Neuronal calcium channels: splicing for optimal performance

Calcium ion channels coordinate an astounding number of cellular functions. Surprisingly, only 10 Ca(V)alpha(1) subunit genes encode the structural cores of all voltage-gated calcium channels. What mechanisms exist to modify the structure of calcium channels and optimize their coupling to the rich spectrum of cellular functions? Growing evidence points to the contribution of post-translational alternative processing of calcium channel RNA as the main mechanism for expanding the functional potential of this important gene family. Alternative splicing of RNA is essential during neuronal development where fine adjustments in protein signaling promote and inhibit cell-cell interactions and underlie axonal guidance. However, attributing a specific functional role to an individual splice isoform or splice site has been difficult. In this regard, studies of ion channels are advantageous because their function can be monitored with precision, allowing even subtle changes in channel activity to be detected. Such studies are especially insightful when coupled with information about isoform expression patterns and cellular localization. In this paper, we focus on two sites of alternative splicing in the N-type calcium channel Ca(V)2.2 gene. We first describe cassette exon 18a that encodes a 21 amino acid segment in the II-III intracellular loop region of Ca(V)2.2. Here, we show that e18a is upregulated in the nervous system during development. We discuss these new data in light of our previous reports showing that e18a protects the N-type channel from cumulative inactivation. Second, we discuss our published data on exons e37a and e37b, which encode 32 amino acids in the intracellular C-terminus of Ca(V)2.2. These exons are expressed in a mutually exclusive manner. Exon e37a-containing Ca(V)2.2 mRNAs and their resultant channels express at higher density in dorsal root ganglia and, as we showed recently, e37a increases N-type channel sensitivity to G-protein-mediated inhibition, as compared to generic e37b-containing N-type channels.     

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 Matters: N-Type Calcium Channels in Nociceptors

How many different calcium channels does it take to make a nervous system? The answer: more than any of us predicted. In 1975 Hagiwara and colleagues published the first evidence that functionally different calcium channels are expressed in cells 1. By 1999, the calcium channel family could boast ten members, each member defined by a unique set of attributes to support their cellular functions and by unique amino acid sequences 2. Although nine of these genes are expressed in the nervous system, that number still seemed insufficient to support the wide spectrum of neuronal functions controlled by voltage-gated calcium channels. This discrepancy is probably explained by alternative pre-messenger RNA splicing which substantially expands the number of protein activities available from a limited number of genes. Like many other ion channel genes, each CaVa1 gene has the capacity to generate perhaps thousands of unique splice isoforms with unique functional properties 3. The high level of conservation among alternatively spliced exons in CaV2.2 genes of different species and in some cases closely related genes implies biological importance. A number of CaVa1 isoforms have been identified from neural tissue but until recently we lacked direct evidence linking a specific splice site in a calcium channel gene to a specific function in an identified neuron population.     

D2 dopamine receptors interact directly with N-type calcium channels and regulate channel surface expression levels

N-type channels are located on dendrites and at pre-synaptic nerve terminals where they play a fundamental role in neurotransmitter release. They are potently regulated by the activation of a number of different types of pertussis toxin (PTX)-sensitive G alpha(i/o) coupled receptors, which results in voltage-dependent inhibition of channel activity via G betagamma subunits. Using heterologous expression in HEK 293T cells, we show via whole cell patch clamp recordings that D2 receptors mediate both G betagamma (i.e., voltage-dependent) and voltage-independent inhibition of channel activity. Furthermore, using co-immunoprecipitation and pull down assays involving the intracellular regions of each protein, we show that D2 receptors and N-type channels form physical signaling complexes. Finally, we use confocal microscopy to demonstrate that D2 receptors regulate N-type channel trafficking to affect the number of calcium channels available at the plasma membrane. Taken together, these data provide evidence for multiple voltage-dependent and voltage-independent mechanisms by which D2 receptor subtypes influence N-type channel activity.     

D2 dopamine receptors interact directly with N-type calcium channels and regulate channel surface expression levels

N-type channels are located on dendrites and at pre-synaptic nerve terminals where they play a fundamental role in neurotransmitter release. They are potently regulated by the activation of a number of different types of pertussis toxin (PTX)-sensitive Gαi/o coupled receptors, which results in voltage-dependent inhibition of channel activity via Gβγ subunits. Using heterologous expression in HEK 293T cells, we show via whole cell patch clamp recordings that D2 receptors mediate both Gβγ (i.e. voltage-dependent) and voltage-independent inhibition of channel activity. Furthermore, using co-immunoprecipitation and pull down assays involving the intracellular regions of each protein, we show that D2 receptors and N-type channels form physical signaling complexes. Finally, we use confocal microscopy to demonstrate that D2 receptors regulate N-type channel trafficking to affect the number of calcium channels available at the plasma membrane. Taken together, these data provide evidence for multiple voltage-dependent and voltage-independent mechanisms by which D2 receptor subtypes influence N-type channel activity.     

N型钙通道与疼痛

N型电压依赖性钙通道(VDCCs)在疼痛的传递与调控中具有重要作用。它们密集分布于脊髓背角伤害感受性神经元突触前末梢,参与主要疼痛介质如谷氨酸和P物质等释放的调节。通过阻断上述通道,选择性N型VDCCs阻断剂表现出强效镇痛作用,N型VDCCs Cav2．2亚基基因敲除小鼠也表现为痛阈提高。N型VDCCs还分布于自主神经系统和中枢神经系统突触部位,现有的N型VDCCs阻断剂用于疼痛治疗时出现的各种副作用与这些部位的突触抑制有关。最近发现,背根节伤害感受性神经元上存在一种特异的N型VDCCs亚型,这为疼痛治疗提供了一个非常有意义的新靶标。     

The novel product of a five-exon stargazin-related gene abolishes Ca(V)2.2 calcium channel expression

We have cloned and characterized a new member of the voltage-dependent Ca(2+) channel gamma subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential gamma subunits identified by their homology to the stargazin gene, CACNG7 is a five-, and not four-exon gene whose mRNA encodes a protein we have designated gamma(7). Expression of human gamma(7) has been localized specifically to brain. N-type current through Ca(V)2.2 channels was almost abolished when co-expressed transiently with gamma(7) in either Xenopus oocytes or COS-7 cells. Furthermore, immunocytochemistry and western blots show that gamma(7) has this effect by causing a large reduction in expression of Ca(V)2.2 rather than by interfering with trafficking or biophysical properties of the channel. No effect of transiently expressed gamma(7) was observed on pre-existing endogenous N-type calcium channels in sympathetic neurones. Low homology to the stargazin-like gamma subunits, different gene structure and the unique functional properties of gamma(7) imply that it represents a distinct subdivision of the family of proteins identified by their structural and sequence homology to stargazin.     

Molecular determinants of syntaxin 1 modulation of N-type calcium channels

We have previously reported that syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation (Jarvis, S. E., and Zamponi, G. W. (2001) J. Neurosci. 21, 2939-2948). Here, we have investigated the molecular determinants that govern the action of syntaxin 1 isoforms on N-type calcium channel function. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein beta subunit and the synaptic protein interaction (synprint) site contained within the N-type calcium channel domain II-III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183-230) binding to Gbeta and the N-terminal (residues 1-69) binding to the synprint motif of the channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH(2)-terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-type calcium channel function. Because syntaxin 1 exists in both "open" and "closed" conformations during exocytosis, we produced a constitutively open form of syntaxin 1A and found that it still promoted G-protein inhibition of the channels, but it did not affect N-type channel availability. This state dependence of the ability of syntaxin 1 to mediate N-type calcium channel availability suggests that syntaxin 1 dynamically regulates N-type channel function during various steps of exocytosis. Finally, syntaxin 1A appeared to compete with Ggamma for the Gbeta subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein gamma subunit-like domain.     

Modulation of neuronal voltage-gated calcium channels by farnesol.

The modulation of presynaptic voltage-dependent calcium channels by classical second messenger molecules such as protein kinase C and G protein betagamma subunits is well established and considered a key factor for the regulation of neurotransmitter release. However, little is known of other endogenous mechanisms that control the activity of these channels. Here, we demonstrate a unique modulation of N-type calcium channels by farnesol, a dephosphorylated intermediate of the mammalian mevalonate pathway. At micromolar concentrations, farnesol acts as a relatively non-discriminatory rapid open channel blocker of all types of high voltage-activated calcium channels, with a mild specificity for L-type channels. However, at 250 nM, farnesol induces an N-type channel-specific hyperpolarizing shift in channel availability that results in approximately 50% inhibition at a typical neuronal resting potential. Additional experiments demonstrated the presence of farnesol in the brain (rodents and humans) at physiologically relevant concentrations (100-800 pmol/g (wet weight)). Altogether, our results indicate that farnesol is a selective, high affinity inhibitor of N-type Ca(2+) channels and raise the possibility that endogenous farnesol and the mevalonate pathway are implicated in neurotransmitter release through regulation of presynaptic voltage-gated Ca(2+) channels.     

Calcium channel beta subunits differentially regulate the inhibition of N-type channels by individual Gbeta isoforms

The direct inhibition of N- and P/Q-type calcium channels by G protein betagamma subunits is considered a key mechanism for regulating presynaptic calcium levels. We have recently reported that a number of features associated with this G protein inhibition are dependent on the G protein beta subunit isoform (Arnot, M. I., Stotz, S. C., Jarvis, S. E., Zamponi, G. W. (2000) J. Physiol. (Lond.) 527, 203-212; Cooper, C. B., Arnot, M. I., Feng, Z.-P., Jarvis, S. E., Hamid, J., Zamponi, G. W. (2000) J. Biol. Chem. 275, 40777-40781). Here, we have examined the abilities of different types of ancillary calcium channel beta subunits to modulate the inhibition of alpha(1B) N-type calcium channels by the five known different Gbeta subunit subtypes. Our data reveal that the degree of inhibition by a particular Gbeta subunit is strongly dependent on the specific calcium channel beta subunit, with N-type channels containing the beta(4) subunit being less susceptible to Gbetagamma-induced inhibition. The calcium channel beta(2a) subunit uniquely slows the kinetics of recovery from G protein inhibition, in addition to mediating a dramatic enhancement of the G protein-induced kinetic slowing. For Gbeta(3)-mediated inhibition, the latter effect is reduced following site-directed mutagenesis of two palmitoylation sites in the beta(2a) N-terminal region, suggesting that the unique membrane tethering of this subunit serves to modulate G protein inhibition of N-type calcium channels. Taken together, our data suggest that the nature of the calcium channel beta subunit present is an important determinant of G protein inhibition of N-type channels, thereby providing a possible mechanism by which the cellular/subcellular expression pattern of the four calcium channel beta subunits may regulate the G protein sensitivity of N-type channels expressed at different loci throughout the brain and possibly within a neuron.     

A single Gbeta subunit locus controls cross-talk between protein kinase C and G protein regulation of N-type calcium channels

The modulation of N-type calcium channels is a key factor in the control of neurotransmitter release. Whereas N-type channels are inhibited by Gbetagamma subunits in a G protein beta-isoform-dependent manner, channel activity is typically stimulated by activation of protein kinase C (PKC). In addition, there is cross-talk among these pathways, such that PKC-dependent phosphorylation of the Gbetagamma target site on the N-type channel antagonizes subsequent G protein inhibition, albeit only for Gbeta(1)-mediated responses. The molecular mechanisms that control this G protein beta subunit subtype-specific regulation have not been described. Here, we show that G protein inhibition of N-type calcium channels is critically dependent on two separate but adjacent approximately 20-amino acid regions of the Gbeta subunit, plus a highly conserved Asn-Tyr-Val motif. These regions are distinct from those implicated previously in Gbetagamma signaling to other effectors such as G protein-coupled inward rectifier potassium channels, phospholipase beta(2), and adenylyl cyclase, thus raising the possibility that the specificity for G protein signaling to calcium channels might rely on unique G protein structural determinants. In addition, we identify a highly specific locus on the Gbeta(1) subunit that serves as a molecular detector of PKC-dependent phosphorylation of the G protein target site on the N-type channel alpha(1) subunit, thus providing for a molecular basis for G protein-PKC cross-talk. Overall, our results significantly advance our understanding of the molecular details underlying the integration of G protein and PKC signaling pathways at the level of the N-type calcium channel alpha(1) subunit.     

Several structural domains contribute to the regulation of N-type calcium channel inactivation by the beta 3 subunit

Calcium channel beta subunits are essential regulatory elements of the gating properties of high voltage-activated calcium channels. Co-expression with beta(3) subunits typically accelerates inactivation, whereas co-expression with beta(4) subunits results in a slowly inactivating phenotype. Here, we have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Ca(v)2.2 + alpha(2)-delta(1) N-type calcium channels by creating a series of 22 chimeric beta subunits that are based on various combinations of variable and conserved regions of the parent beta subunit isoforms. Our data show that replacement of the N terminus region of beta(4) with a corresponding 14-amino acid stretch of beta(3) sequence accelerates the inactivation kinetics to levels seen with wild type beta(3). A similar kinetic speeding is observed by a concomitant substitution of the second conserved and variable regions, but not when these regions are substituted individually, suggesting that 1) the second variable and conserved regions cooperatively regulate N-type calcium channel inactivation and 2) that there are two redundant mechanisms that allow the beta(3) subunit to accelerate N-type channel inactivation. In contrast with previous reports in Ca(v)2.1 calcium channels, deletion of the C-terminal region of Ca(v)2.2 did not alter the regulation of the channel by wild type and chimeric beta subunits. Hence, the molecular underpinnings of beta subunit regulation of voltage-gated calcium channels appear to vary with calcium channel subtype.     

Interactions among toxins that inhibit N-type and P-type calcium channels

A number of peptide toxins from venoms of spiders and cone snails are high affinity ligands for voltage-gated calcium channels and are useful tools for studying calcium channel function and structure. Using whole-cell recordings from rat sympathetic ganglion and cerebellar Purkinje neurons, we studied toxins that target neuronal N-type (Ca(V)2.2) and P-type (Ca(V)2.1) calcium channels. We asked whether different toxins targeting the same channels bind to the same or different sites on the channel. Five toxins (omega-conotoxin-GVIA, omega-conotoxin MVIIC, omega-agatoxin-IIIA, omega-grammotoxin-SIA, and omega-agatoxin-IVA) were applied in pairwise combinations to either N- or P-type channels. Differences in the characteristics of inhibition, including voltage dependence, reversal kinetics, and fractional inhibition of current, were used to detect additive or mutually occlusive effects of toxins. Results suggest at least two distinct toxin binding sites on the N-type channel and three on the P-type channel. On N-type channels, results are consistent with blockade of the channel pore by omega-CgTx-GVIA, omega-Aga-IIIA, and omega-CTx-MVIIC, whereas grammotoxin likely binds to a separate region coupled to channel gating. omega-Aga-IIIA produces partial channel block by decreasing single-channel conductance. On P-type channels, omega-CTx-MVIIC and omega-Aga-IIIA both likely bind near the mouth of the pore. omega-Aga-IVA and grammotoxin each bind to distinct regions associated with channel gating that do not overlap with the binding region of pore blockers. For both N- and P-type channels, omega-CTx-MVIIC binding produces complete channel block, but is prevented by previous partial channel block by omega-Aga-IIIA, suggesting that omega-CTx-MVIIC binds closer to the external mouth of the pore than does omega-Aga-IIIA.     

Block of voltage-dependent calcium channels by aliphatic monoamines

We have recently identified farnesol, an intermediate in the mevalonate pathway, as a potent endogenous modulator and blocker of N-type calcium channels (Roullet, J. B., R. L. Spaetgens, T. Burlingame, and G. W. Zamponi. 1999. J. Biol. Chem. 274:25439-25446). Here, we investigate the action of structurally related compounds on various types of voltage-dependent Ca(2+) channels transiently expressed in human embryonic kidney cells. 1-Dodecanol, despite sharing the 12-carbon backbone and headgroup of farnesol, exhibited a significantly lower blocking affinity for N-type Ca(2+) channels. Among several additional 12-carbon compounds tested, dodecylamine (DDA) mediated the highest affinity inhibition of N-type channels, indicating that the functional headgroup is a critical determinant of blocking affinity. This inhibition was concentration-dependent and relatively non-discriminatory among N-, L-, P/Q-, and R-Ca(2+) channel subtypes. However, whereas L-type channels exhibited predominantly resting channel block, the non-L-type isoforms showed substantial rapid open channel block manifested by a speeding of the apparent time course of current decay and block of the inactivated state. Consistent with these findings, we observed significant frequency-dependence of block and dependence on external Ba(2+) concentration for N-type, but not L-type, channels. We also systematically investigated the drug structural requirements for N-type channel inhibition. Blocking affinity varied with carbon chain length and showed a clear maximum at C12 and C13, with shorter and longer molecules producing progressively weaker peak current block. Overall, our data indicate that aliphatic monoamines may constitute a novel class of potent inhibitors of voltage-dependent Ca(2+) channels, with block being governed by rigid structural requirements and channel-specific state dependencies.     

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_vα₁ 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_vα₁ 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”.     

Voltage-independent inhibition of Ca(V)2.2 channels is delimited to a specific region of the membrane potential in rat SCG neurons

Neurotransmitters and hormones regulate Ca(V)2.2 channels through a voltage-independent pathway which is not well understood. It has been suggested that this voltage-independent inhibition is constant at all membrane voltages. However, changes in the percent of voltage-independent inhibition of Ca(V)2.2 have not been tested within a physiological voltage range. Here, we used a double-pulse protocol to isolate the voltage-independent inhibition of Ca(V)2.2 channels induced by noradrenaline in rat superior cervical ganglion neurons. To assess changes in the percent of the voltage-independent inhibition, the activation voltage of the channels was tested between -40 and +40 mV. We found that the percent of voltage-independent inhibition induced by noradrenaline changed with the activation voltage used. In addition, voltage-independent inhibition induced by oxo-M, a muscarinic agonist, exhibited the same dependence on activation voltage, which supports that this pattern is not exclusive for adrenergic activation. Our results suggested that voltage-independent inhibition of Ca(V)2.2 channels depends on the activation voltage of the channel in a physiological voltage range. This may have relevant implications in the understanding of the mechanism involved in voltage-independent inhibition.     

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.     

Modes of operation of the BKCa channel beta2 subunit

The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.