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Biomedical subjects

Diane Lipscombe

Publications and source records attributed to Diane Lipscombe.

11 recordsLinked to original sources
PubMed2006

Alternative splicing in the C-terminus of CaV2.2 controls expression and gating of N-type calcium channels.

N-type Ca(V)2.2 calcium channels localize to presynaptic nerve terminals of nociceptors where they control neurotransmitter release. Nociceptive neurons express a unique set of ion channels and receptors important for optimizing their role in transmission of noxious stimuli. Included among these is a structurally and functionally distinct N-type calcium channel splice isoform, Ca(V)2.2e[37a], expressed in a subset of nociceptors and with limited expression in other parts of the nervous system. Ca(V)2.2[e37a] arises from the mutually exclusive replacement of e37a for e37b in the C-terminus of Ca(V)2.2 mRNA. N-type current densities in nociceptors that express a combination of Ca(V)2.2e[37a] and Ca(V)2.2e[37b] mRNAs are significantly larger compared to cells that express only Ca(V)2.2e[37b]. Here we show that e37a supports increased expression of functional N-type channels and an increase in channel open time as compared to Ca(V)2.2 channels that contain e37b. To understand how e37a affects N-type currents we compared macroscopic and single-channel ionic currents as well as gating currents in tsA201 cells expressing Ca(V)2.2e[37a] and Ca(V)2.2e[37b]. When activated, Ca(V)2.2e[37a] channels remain open for longer and are expressed at higher density than Ca(V)2.2e[37b] channels. These unique features of the Ca(V)2.2e[37a] isoform combine to augment substantially the amount of calcium that enters cells in response to action potentials. Our studies of the e37a/e37b splice site reveal a multifunctional domain in the C-terminus of Ca(V)2.2 that regulates the overall activity of N-type calcium channels in nociceptors.

Action Potentials↗
PubMed2006

Single-channel analysis of a point mutation of a conserved serine residue in the S2 ligand-binding domain of the NR2A NMDA receptor subunit.

We have examined the function of a conserved serine residue (Ser670) in the S2 ligand-binding region of the NR2A N-methyl-d-aspartate (NMDA) receptor subunit, using recombinant NR1/NR2A receptors expressed in Xenopus laevis oocytes. Mutation of Ser670 to glycine (S670G) in NR2A reduced the potency of glutamate by 124-fold. Single-channel conductance and the duration of apparent open periods of NR2A(S670G) receptor mutants were, however, indistinguishable from wild-type NMDA receptors. NR1/NR2A(S670G) shut-time distributions were best described by a mixture of six exponential components, and the four shortest shut intervals of each distribution were considered to occur within a channel activation (burst). Bursts of single-channel openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer and had mean durations of 9.6 +/- 0.5 and 29.6 +/- 1.5 ms. The overall channel open probability during a burst was high (mean, 0.83 +/- 0.06). Consistent with a shortening of NMDA receptor-channel burst lengths was the observation of an increased deactivation rate of macroscopic currents evoked by brief applications of glutamate to outside-out membrane patches. Correlations between shut times and adjacent open times were observed in all data records. Noticeably, shorter than average openings tended to occur next to long closed periods, whereas longer than average openings tended to occur next to short closings. Our single-channel data, together with modelling using a kinetic scheme to describe channel activations, support our hypothesis that the S670G point mutation reduces the dwell time of glutamate in its binding site.

Animals↗
PubMed2005

Neuronal L-type calcium channels open quickly and are inhibited slowly.

Neuronal L-type calcium channels are essential for regulating activity-dependent gene expression, but they are thought to open too slowly to contribute to action potential-dependent calcium entry. A complication of studying native L-type channels is that they represent a minor fraction of the whole-cell calcium current in most neurons. Dihydropyridine antagonists are therefore widely used to establish the contribution of L-type channels to various neuronal processes and to study their underlying biophysical properties. The effectiveness of these antagonists on L-type channels, however, varies with stimulus and channel subtype. Here, we study recombinant neuronal L-type calcium channels, CaV1.2 and CaV1.3. We show that these channels open with fast kinetics and carry substantial calcium entry in response to individual action potential waveforms, contrary to most studies of native L-type currents. Neuronal CaV1.3 L-type channels were as efficient as CaV2.2 N-type channels at supporting calcium entry during action potential-like stimuli. We conclude that the apparent slow activation of native L-type currents and their lack of contribution to single action potentials reflect the state-dependent nature of the dihydropyridine antagonists used to study them, not the underlying properties of L-type channels.

Action Potentials↗
PubMed2005

Neuronal proteins custom designed by alternative splicing.

The evolution of alternative splicing in eukaryotes greatly expanded the number of functionally distinct proteins that could be produced from a finite gene pool. Extensive in the brains of higher organisms, alternative splicing might be the primary mechanism for generating the spectrum of protein activities that support complex brain functions. Alternative splicing is controlled at the level of individual neurons to custom design proteins for optimal performance. The expression profiles of splice isoforms are modified during development and as neuronal activity changes. Alternative splicing can lead to incremental, long lasting changes in ion channel and receptor activities, independent of changes in gene transcription. Recent studies of tissue-specific splicing factors are revealing how coordinated alterations in alternative splicing of RNA transcripts control synaptic function.

Alternative Splicing↗
PubMed2004

Alternative splicing in the voltage-sensing region of N-Type CaV2.2 channels modulates channel kinetics.

The CaV2.2 gene encodes the functional core of the N-type calcium channel. This gene has the potential to generate thousands of CaV2.2 splice isoforms with different properties. However, the functional significance of most sites of alternative splicing is not established. The IVS3-IVS4 region contains an alternative splice site that is conserved evolutionarily among CaValpha1 genes from Drosophila to human. In CaV2.2, inclusion of exon 31a in the IVS3-IVS4 region is restricted to the peripheral nervous system, and its inclusion slows the speed of channel activation. To investigate the effects of exon 31a in more detail, we generated four tsA201 cell lines stably expressing CaV2.2 splice isoforms. Coexpression of auxiliary CaVbeta and CaValpha2delta subunits was required to reconstitute currents with the kinetics of N-type channels from neurons. Channels including exon 31a activated and deactivated more slowly at all voltages. Current densities were high enough in the stable cell lines co-expressing CaValpha2delta to resolve gating currents. The steady-state voltage dependence of charge movement was not consistently different between splice isoforms, but on gating currents from the exon 31a-containing CaV2.2 isoform decayed with a slower time course, corresponding to slower movement of the charge sensor. Exon 31a-containing CaV2.2 is restricted to peripheral ganglia; and the slower gating kinetics of CaV2.2 splice isoforms containing exon 31a correlated reasonably well with the properties of native N-type currents in sympathetic neurons. Our results suggest that alternative splicing in the S3-S4 linker influences the kinetics but not the voltage dependence of N-type channel gating.

Alternative Splicing↗
PubMed2004

Cumulative inactivation of N-type CaV2.2 calcium channels modified by alternative splicing.

The Ca(V)2 family of voltage-gated calcium channels, present in presynaptic nerve terminals, regulates exocytosis and synaptic transmission. Cumulative inactivation of these channels occurs during trains of action potentials, and this may control short-term dynamics at the synapse. Inactivation during brief, repetitive stimulation is primarily attributed to closed-state inactivation, and several factors modulate the susceptibility of voltage-gated calcium channels to this form of inactivation. We show that alternative splicing of an exon in a cytoplasmic region of the Ca(V)2.2 channel modulates its sensitivity to inactivation during trains of action potential waveforms. The presence of this exon, exon 18a, protects the Ca(V)2.2 channel from entry into closed-state inactivation specifically during short (10 ms to 3 s) and small depolarizations of the membrane potential (-60 mV to -50 mV). The reduced sensitivity to closed-state inactivation within this dynamic range likely underlies the differential responsiveness of Ca(V)2.2 splice isoforms to trains of action potential waveforms. Regulated alternative splicing of Ca(V)2.2 represents a possible mechanism for modulating short-term dynamics of synaptic efficacy in different regions of the nervous system.

Action Potentials↗
PubMed2004

Cell-specific alternative splicing increases calcium channel current density in the pain pathway.

N-type calcium channels are critical for pain transduction. Inhibitors of these channels are powerful analgesics, but clinical use of current N-type blockers remains limited by undesirable actions in other regions of the nervous system. We now demonstrate that a unique splice isoform of the N-type channel is restricted exclusively to dorsal root ganglia. By a combination of functional and molecular analyses at the single-cell level, we show that the DRG-specific exon, e37a, is preferentially present in Ca(V)2.2 mRNAs expressed in neurons that contain nociceptive markers, VR1 and Na(V)1.8. Cell-specific inclusion of exon 37a correlates closely with significantly larger N-type currents in nociceptive neurons. This unique splice isoform of the N-type channel could represent a novel target for pain management.

Alternative Splicing↗
PubMed2004

L-type calcium channels: the low down.

L-type calcium channels couple membrane depolarization in neurons to numerous processes including gene expression, synaptic efficacy, and cell survival. To establish the contribution of L-type calcium channels to various signaling cascades, investigators have relied on their unique pharmacological sensitivity to dihydropyridines. The traditional view of dihydropyridine-sensitive L-type calcium channels is that they are high-voltage-activating and have slow activation kinetics. These properties limit the involvement of L-type calcium channels to neuronal functions triggered by strong and sustained depolarizations. This review highlights literature, both long-standing and recent, that points to significant functional diversity among L-type calcium channels expressed in neurons and other excitable cells. Past literature contains several reports of low-voltage-activated neuronal L-type calcium channels that parallel the unique properties of recently cloned CaV1.3 L-type channels. The fast kinetics and low activation thresholds of CaV1.3 channels stand in stark contrast to criteria currently used to describe L-type calcium channels. A more accurate view of neuronal L-type calcium channels encompasses a broad range of activation thresholds and recognizes their potential contribution to signaling cascades triggered by subthreshold depolarizations.

Animals↗
PubMed2002

Functional diversity in neuronal voltage-gated calcium channels by alternative splicing of Ca(v)alpha1.

Alternative splicing is a critical mechanism used extensively in the mammalian nervous system to increase the level of diversity that can be achieved by a set of genes. This review focuses on recent studies of voltage-gated calcium (Ca) channel Ca(v)alpha1 subunit splice isoforms in neurons. Voltage-gated Ca channels couple changes in neuronal activity to rapid changes in intracellular Ca levels that in turn regulate an astounding range of cellular processes. Only ten genes have been identified that encode Ca(v)alpha1 subunits, an insufficient number to account for the level of functional diversity among voltage-gated Ca channels. The consequences of regulated alternative splicing among the genes that comprise voltage-gated Ca channels permits specialization of channel function, optimizing Ca signaling in different regions of the brain and in different cellular compartments. Although the full extent of alternative splicing is not yet known for any of the major subtypes of voltage-gated Ca channels, it is already clear that it adds a rich layer of structural and functional diversity".

Alternative Splicing↗