Andrew J. Castiglioni

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

Neurotransmitter release from mammalian sensory neurons is controlled by CaV2.2 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 Gβγ, 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 CaV2.2) creates, de novo, a C-terminal module that mediates voltage-independent inhibition. This inhibitory pathway requires tyrosine kinase activation but not Gβγ. 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.

Differential Role of N-Type Calcium Channel Splice Isoforms in Pain

N-type calcium channels are essential mediators of spinal nociceptive transmission. The core subunit of the N-type channel is encoded by a single gene, and multiple N-type channel isoforms can be generated by alternate splicing. In particular, cell-specific inclusion of an alternatively spliced exon 37a generates a novel form of the N-type channel that is highly enriched in nociceptive neurons and, as we show here, downregulated in a neuropathic pain model. Splice isoform-specific small interfering RNA silencing in vivo reveals that channels containing exon 37a are specifically required for mediating basal thermal nociception and for developing thermal and mechanical hyperalgesia during inflammatory and neuropathic pain. In contrast, both N-type channel isoforms (e37a- and e37b-containing) contribute to tactile neuropathic allodynia. Hence, exon 37a acts as a molecular switch that tailors the channels toward specific roles in pain.

The varitint-waddler (Va) deafness mutation in TRPML3 generates constitutive, inward rectifying currents and causes cell degeneration

Varitint-waddler (Va and VaJ) mice are deaf and have vestibular impairment, with inner ear defects that include the degeneration and loss of sensory hair cells. The semidominant Va mutation results in an alanine-to-proline substitution at residue 419 (A419P) of the presumed ion channel TRPML3. Another allele, VaJ, has the A419P mutation in addition to an I362T mutation. We found that hair cells, marginal cells of stria vascularis, and other cells lining the cochlear and vestibular endolymphatic compartments express TRPML3. When heterologously expressed in LLC-PK1-CL4 epithelial cells, a culture model for hair cells, TRPML3 accumulated in lysosomes and in espin-enlarged microvilli that resemble stereocilia. We also demonstrated that wild-type TRPML3 forms channels that are blocked by Gd3+, have a conductance of 50–70 pS and, like many other TRP channels, open at very positive potentials and thus rectify outwardly. In addition to this outward current, TRPML3(419P) and (I362T+A419P) generated a constitutive inwardly rectifying current that suggests a sensitivity to hyperpolarizing negative potentials and that depolarized the cells. Cells expressing TRPML3(A419P) or (I362T+A419P), but not wild-type TRPML3, died and were extruded from the epithelium in a manner reminiscent of degenerating hair cells in Va mice. The increased open probability of TRPML3(A419P) and (I362T+A419P) at physiological potentials likely underlies hair cell degeneration and deafness in Va and VaJ mice.

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

N‐type CaV2.2 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, CaV2.2e[37a], expressed in a subset of nociceptors and with limited expression in other parts of the nervous system. CaV2.2[e37a] arises from the mutually exclusive replacement of e37a for e37b in the C‐terminus of CaV2.2 mRNA. N‐type current densities in nociceptors that express a combination of CaV2.2e[37a] and CaV2.2e[37b] mRNAs are significantly larger compared to cells that express only CaV2.2e[37b]. Here we show that e37a supports increased expression of functional N‐type channels and an increase in channel open time as compared to CaV2.2 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 CaV2.2e[37a] and CaV2.2e[37b]. When activated, CaV2.2e[37a] channels remain open for longer and are expressed at higher density than CaV2.2e[37b] channels. These unique features of the CaV2.2e[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 CaV2.2 that regulates the overall activity of N‐type calcium channels in nociceptors.

Expression and vesicular localization of mouse Trpml3 in stria vascularis, hair cells, and vomeronasal and olfactory receptor neurons

TRPML3 is a member of the mucolipin branch of the transient receptor potential cation channel family. A dominant missense mutation in Trpml3 (also known as Mcoln3) causes deafness and vestibular impairment characterized by stereocilia disorganization, hair cell loss, and endocochlear potential reduction. Both marginal cells of the stria vascularis and hair cells express Trpml3 mRNA. Here we used in situ hybridization, quantitative RT‐qPCR, and immunohistochemistry with several antisera raised against TRPML3 to determine the expression and subcellular distribution of TRPML3 in the inner ear as well as in other sensory organs. We also use Trpml3 knockout tissues to distinguish TRPML3‐specific from nonspecific immunoreactivities. We find that TRPML3 localizes to vesicles of hair cells and strial marginal cells but not to stereociliary ankle links or pillar cells, which nonspecifically react with two antisera raised against TRPML3. Upon cochlear maturation, TRPML3 protein is redistributed to perinuclear vesicles of strial marginal cells and is augmented in inner hair cells vs. outer hair cells. Mouse somatosensory neurons, retinal neurons, and taste receptor cells do not appear to express physiologically relevant levels of TRPML3. Finally, we found that vomeronasal and olfactory sensory receptor cells do express TRPML3 mRNA and protein, which localizes to vesicles in their somas and dendrites as well as at apical dendritic knobs. J. Comp. Neurol. 519:1095–1114, 2011.

Differential Ubiquitination and Proteasome Regulation of CaV2.2 N-Type Channel Splice Isoforms

CaV2.2 (N-type) calcium channels control the entry of calcium into neurons to regulate essential functions but most notably presynaptic transmitter release. CaV2.2 channel expression levels are precisely controlled, but we know little of the cellular mechanisms involved. The ubiquitin proteasome system (UPS) is known to regulate expression of many synaptic proteins, including presynaptic elements, to optimize synaptic efficiency. However, we have limited information about ubiquitination of CaV2 channels. Here we show that CaV2.2 proteins are ubiquitinated, and that elements in the proximal C terminus of CaV2.2 encoded by exon 37b of the mouse Cacna1b gene predispose cloned and native channels to downregulation by the UPS. CaV2.2 channels containing e37b are expressed throughout the mammalian nervous system, but in some cells, notably nociceptors, sometimes e37a—not e37b—is selected during alternative splicing of CaV2.2 pre-mRNA. By a combination of biochemical and functional analyses we show e37b promotes a form of ubiquitination that is coupled to reduced CaV2.2 current density and increased sensitivity to the UPS. Cell-specific alternative splicing of e37a in nociceptors reduces CaV2.2 channel ubiquitination and sensitivity to the UPS, suggesting a role in pain processing.

Alternative Splicing in Voltage Gated Calcium Channels

The mammalian nervous system utilizes extensive alternative splicing to support an astounding level of structural and functional diversity in the voltage-gated calcium channel family. All major calcium channel subunits, Cavα1, Cavβ, and Cavα2γ, are subject to alternative splicing. The structural changes generated by this form of RNA processing are often quite subtle but clearly critical for highly effective coupling to specific signaling pathways. The inclusion or exclusion of certain alternatively spliced exons can be differentially regulated according to tissue type, cell type, and stage of development. In certain instances, structural and functional differences generated by alternative splicing translate into unique pharmacological phenotypes. Pharmacologically distinct splice isoforms preferentially expressed in specific tissues or cell types offer exciting new targets for the design of highly specific drugs.

Mucolipin co-deficiency causes accelerated endolysosomal vacuolation of enterocytes and failure-to-thrive from birth to weaning.

During the suckling period, intestinal enterocytes are richly endowed with endosomes and lysosomes, which they presumably utilize for the uptake and intracellular digestion of milk proteins. By weaning, mature intestinal enterocytes replace those rich in lysosomes. We found that mouse enterocytes before weaning express high levels of two endolysosomal cation channels, mucolipins 3 and 1 -products of Trpml3 and Trpml1 genes; moreover neonatal enterocytes of mice lacking both mucolipins (Trpml3−/−;Trpml1−/−) vacuolated pathologically within hours of birth and remained so until weaning. Ultrastructurally and chemically these fast-forming vacuoles resembled those that systemically appear in epithelial cells of mucolipidosis type IV (MLIV) patients, which bear mutations in Trpml1. Hence, lack of both mucolipins 1 and 3 causes an accelerated MLIV-type of vacuolation in enterocytes. The vacuoles were aberrant hybrid organelles with both endosomal and lysosomal components, and were not generated by alterations in endocytosis or exocytosis, but likely by an imbalance between fusion of lysosomes and endosomes and their subsequent scission. However, upon extensive vacuolation enterocytes displayed reduced endocytosis from the intestinal lumen, a defect expected to compromise nutrient uptake. Mice lacking both mucolipins suffered a growth delay that began after birth and continued through the suckling period but recovered after weaning, coinciding with the developmental period of enterocyte vacuolation. Our results demonstrate genetic redundancy between lysosomal mucolipins 3 and 1 in neonatal enterocytes. Furthermore, our Trpml3−/−;Trpml1−/− mice represent a polygenic animal model of the poorly-understood, and often intractable, neonatal failure-to-thrive with intestinal pathology. Our results implicate lysosomes in neonatal intestinal pathologies, a major cause of infant mortality worldwide, and suggest transient intestinal dysfunction might affect newborns with lysosomal storage disorders. Finally, we conclude that mucolipin-endowed lysosomes in the young play an evolutionarily-conserved role in the intracellular digestion of maternally-provided nutrients, whether milk in mammals or yolk in oviparous species.

MechanoTRPs and TRPA1.

Genetic and molecular searches in animals identify two families of ion channels used by specialized mechanosensory cells. These are the degenerin/epithelial Na+ channels (Deg/ENaCs) and transient receptor potential (TRP) channels. Some of these channels open in response to mechanical forces and/or mediate cellular responses to mechanical stimulation. TRPA1 is expressed in nociceptive neurons of peripheral ganglia and in the sensory epithelia of the inner ear. In nociceptors, TRPA1 forms chemosensitive channels that mediate the response to exogenous pain-producing chemicals as well as to the endogenous proalgesic bradykinin (BK). More indirect evidence suggests that TRPA1 might also form mechanosensory channels. Some of the TRP channels that mediate mechanical responses are not necessarily mechanically gated. For example, TRPV4 mutant mice have reduced sensitivity to noxious tactile stimulation, and heterologously expressed TRPV4 opens in response to hypotonic solution (which induces cell swelling and thus stretches membranes). TRPA1 genes in mammals are large, occupy around 50kb of chromosomal DNA and are encoded by at least 27 exons. In humans, the TRPA1 gene is located on chromosome 8q13.