Functional impact of alternative splicing of human T-type Cav3.3 calcium channels

Low-voltage-activated T-type (Cav3) Ca2+ channels produce low-threshold spikes that trigger burst firing in many neurons. The CACNA1I gene encodes the Cav3.3 isoform, which activates and inactivates much more slowly than the other Cav3 channels. These distinctive kinetic features, along with its brain-region-specific expression, suggest that Cav3.3 channels endow neurons with the ability to generate long-lasting bursts of firing. The human CACNA1I gene contains two regions of alternative splicing: variable inclusion of exon 9 and an alternative acceptor site within exon 33, which leads to deletion of 13 amino acids (Delta33). The goal of this study is to determine the functional consequences of these variations in the full-length channel. The cDNA encoding these regions were cloned using RT-PCR from human brain, and currents were recorded by whole cell patch clamp. Introduction of the Delta33 deletion slowed the rate of channel opening. Addition of exon 9 had little effect on kinetics, whereas its addition to Delta33 channels unexpectedly slowed both activation and inactivation kinetics. Modeling of neuronal firing showed that exon 9 or Delta33 alone reduced burst firing, whereas the combination enhanced firing. The major conclusions of this study are that the intracellular regions after repeats I and IV play a role in channel gating, that their effects are interdependent, suggesting a direct interaction, and that splice variation of Cav3.3 channels provides a mechanism for fine-tuning the latency and duration of low-threshold spikes.     

Long splice variant N type calcium channels are clustered at presynaptic transmitter release sites without modular adaptor proteins

The presynaptic N type Ca channel (CaV2.2) is associated with the transmitter release site apparatus and plays a critical role in the gating of transmitter release. It has been suggested that a distinct CaV2.2 long C terminal splice variant is targeted to the nerve terminal and is anchored at the release face by calcium/calmodulin-dependent serine protein kinase (CASK) and Munc-18-interacting protein (MINT), two modular adaptor proteins. We used the isolated chick ciliary ganglion calyx terminal together with two new antibodies (L4569, L4570) selective for CaV2.2 long C terminal splice variant to test these hypotheses. CaV2.2 long C terminal splice variant was present at the presynaptic transmitter release sites, as identified by Rab3a-interacting molecule (RIM) co-staining and quantitative immunocytochemistry. CASK was also present at the terminal both in conjunction with, and independent of its binding partner, MINT. Immunoprecipitation of CaV2.2 long C terminal splice variant from brain lysate coprecipitated CASK, confirming that these two proteins can form a complex. However, CASK was not colocalized either with CaV2.2 long C terminal splice variant or the transmitter release site marker RIM at the calyx terminal release face. Neither was MINT colocalized with CaV2.2 long C terminal splice variant. Our results show that native CaV2.2 long C terminal splice variant is targeted to the transmitter release sites at an intact presynaptic terminal. However, the lack of enrichment of CASK at the release site combined with the failure of this protein or its partner MINT to colocalize with CaV2.2 argues against the idea that these modular adaptor proteins anchor CaV2.2 at presynaptic nerve terminals.     

omega-conotoxin GVIA alters gating charge movement of N-type (CaV2.2) calcium channels

omega-conotoxin GVIA (omegaCTX) is a specific blocker of N-type calcium (CaV2.2) channels that inhibits neuropathic pain. While the toxin appears to be an open channel blocker, we show that N-channel gating charge movement is modulated. Gating currents were recorded from N-channels expressed along with beta2a and alpha2delta subunits in HEK293 cells in external solutions containing either lanthanum and magnesium (La-Mg) or 5 mM Ca2+ plus omegaCTX (omegaCTX-Ca). A comparison showed that omegaCTX induced a 10-mV right shift in the gating charge versus voltage (Q-V) relationship, smaller off-gating current time constant (tau Q(Off)), a lower tau Q(Off) voltage dependence, and smaller on-gating current (Q(On)) tau. We also examined gating current in La-Mg plus omegaCTX and found no significant difference from that in omegaCTX-Ca; this demonstrates that the modulation was induced by the toxin. A model with strongly reduced open-state occupancy reproduced the omegaCTX effect on gating current and showed that the gating modulation alone would inhibit N-current by 50%. This mechanism of N-channel inhibition could be exploited to develop novel analgesics that induce only a partial block of N-current, which may limit some of the side effects associated with the toxin analgesic currently approved for human use (i.e., Prialt).     

Biophysical properties of human Na v1.7 splice variants and their regulation by protein kinase A

The sodium channel Na(v)1.7 is preferentially expressed in nociceptive neurons and is believed to play a crucial role in pain sensation. Four alternative splice variants are expressed in human dorsal root ganglion neurons, two of which differ in exon 5 by two amino acids in the S3 segment of domain I (exons 5A and 5N). Two others differ in exon 11 by the presence (11L) or absence (11S) of an 11 amino acid sequence in the loop between domains I and II, an important region for PKA regulation. In the present study, we used the whole cell configuration of the patch-clamp technique to investigate the biophysical properties and 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) modulation of these splice variants expressed in tsA201 cells in the presence of the beta(1)-subunit. The alternative splicing of Na(v)1.7 had no effect on most of the biophysical properties of this channel, including activation, inactivation, and recovery from inactivation. However, development of inactivation experiments revealed that the isoform containing exon 5A had slower kinetics of inactivation for negative potentials than that of the variant containing exon 5N. This difference was associated with higher ramp current amplitudes for isoforms containing exon 5A. Moreover, 8Br-cAMP-mediated phosphorylation induced a negative shift of the activation curve of variants containing exon 11S, whereas inactivation properties were unchanged. Isoforms with exon 11L were not modulated by 8Br-cAMP-induced phosphorylation. We conclude that alternative splicing of human Na(v)1.7 can specifically modulate the biophysical properties and cAMP-mediated regulation of this channel. Changing the proportions of these variants may thus influence neuronal excitability and pain sensation.     

A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA

To explore the structural basis of alternative splicing, we have analyzed the splicing of pre-mRNAs containing an optional exon, E4, from the preprotachykinin gene. This gene encodes substance P and related tachykinin peptides by alternative splicing of a common pre-mRNA. We have shown that alternative splicing of preprotachykinin pre-mRNA occurs by preferential skipping of optional E4. The competing mechanism that incorporates E4 into the final spliced RNA is constrained by an initial block to splicing of the immediate upstream intervening sequence (IVS), IVS3. This block is relieved by sequential splicing, in which the immediate downstream IVS4 is removed first. The structural change resulting from the first splicing event is directly responsible for activation of IVS3 splicing. This structural rearrangement replaces IVS4 sequences with E5 and its adjacent IVS5 sequences. To determine how this structural change promoted IVS3 splicing, we asked what structural change(s) would restore activity of IVS3 splicing-defective mutants. The most significant effect was observed by a 2-nucleotide substitution that converted the 5' splice site of E4 to an exact consensus match, GUAAGU. Exon 5 sequences alone were found not to promote splicing when present in one or multiple copies. However, when a 15-nucleotide segment of IVS5 containing GUAAGU was inserted into a splicing-defective mutant just downstream of the hybrid exon segment E4E5, splicing activity was recovered. Curiously, the 72-nucleotide L2 exon of adenovirus, without its associated 5' splice site, activates splicing when juxtaposed to E4. Models for the activation of splicing by an RNA structural change are discussed.     

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

N-type Ca_V2.2 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_V2.2e[37a], expressed in a subset of nociceptors and with limited expression in other parts of the nervous system. Ca_V2.2[e37a] arises from the mutually exclusive replacement of e37a for e37b in the C-terminus of Ca_V2.2 mRNA. N-type current densities in nociceptors that express a combination of Ca_V2.2e[37a] and Ca_V2.2e[37b] mRNAs are significantly larger compared to cells that express only Ca_V2.2e[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_V2.2 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_V2.2e[37a] and Ca_V2.2e[37b]. When activated, Ca_V2.2e[37a] channels remain open for longer and are expressed at higher density than Ca_V2.2e[37b] channels. These unique features of the Ca_V2.2e[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_V2.2 that regulates the overall activity of N-type calcium channels in nociceptors.     

A118G Mu Opioid Receptor polymorphism increases inhibitory effects on Ca(V)2.2 channels

Single nucleotide polymorphisms (SNPs) in the human OPRM1 gene result in common variants of Mu Opioid Receptors (hMORs). The A118G SNP occurs at high frequency in certain human populations and produces an aminoacidic substitution: N40D (hMOR-N to hMOR-D) at protein level. N40D is reported to alter pain thresholds and morphine efficacy. hMORs inhibit Ca(V)2.2 channels (N-type currents) at presynaptic nociceptor terminals in dorsal horn, thus reducing calcium influx, transmitter release, and transmission of noxious signals. Nociceptors express different splice isoforms of Ca(V)2.2. Isoforms distinguished by the presence of alternatively spliced exon e37a are of interest because channels containing e37a are particularly enriched in nociceptors. Recent studies showed that Ca(V)2.2e37a is more sensitive to inhibition by Mu Opioid Receptors than the ubiquitous splice variant Ca(V)2.2e37b. Here, we evaluate the effect of hMOR-N and hMOR-D on cloned Ca(V)2.2e37a channels expressed in mammalian cells. We observe that hMOR-D inhibits Ca(V)2.2e37a currents at agonist concentrations 4-fold lower than those needed to inhibit Ca(V)2.2e37a currents by the same degree via hMOR-N. We observe little difference in hMOR-D and hMOR-N inhibition of Ca(V)2.2e37b currents. Our study demonstrates that this common site of OPRM1 polymorphism affects the inhibitory actions of MORs on both major Ca(V)2.2 isoforms expressed in nociceptors.     

Alternative splicing controls G protein-dependent inhibition of N-type calcium channels in nociceptors

Neurotransmitter release from mammalian sensory neurons is controlled by Ca(V)2.2 N-type calcium channels. N-type channels are a major target of neurotransmitters and drugs that inhibit calcium entry, transmitter release and nociception through their specific G protein-coupled receptors. G protein-coupled receptor inhibition of these channels is typically voltage-dependent and mediated by Gbetagamma, whereas N-type channels in sensory neurons are sensitive to a second G protein-coupled receptor pathway that inhibits the channel independent of voltage. Here we show that preferential inclusion in nociceptors of exon 37a in rat Cacna1b (encoding Ca(V)2.2) creates, de novo, a C-terminal module that mediates voltage-independent inhibition. This inhibitory pathway requires tyrosine kinase activation but not Gbetagamma. A tyrosine encoded within exon 37a constitutes a critical part of a molecular switch controlling N-type current density and G protein-mediated voltage-independent inhibition. Our data define the molecular origins of voltage-independent inhibition of N-type channels in the pain pathway.     

Functional significance of a deep intronic mutation in the ATM gene and evidence for an alternative exon 28a

Screening for ATM mutations is usually performed using genomic DNA as a template for PCR amplification across exonic regions, with the consequence that deep intronic sequences are not analyzed. Here we report a novel pseudoexon-retaining deep intronic mutation (IVS28-159A>G; g.75117A>G based on GenBank U82828.1) in a patient with ataxia-telangiectasia (A-T), as well as the identification of a previously unrecognized alternative exon in the ATM gene (exon 28a) expressed in lymphoblastoid cell lines (LCL) derived from normal individuals. cDNA analysis using the A-T patient's LCL showed the retention of two aberrant intronic segments of 112 and 190 nt between exons 28 and 29. Minigenes were constructed to determine the functional significance of two genomic changes in the region of aberrant splicing: IVS28-193C>T (g.75083C>T) and IVS28-159A>G, revealing that: 1) the first is a polymorphism; 2) IVS28-159A>G weakens the 5' splice site of the alternative exon 28a and activates a cryptic 5' splice site (ss) 83 nt downstream; and 3) wild-type constructs also retain a 29-nt segment (exon 28a) as part of both the 112- and 190-nt segments. Maximum entropy estimates of ss strengths corroborate the cDNA and minigene findings. Such mutations may prove relevant in planning therapy that targets specific splicing aberrations.     

Prediction of Mutant mRNA Splice Isoforms by Information Theory‐Based Exon Definition

Mutations that affect mRNA splicing often produce multiple mRNA isoforms, resulting in complex molecular phenotypes. Definition of an exon and its inclusion in mature mRNA relies on joint recognition of both acceptor and donor splice sites. This study predicts cryptic and exon‐skipping isoforms in mRNA produced by splicing mutations from the combined information contents (R_i, which measures binding‐site strength, in bits) and distribution of the splice sites defining these exons. The total information content of an exon (R_i,total) is the sum of the R_i values of its acceptor and donor splice sites, adjusted for the self‐information of the distance separating these sites, that is, the gap surprisal. Differences between total information contents of an exon (ΔR_i,total) are predictive of the relative abundance of these exons in distinct processed mRNAs. Constraints on splice site and exon selection are used to eliminate nonconforming and poorly expressed isoforms. Molecular phenotypes are computed by the Automated Splice Site and Exon Definition Analysis ( http://splice.uwo.ca ) server. Predictions of splicing mutations were highly concordant (85.2%; n = 61) with published expression data. In silico exon definition analysis will contribute to streamlining assessment of abnormal and normal splice isoforms resulting from mutations.     

Voltage-gated calcium channels in chronic pain: emerging role of alternative splicing

N- and T-type voltage-gated calcium channels are key established players in chronic pain. Current work suggests that alternative splicing of these channels constitutes an important aspect in the investigation of their roles in the pathogenesis of chronic pain. Recent N-type channel studies describe a nociceptor-enriched alternatively spliced module responsible for voltage-independent G protein modulation and internalization, which is implicated in the control of distinct nociceptive pathways. On the contrary, although a large body of work has demonstrated that peripheral Ca_v3.2-encoded T-type currents are involved in several types of chronic pain, little is known with respect to the expression of numerous newly discovered splice variants in specific pain pathways. The elucidation of the new layers of molecular complexity uncovered in N- and T-type channel splice variants and their respective locations and roles in different pain pathways will allow for the development of better therapeutic strategies for the treatment of chronic pain.     

Alternative splicing in the neural cell adhesion molecule pre-mRNA: regulation of exon 18 skipping depends on the 5'-splice site

Two isoforms of the neural cell adhesion molecule (NCAM), termed NCAM-180 and NCAM-140, derive from a single gene via inclusion or exclusion of the penultimate exon 18 (E18). This alternative splicing event is tissue-specific and regulated during differentiation. To explore its structural basis, we have analyzed the pattern of spliced mRNA generated from transiently transfected minigenes construct containing this exon and portions of the adjacent introns and exons faithfully reproduces the differentiation state-dependent alternative splicing of the endogenous pre-mRNA. By systematic deletion and replacement analysis, we scanned the minigene for the presence of functionally important cis-elements. We identified two sequences that affected differentiation state-dependent regulation. One, the central part of E18, does not seem to contain a specific cis-element essential for proper splice site choice, because extending the deletion restored correctly regulated expression of the splicing products. In contrast, the 5'-splice site is an important element for regulation. Replacing it with a corresponding sequence from the alpha-globin gene resulted in constitutive use of the optional exon. When placed in the alpha-globin gene it did not promote alternative splicing. Instead, we observed a strongly decreased efficiency of splicing of the downstream intron in undifferentiated cells. This block of splicing was partially relieved after differentiation. The results are consistent with a model in which skipping of E18 is controlled in part at the associated 5'-splice site by trans-acting factors that undergo quantitative or qualitative changes during differentiation of N2a cells.