabolism , Homeostasis , Myocardium/cytology , N-Acetylneuraminic Acid/deficiency , Neuraminidase/pharmacology , Potassium Channels/drug effects , Reaction Time/drug effects , Shal Potassium ChannelsABSTRACT
We investigated the cellular and molecular mechanisms underlying arrhythmias in heart failure. A genetically engineered mouse lacking the expression of the muscle LIM protein (MLP-/-) was used in this study as a model of heart failure. We used electrocardiography and patch clamp techniques to examine the electrophysiological properties of MLP-/- hearts. We found that MLP-/- myocytes had smaller Na+ currents with altered voltage dependencies of activation and inactivation and slower rates of inactivation than control myocytes. These changes in Na+ currents contributed to longer action potentials and to a higher probability of early afterdepolarizations in MLP-/- than in control myocytes. Western blot analysis suggested that the smaller Na+ current in MLP-/- myocytes resulted from a reduction in Na+ channel protein. Interestingly, the blots also revealed that the alpha-subunit of the Na+ channel from the MLP-/- heart had a lower average molecular weight than in the control heart. Treating control myocytes with the sialidase neuraminidase mimicked the changes in voltage dependence and rate of inactivation of Na+ currents observed in MLP-/- myocytes. Neuraminidase had no effect on MLP-/- cells thus suggesting that Na+ channels in these cells were sialic acid-deficient. We conclude that deficient glycosylation of Na+ channel contributes to Na+ current-dependent arrhythmogenesis in heart failure.
Subject(s)
Arrhythmias, Cardiac/etiology , Arrhythmias, Cardiac/metabolism , Heart Failure/etiology , Heart Failure/metabolism , Sodium Channels/metabolism , Action Potentials , Animals , Blotting, Western , Disease Models, Animal , Electrocardiography , Glycosylation , Humans , Mice , Mice, Transgenic , Neuraminidase/pharmacology , Patch-Clamp Techniques , Protein Processing, Post-Translational , Sodium/metabolismABSTRACT
The patterned activity generated by the pyloric circuit in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus, results not only from the synaptic connectivity between the 14 component neurons but also from differences in the intrinsic properties of the neurons. Presumably, differences in the complement and distribution of expressed ion channels endow these neurons with many of their distinct attributes. Each pyloric cell type possesses a unique, modulatable transient potassium current, or A-current (I(A)), that is instrumental in determining the output of the network. Two genes encode A-channels in this system, shaker and shal. We examined the hypothesis that cell-specific differences in shaker and shal channel distribution contribute to diversity among pyloric neurons. We found a stereotypic distribution of channels in the cells, such that each channel type could contribute to different aspects of the firing properties of a cell. Shal is predominantly found in the somatodendritic compartment in which it influences oscillatory behavior and spike frequency. Shaker channels are exclusively localized to the membranes of the distal axonal compartments and most likely affect distal spike propagation. Neither channel is detectably inserted into the preaxonal or proximal portions of the axonal membrane. Both channel types are targeted to synaptic contacts at the neuromuscular junction. We conclude that the differential targeting of shaker and shal to different compartments is conserved among all the pyloric neurons and that the channels most likely subserve different functions in the neuron.
Subject(s)
Ganglia, Invertebrate/physiology , Neurons/physiology , Potassium Channels/physiology , Amino Acid Sequence , Animals , Axons/physiology , Axons/ultrastructure , Cell Membrane/physiology , Cell Membrane/ultrastructure , Molecular Sequence Data , Nephropidae , Neurites/physiology , Neurites/ultrastructure , Neurons/ultrastructure , Potassium Channels/analysis , Potassium Channels/genetics , Shaker Superfamily of Potassium Channels , Synapses/physiologyABSTRACT
The patterns of expression of voltage gated potassium channel genes of the Shaker family have been mapped in identified neurons of the lobster (Homarus americanus) ventral nerve cord using a single cell reverse transcriptase polymerase chain reaction procedure. Using specific oligonucleotides derived from the sequences of the shaker, shab, and shaw genes of the spiny lobster, Panulirus interruptus, we detected the corresponding potassium channel DNA fragments from Homarus americanus. The Homarus DNA fragments are 87-98% identical at the nucleotide level to the Panulirus DNA fragments. We used the Panulirus primers to measure the complement of RNAs for shaker, shab, and shaw in single identified cells that use GABA, glutamate, octopamine or serotonin as chemical messengers. Shaker and shaw RNAs were found in all four identified neuron types but shab RNA was not detected in serotonin cells under the present experimental conditions. All cells expressed alpha-tubulin RNA, which serves as an internal control suggesting that cells are intact after dissection. In glial cells that surround the neuronal cell bodies, the potassium channel genes are expressed at low to non-detectable levels.
Subject(s)
Gene Expression , Nephropidae/metabolism , Neurons/metabolism , Potassium Channels/genetics , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/genetics , Sequence Homology, Nucleic Acid , Shaker Superfamily of Potassium ChannelsABSTRACT
Technological advances in electrophysiology and molecular biology in the last two decades have led to great progress in ion channel research. The invention of the patch-clamp recording technique has enabled the characterization of the biophysical and pharmacological properties of single channels. Rapid progress in the development of molecular biology techniques and their application to ion channel research led to the cloning, in the 1980s, of genes encoding all major classes of voltage- and ligand-gated ionic channels. It has become clear that operationally defined channel types represent extended families of ionic channels. Several experimental approaches have been developed to test whether there is a correlation between the detection of particular ion channel subunit mRNAs and the electrophysiological response to a pharmacological or electrical stimulus in a cell. In one method, whole-cell patch-clamp recording is performed on a cell in culture or tissue-slice preparation. The biophysical and pharmacological properties of the ionic channels of interest are characterized and the cytoplasmic contents of the recorded cell are then harvested into the patch pipette. In a variant of this method, the physiological properties of a cell are characterized with a two-electrode voltage clamp and, following the recording, the entire cell is harvested for its RNA. In both methods, the RNA from a single cell is reverse-transcribed into cDNA by a reverse transcriptase and subsequently amplified by the polymerase chain reaction, i.e. by the so-called single-cell/reverse transcription/polymerase chain reaction method (SC-RT-PCR). This review presents an analysis of the results of work obtained by using a combination of whole-cell patch-clamp recording or two-electrode voltage clamp and SC-RT-PCR with emphasis on its potential and limitations for quantitative analysis.
Subject(s)
Ion Channels/genetics , Patch-Clamp Techniques/methods , Polymerase Chain Reaction/methods , Transcriptional Activation , Animals , DNA Primers , Ion Channels/metabolism , Neurons/metabolism , RNA/biosynthesis , RNA/isolation & purification , RNA Processing, Post-Transcriptional , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We consider whole-cell voltage-clamp data of isolated currents characterized by the Hodgkin-Huxley paradigm. We examine the errors associated with the typical parameter estimation method for these data and show them to be unsatisfactorally large especially if the time constants of activation and inactivation are not sufficiently separated. The size of these errors is due to the fact that the steady-state and kinetic properties of the current are estimated disjointly. We present an improved parameter estimation method that utilizes all of the information in the voltage-clamp conductance data to estimate steady-state and kinetic properties simultaneously and illustrate its success compared to the standard method using simulated data and data from P. interruptus shal channels expressed in oocytes.
Subject(s)
Models, Neurological , Neurons/physiology , Patch-Clamp Techniques/standards , Animals , DNA, Complementary/pharmacology , Electric Conductivity , Ion Channels/genetics , Ion Channels/metabolism , Least-Squares Analysis , Membrane Potentials/physiology , Oocytes/physiologyABSTRACT
In Drosophila shaker voltage-dependent potassium channels, alternative splicing at the amino and carboxy termini produces currents with different electrophysiological characteristics. We have cloned alternatively spliced forms of shaker from the spiny lobster Panulirus interruptus. Alternative exons were found at three sites of the gene; eight different 5' exons, two alternative exons encoding the pore-forming P region, and an alternative 3' exon. Two of the different amino terminal splice forms were expressed with two alternatively spliced pore forms to produce channels with markedly different characteristics. One of the amino termini produced a channel with transient characteristics while the other produced a delayed rectifier-type channel. The effects of alternative exons at the amino terminus and in the P region appear to be additive. Our results provide new information on the structural requirements for rapid N-type inactivation.
Subject(s)
Alternative Splicing , Nephropidae/physiology , Potassium Channels/physiology , Amino Acid Sequence , Animals , Cloning, Molecular , Molecular Sequence DataABSTRACT
A molecular analysis of motor pattern generation is an essential complement to electrophysiological and computational investigations. In arthropods, A-channels are posttranslationally modified multimeric proteins containing Shaker family alpha-subunits that may interact with beta-subunits, gamma-subunits, and other auxiliary proteins. One consequence of A-channel structure is that several mechanisms could underlie the cell-specific differences in pyloric IAs including differential gene expression, alternate splicing, and posttranslational modifications. Oocyte expression studies, single-cell RT-PCR, and immunocytochemistry suggest that differential alpha-subunit gene expression is not a mechanism for creating pyloric IA heterogeneity, and that the same gene, shal, encodes the alpha-subunits for the entire family of somatic IAs in the pyloric network. Changes in the level of shal gene expression alter A-channel density between cells, but cannot account for the differences in the biophysical properties of the six pyloric IAs. Preliminary data suggest that the shal gene also encodes the A-channel alpha-subunits for the coarse and fine neuropil but not for most axons. A second gene, shaker, encodes the A-channel alpha-subunits in the majority of axons and at the neuromuscular junction. The distinct properties of the two types of A-channels are consistent with the different roles of IA at the different locations. Both the shaker and shal genes are alternately spliced, and investigations are under way to determine whether alternate splicing is a mechanism for generating pyloric IA heterogeneity.
Subject(s)
Motor Neurons/chemistry , Motor Neurons/physiology , Periodicity , Potassium Channels/genetics , Animals , Ganglia, Invertebrate/chemistry , Ganglia, Invertebrate/cytology , Ganglia, Invertebrate/physiology , Gene Expression/physiology , Nephropidae , Pylorus/innervationABSTRACT
We have cloned cDNAs for the shaker potassium channel gene from the spiny lobster Panulirus interruptus. As previously found in Drosophila, there is alternative splicing at the 5' and 3' ends of the coding region. However, in Panulirus shaker, alternative splicing also occurs within the pore-forming region of the protein. Three different splice variants were found within the P region, two of which bestow unique electrophysiological characteristics to channel function. Pore I and pore II variants differ in voltage dependence for activation, kinetics of inactivation, current rectification, and drug resistance. The pore 0 variant lacks a P region exon and does not produce a functional channel. This is the first example of alternative splicing within the pore-forming region of a voltage-dependent ion channel. We used a recently identified potassium channel blocker, kappa-conotoxin PVIIA, to study the physiological role of the two pore forms. The toxin selectively blocked one pore form, whereas the other form, heteromers between the two pore forms, and Panulirus shal were not blocked. When it was tested in the Panulirus stomatogastric ganglion, the toxin produced no effects on transient K+ currents or synaptic transmission between neurons.
Subject(s)
Conotoxins , Potassium Channels/genetics , RNA Splicing , Amino Acid Sequence , Animals , Cell Membrane Permeability/drug effects , DNA, Complementary/genetics , Drosophila Proteins , Drosophila melanogaster/genetics , Ganglia, Invertebrate/drug effects , Ganglia, Invertebrate/metabolism , Ion Channel Gating/drug effects , Ion Transport/drug effects , Molecular Sequence Data , Mollusk Venoms/pharmacology , Nephropidae/genetics , Neurotoxins/pharmacology , Oocytes , Potassium Channel Blockers , Potassium Channels/chemistry , Protein Conformation , Sequence Alignment , Sequence Homology, Amino Acid , Shaker Superfamily of Potassium Channels , Species Specificity , Transfection , Xenopus laevisABSTRACT
Different Shaker family alpha-subunit genes generate distinct voltage-dependent K+ currents when expressed in heterologous expression systems. Thus it generally is believed that diverse neuronal K+ current phenotypes arise, in part, from differences in Shaker family gene expression among neurons. It is difficult to evaluate the extent to which differential Shaker family gene expression contributes to endogenous K+ current diversity, because the specific Shaker family gene or genes responsible for a given K+ current are still unknown for nearly all adult neurons. In this paper we explore the role of differential Shaker family gene expression in creating transient K+ current (IA) diversity in the 14-neuron pyloric network of the spiny lobster, Panulirus interruptus. We used two-electrode voltage clamp to characterize the somatic IA in each of the six different cell types of the pyloric network. The size, voltage-dependent properties, and kinetic properties of the somatic IA vary significantly among pyloric neurons such that the somatic IA is unique in each pyloric cell type. Comparing these currents with the IAs obtained from oocytes injected with Panulirus shaker and shal cRNA (lobster Ishaker and lobster Ishal, respectively) reveals that the pyloric cell IAs more closely resemble lobster Ishal than lobster Ishaker. Using a novel, quantitative single-cell-reverse transcription-PCR method to count the number of shal transcripts in individual identified pyloric neurons, we found that the size of the somatic IA varies linearly with the number of endogenous shal transcripts. These data suggest that the shal gene contributes substantially to the peak somatic IA in all neurons of the pyloric network.
Subject(s)
Drosophila/genetics , Ganglia, Invertebrate/physiology , Gene Expression , Mutation , Neurons/physiology , Potassium/physiology , Pylorus/innervation , Pylorus/physiology , Animals , Cell Separation , Electric Conductivity , Ganglia, Invertebrate/cytology , Nephropidae , Oocytes , Patch-Clamp Techniques , Polymerase Chain Reaction , Transcription, Genetic , XenopusABSTRACT
We cloned and sequenced the cDNA for the shaw gene, encoding a voltage-dependent potassium (K+) channel, from the spiny lobster, Panulirus interruptus. The deduced amino acid sequence has a high degree of homology to the Drosophila melanogaster Shaw protein. In addition, lobster Shaw has several putative sites for post-translational modifications.
Subject(s)
Nephropidae/genetics , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA , Drosophila melanogaster/genetics , Molecular Sequence Data , Shaw Potassium ChannelsABSTRACT
The transient potassium (K+) current, or A-current (IA), plays an essential role in shaping the firing properties of identified neurons in the 14-cell pyloric network in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. The different cells in the pyloric network have distinct IAs. To begin to understand the molecular basis for IA heterogeneity, we examined the relationship between the Panulirus shal current, the IAs in the lateral pyloric (LP) and pyloric dilator (PY) cells, and the Drosophila shal current. After isolating a complete open reading frame for lobster shal 1, which shows significant sequence homology to the fly, mouse, and rat shal homologs, we used a single-cell reverse transcription polymerase chain reaction method to demonstrate that the shal 1 gene was expressed in the LP and PY cells. Next, we compared the lobster shal 1 current generated in a Xenopus oocyte expression system to the IAs in the LP and PY neurons as well as to the Drosophila shal current in Xenopus oocytes. While the transient K+ lobster shal 1 current was similar to the IAs in pyloric neurons, a detailed comparison shows that they are not identical and differ in kinetic and voltage-dependent parameters. The highly homologous lobster and fly shal genes also produce currents with some significant similarities and differences in an oocyte expression system.
Subject(s)
Drosophila/genetics , Nephropidae/genetics , Neurons/metabolism , Potassium Channels/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , Electrophysiology , Gene Expression , Introns , Molecular Probes/genetics , Molecular Sequence Data , Oocytes/metabolism , Potassium Channels/metabolism , Potassium Channels/physiology , Pylorus/innervation , Xenopus/metabolismABSTRACT
We have developed a reverse transcription-polymerase chain reaction (RT-PCR) method to examine single neurons and glial cells in the stomatogastric ganglion of the spiny lobster Panulirus interruptus for the expression of four members of the Shaker family of potassium channel genes. Using this technique we found that shaker, shab, shaw, and shal are expressed in 100%, 78%, 100%, and 94% of stomatogastric neurons. Furthermore, neuronal shab, shaw, and shal transcript levels vary among cells in a manner which is independent of cell size. We also detected Shaker family gene expression in glial cells. Shaker, shaw, and shal are detectably expressed in 100%, 63%, and 100% of the glial caps examined, respectively, while shab gene expression could not be detected in glial cells.
Subject(s)
Gene Expression/physiology , Neuroglia/metabolism , Neurons/metabolism , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Animals , Delayed Rectifier Potassium Channels , Digestive System/chemistry , Digestive System/innervation , Multigene Family , Nephropidae , Neurons/chemistry , Polymerase Chain Reaction/methods , Shab Potassium Channels , Shaker Superfamily of Potassium Channels , Shal Potassium Channels , Shaw Potassium ChannelsABSTRACT
A single shab gene exists in the lobster, Panulirus interruptus, and undergoes alternate splicing to produce multiple transcripts. Using in situ hybridization we have determined the expression pattern of the shab gene in identified neurons of the pyloric network. The shab gene is consistently expressed at a low level in the Ventricular Dilator cell, a high level in the Pyloric Dilator cell, and is not detectably expressed in the Lateral Pyloric or Inferior Cardiac cells. Shab gene expression in the Anterior Burster cell varies from animal to animal. The electrophysiologically heterogeneous group of eight Pyloric Constrictor cells also shows differences in shab gene expression. These results support the idea that differences in shab gene expression contribute to the unique electrophysiological phenotypes displayed by each cell type.