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A B Ribera

Publications and source records attributed to A B Ribera.

At least 19 recordsLinked to original sources
PubMed2001

Kv2 channels form delayed-rectifier potassium channels in situ.

A non inactivating potassium current known as the delayed rectifier plays a major role in membrane repolarization during an action potential. Whereas several candidate genes exist that code for potassium current, the identities of the molecular isotypes that are responsible in situ for membrane repolarization remain unidentified. We report that Kv2 channels play a major role in action potential repolarization. Kv2 channel elimination resulted in a reduction of the density of noninactivating potassium current and a prolonged impulse duration. In contrast, suppression of noninactivating current carried by Kv1 channels was much less effective in increasing action potential durations. Thus, whereas different potassium channels encode sustained potassium current, their contributions to action potential repolarization vary and require direct examination in situ. Our results indicate that Kv2 subunits function as classic delayed-rectifier channels in vertebrate neurons.

Action Potentials↗
PubMed2001

Activity regulates programmed cell death of zebrafish Rohon-Beard neurons.

Programmed cell death is a normal aspect of neuronal development. Typically, twice as many neurons are generated than survive. In extreme cases, all neurons within a population disappear during embryogenesis or by early stages of postnatal development. Examples of transient neuronal populations include Cajal-Retzius cells of the cerebral cortex and Rohon-Beard cells of the spinal cord. The novel mechanisms that lead to such massive cell death have not yet been identified. We provide evidence that electrical activity regulates the cell death program of zebrafish Rohon-Beard cells. Activity was inhibited by reducing Na+ current in Rohon-Beard cells either genetically (the macho mutation) or pharmacologically (tricaine). We examined the effects of activity block on three different reporters of cell death: DNA fragmentation, cytoskeletal rearrangements and cell body loss. Both the mao mutation and pharmacological blockade of Na+ current reduced these signatures of the cell death program. Moreover, the mao mutation and pharmacological blockade of Na+ current produced similar reductions in Rohon-Beard cell death. The results indicate that electrical activity provides signals that are required for the normal elimination of Rohon-Beard cells.

Acetylation↗
PubMed2000

Synaptic activity modulates presynaptic excitability.

Synaptic activity modulates synaptic efficacy and is important in learning and development. Here we show that development of excitability in presynaptic motor neurons required synaptic activation of postsynaptic muscle cells. Synaptic blockade broadened action potentials and decreased repetitive firing of presynaptic neurons. Consistent with these findings, synaptic blockade also decreased potassium-current density in the presynaptic cell. Application of neurotrophin-3, but not related neurotrophins, prevented these changes. Recordings from patches of somatic membrane indicated that modifications of presynaptic potassium and sodium currents occurred in a remote, nonsynaptic compartment. Thus, activity-dependent postsynaptic signals modulated presynaptic excitability, potentially regulating transmission at all synapses of the presynaptic cell.

Action Potentials↗
PubMed1999

Xenopus embryonic spinal neurons express potassium channel Kvbeta subunits.

Developmental regulation of voltage-dependent delayed rectifier potassium current (I(Kv)) of Xenopus primary spinal neurons regulates the waveform of the action potential. I(Kv) undergoes a tripling in density and acceleration of it activation kinetics during the initial day of its appearance. Another voltage-dependent potassium current, the A current, is acquired during the subsequent day and contributes to further shortening of the impulse duration. To decipher the molecular mechanisms underlying this functional differentiation, we are identifying potassium channel genes expressed in the embryonic amphibian nervous system. Potassium channels consist of pore-forming (alpha) as well as auxiliary (beta) subunits. Here, we report the primary sequence, developmental localization, and functional properties of two Xenopus Kvbeta genes. On the basis of primary sequence, one of these (xKvbeta2) is highly conserved with Kvbeta2 genes identified in other species, whereas the other (xKvbeta4) appears to identify a new member of the Kvbeta family. Both are expressed in developing spinal neurons during the period of impulse maturation but in different neuronal populations. In a heterologous system, coexpression of xKvbeta subunits modulates properties of potassium current that are developmentally regulated in the endogenous I(Kv). Consistent with xKvbeta4's unique primary sequence, the repertoire of functional effects it has on coexpressed Kv1alpha subunits is novel. Taken together, the results implicate auxiliary subunits in regulation of potassium current function and action potential waveforms in subpopulations of embryonic primary spinal neurons.

Amino Acid Sequence↗
PubMed1999

Potassium currents in developing neurons.

In Xenopus spinal neurons, delayed rectifier type voltage-dependent potassium currents (IKv) are developmentally regulated. These currents play a pivotal role in maturation of the action potential from a long-duration calcium-dependent impulse to a brief sodium-dependent one. Although spinal neurons are heterogeneous, IKv undergoes a synchronized and homogeneous developmental functional up-regulation across this diverse population of motor, sensory, and interneurons. This finding suggested that the diverse population of neurons expressed a common potassium channel. Thus, recent efforts have been directed towards cloning the relevant potassium channel gene. However, these molecular studies reveal an unsuspected heterogeneity in the molecular components of voltage-dependent potassium channels. Further, synchronous differentiation of IKv is achieved via heterogeneous Kv channel gene expression.

Action Potentials↗
PubMed1998

Heteromultimeric potassium channels formed by members of the Kv2 subfamily.

Four alpha-subunits are thought to coassemble and form a voltage-dependent potassium (Kv) channel. Kv alpha-subunits belong to one of four major subfamilies (Kv1, Kv2, Kv3, Kv4). Within a subfamily up to eight different genetic isotypes exist (e.g., Kv1.1, Kv1.2). Different isotypes within the Kv1 or Kv3 subfamily coassemble. It is not known, however, whether the only two members of the vertebrate Kv2 subfamily identified thus far, Kv2.1 and Kv2.2, heteromultimerize. This might account for the lack of detection of heteromultimeric Kv2 channels in situ despite the coexpression of Kv2.1 and Kv2.2 mRNAs within the same cell. To probe whether Kv2 isotypes can form heteromultimers, we developed a dominant-negative mutant Kv2.2 subunit to act as a molecular poison of Kv2 subunit-containing channels. The dominant-negative Kv2.2 suppresses formation of functional channels when it is coexpressed in oocytes with either wild-type Kv2.2 or Kv2.1 subunits. These results indicate that Kv2.1 and Kv2.2 subunits are capable of heteromultimerization. Thus, in native cells either Kv2.1 and Kv2.2 subunits are targeted at an early stage to different biosynthetic compartments or heteromultimerization otherwise is inhibited.

Amino Acid Sequence↗
PubMed1998

Zebrafish touch-insensitive mutants reveal an essential role for the developmental regulation of sodium current.

Developmental changes in neuronal connectivity and membrane properties underlie the stage-specific appearance of embryonic behaviors. The behavioral response of embryonic zebrafish to tactile stimulation first appears at 27 hr postfertilization. Because the touch response requires the activation of mechanosensory Rohon-Beard neurons, we have used whole-cell recordings in semi-intact preparations to characterize Rohon-Beard cell electrical membrane properties in several touch-insensitive mutants and then to correlate the development of excitability in these cells with changes in wild-type behavior. Electrophysiological analysis of mechanosensory neurons of touch-insensitive zebrafish mutants indicates that in three mutant lines that have been examined the sodium current amplitudes are reduced, and action potentials either have diminished overshoots or are not generated. In macho mutants the action potential never overshoots, and the sodium current remains small; alligator and steifftier show similar but weaker effects. The effects are specific to sodium channel function; resting membrane potentials are unaffected, and outward currents of normal amplitude are present. Developmental analysis of sodium current expression in mechanosensory neurons of wild-type embryos indicates that, during the transition from a touch-insensitive to a touch-sensitive embryo, action potentials acquire larger overshoots and briefer durations as both sodium and potassium currents increase in amplitude. However, in macho touch-insensitive mutants, developmental changes in action potential overshoot and sodium current are absent despite the normal regulation of action potential duration and potassium current. Thus, the maturation of a voltage-dependent sodium current promotes a behavioral response to touch. A study of these mutants will allow insight into the genes controlling the maturation of the affected sodium current.

Action Potentials↗
PubMed1998

Development of electrical excitability in embryonic neurons: mechanisms and roles.

Xenopus spinal neurons serve as a nearly ideal population of excitable cells for study of developmental regulation of electrical excitability. On the one hand, the firing properties of these neurons can be directly examined at early stages of differentiation and membrane excitability changes as neurons mature. Underlying changes in voltage-dependent ion channels have been characterized and the mechanisms that bring about these changes are being defined. On the other hand, these neurons have been shown to be spontaneously active at stages when action potentials provide significant calcium entry. Calcium entry provokes further elevation of intracellular calcium via release from intracellular stores. The resultant transient elevations of intracellular calcium encode differentiation in their frequency. Recent studies have shown that different neuronal subpopulations enlist distinct mechanisms for regulation of excitability and recruit specific programs of differentiation by particular patterns of activity.

Action Potentials↗
PubMed1996

Temporal regulation of Shaker- and Shab-like potassium channel gene expression in single embryonic spinal neurons during K+ current development.

A developmental increase in density of delayed rectifier potassium current (IKv) in embryonic Xenopus spinal neurons shortens action potential durations and limits calcium influx governing neuronal differentiation. Although previous work demonstrates that maturation of IKv depends on general mRNA synthesis, it is not known whether increases in K+ channel gene transcripts direct maturation of the current. Accordingly, the developmental appearance of specific Kv potassium channel genes was determined using single-cell reverse transcription-PCR techniques after whole-cell recording of IKv during the period of its development. Detection of a coexpressed housekeeping gene along with the potassium channel gene controlled for successful aspiration of cellular mRNA and allowed scoring of cells in which Kv gene transcripts were not detected. Diverse types of Xenopus spinal neurons exhibit homogeneous development of IKv both in vivo and in culture. In contrast, transcripts of two genes encoding delayed rectifier current, Kv1.1 (Shaker) and Kv2.2 (Shab), are expressed heterogeneously during the period in which the current develops. Kv1.1 mRNA achieves maximal appearance in approximately 30% of cells, while IKv is immature; Kv2.2 mRNA appears later in approximately 60% of mature neurons. Kv1.1 and 2.2 are thus candidates for generation of IKv, and spinal neurons are a heterogeneous population with respect to potassium channel gene expression. Moreover, correlation of gene expression with current properties shows that neurons lacking Kv2.2 have a characteristic voltage dependence of activation of IKv.

Animals↗
PubMed1996

Xenopus spinal neurons express Kv2 potassium channel transcripts during embryonic development.

Developmentally regulated delayed rectifier potassium currents determine the waveform of the action potential in all Xenopus embryonic primary spinal neurons. To examine this developmental program at the molecular level, we have isolated Xenopus Kv2 potassium channel genes Kv2.1 and Kv2.2. Both genes induce functional heterologous expression of delayed rectifier potassium currents. Transcripts from both Kv2 genes are present in developing embryos; however, only Kv2.2 mRNA is detectable in embryonic spinal neurons. Notably, Kv2.2 transcripts localize to ventral spinal neurons, whereas previously described Kv1.1 transcripts are found in dorsal spinal neurons. Thus, spinal neuron subtypes express distinct potassium channel genes, yet they temporally coordinate functional expression of delayed rectifier potassium currents.

Amino Acid Sequence↗
PubMed1996

Homogeneous development of electrical excitability via heterogeneous ion channel expression.

Synchronous differentiation of delayed-rectifier potassium current regulates electrical excitability and calcium entry in motor, sensory, and interneurons of the developing amphibian spinal cord. Although Kv1 and Kv2 potassium channel transcripts are detectable in these cells, it is not known which transcript contributes to functional expression. Overexpression of a Kv1 dominant-negative subunit indicates that 20% of neurons have only Kv1 potassium currents. In other neurons, non-Kv1 channels function because the dominant-negative subunit either only partially suppresses or has no effect on current. Thus, diverse embryonic neurons coordinate differentiation of excitability yet rely on heterogeneous potassium channel gene expression.

Animals↗
PubMed1996

Probing molecular identity of native single potassium channels by overexpression of dominant negative subunits.

Overexpression of dominant negative subunits previously has been shown to affect the whole cell delayed-rectifier potassium current (Ikv) in Xenopus spinal neurons. Here, we show that effects of overexpression of wild-type and dominant negative Kv1 channels are evident at the single channel level. The goal of these studies was to match molecular species of Kv subunits to specific, functionally identified single voltage-dependent potassium channels. In a heterologous system (the Xenopus oocyte), co-expression of wild-type and dominant negative mutant Kv1.1 subunits results in loss of active channels rather than channels of altered conductance. However, in situ overexpression studies are difficult to interpret due to the diversity in the control population of channels. Therefore, identification of endogenous channel populations containing Kv1 subunits is limited. Future work will reduce the endogenous diversity of potassium channels by study of single channels in identified subtypes of neurons.

Animals↗
PubMed1995

Overexpression of potassium channel RNA: in vivo development rescues neurons from suppression of morphological differentiation in vitro.

Neuronal differentiation often proceeds differently in vitro than it does in vivo. Previous work demonstrated that overexpression of potassium channel RNA reduces the number of morphologically identifiable neurons that appear in cultures prepared from neural plate stage (17-1/2 hr) embryos (Jones and Ribera, 1994). Here, we report that morphological differentiation of neurons in situ is only slightly affected by overexpression of potassium channels. Endogenous factors appear to compensate for the effect of channel overexpression. Consistent with this view, when cultures are prepared from older neural tube embryos (22-24 hr), more neurons containing excess potassium channel RNA differentiate morphologically in vitro. Exposure in situ to a rapid intracellular calcium chelator, but not to tetrodotoxin, omega-conotoxin or a slow calcium chelator, prevents the compensation provided by extended development in vivo. Typically, RNA overexpression is limited to half of the embryo in order to provide an internal control. However, when potassium channel RNA is overexpressed throughout the embryo, few neurons differentiate morphologically in vitro, even if cultures are prepared from older neural tube embryos. Thus, recovery is possible if a minimum of 5 hr of further development in vivo is allowed under conditions in which rapid elevations of intracellular calcium are permitted and half of the nervous system has normal levels of potassium channel RNA. These results suggest that different or additional mechanisms operate in situ than in vitro to promote morphological differentiation of neurons.

Animals↗
PubMed1994

Overexpression of a potassium channel gene perturbs neural differentiation.

Functional regulation of potassium currents in developing neurons is pivotal for changes in excitability and action potential waveform. Here, we test whether an excess of potassium channel transcripts is sufficient to drive functional expression of potassium current and shortening of the duration of the action potential. Injection of Shaker-like potassium channel transcripts into two-cell stage embryos achieves increases in RNA levels. The elevated levels of potassium channel RNA produce larger delayed-rectifier currents. Action potential durations are briefer, indicating that larger potassium currents are not compensated by changes in inward currents. Strikingly, overexpression of potassium current RNA leads to a reduction in the number of morphologically differentiated neurons in culture. We suggest that, by prematurely reducing the duration of the impulse, early overexpression of potassium channel activity suppresses normal developmental cues.

Action Potentials↗
PubMed1993

Primary sensory neurons express a Shaker-like potassium channel gene.

Developmentally regulated action potentials are a hallmark of Rohon-Beard cells, a class of sensory neurons. In these neurons as well as other primary spinal neurons of Xenopus laevis, the functional differentiation of delayed-rectifier potassium current regulates the waveform of the action potential during the initial day of its appearance. Later, the acquisition of another voltage-dependent potassium current--the A current--plays a major role in regulating excitability. In order to understand the molecular basis of this functional differentiation, genes encoding voltage-dependent potassium currents expressed in the embryonic amphibian nervous system are being cloned. Here, we report the functional properties and developmental localization of a second Xenopus Shaker-like gene (Xenopus Kv 1.1; XSha1; GenBank accession number M94258) encoding a potassium current. Homology screening with the mouse gene MBK1 led to its isolation. Functional expression in oocytes identifies it as a delayed-rectifier current when assembled as a homooligomeric structure. Specific transcripts corresponding to XSha1 and to the previously cloned gene XSha2 are both detectable by RNase protection in RNA isolated from the embryonic nervous system. However, whole-mount in situ hybridization reveals the temporal pattern and cellular localization of XSha1 but not XSha2 mRNA, suggesting that the concentration of XSha2 transcripts in individual cells is lower than the threshold for detection by this method. Of particular interest, Rohon-Beard cells express XSha1 mRNA. In addition, XSha1 mRNA is detected in several structures containing neural crest derivatives including spinal ganglia, the trigeminal ganglion, and branchial arches; its presence in motor nerves and lateral spinal tracts suggests that both CNS and PNS glia express the mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗