High-voltage-activated Research Articles

A Short Basic Segment within a Non-Conserved Region of the Beta-Subunit Modulates the Rate of Inactivation of the R-Type Calcium Channel

Besides opening and closing, high-voltage activated (HVA) calcium channels transit to an inactivated state from which they do not re-open unless the plasma membrane is repolarized. This process is critical for the temporal regulation of intracellular calcium signaling. CaV2.3, a particular class of HVA channels supporting R-type calcium currents, inactivates fully in few hundred milliseconds when expressed alone but when co-expressed with the palmitoylated form of the regulatory β-subunit (CaVβ2a), this process is slowed several fold and is incomplete. The widely accepted view is that membrane-anchoring of the CaVβ2a immobilizes the channel inactivation machinery. Some evidence suggests that an additional structural determinant may be coded in the linker peptide that joins the two highly conserved domains composing CaVβ but whether these determinants target the subunit to the membrane has not been shown. Here we indentify a short positively charged segment lying at the boundary of the guanylate kinase domain of CaVβ2a that slows down channel inactivation without relocating the protein to the plasma membrane. Deletion analysis demonstrates that while neither the N-terminal nor the C-terminal variable region of the protein affects the regulatory effect of the basic segment the presence of the remaining residues within the linker does. These results demonstrate that membrane anchoring is not the only factor modulating inactivation rate CaV2.3 calcium channels and suggest the presence of intralinker interactions or posttranslational modifications that counteract the effect of this segment.

Gem Stabilizes Voltage-Gated Calcium Channels in the Inactivated State: Implications for Human Disease

RGK proteins (Rad, Rem1, Rem2 and Gem/Kir) are small GTPases that often colocalize with and strongly inhibit high-voltage activated (HVA) calcium channels (L-, N-, P/Q- and R-type). The mechanism of inhibition is largely unclear. The pore-forming alpha-1 subunit of HVA calcium channels requires the beta subunit for cell surface trafficking and gating. We previously showed direct binding between alpha-1 and Gem and obtained a Gem-insensitive chimeric P/Q alpha-1containing the IIS1-S3 region of a Gem-resistant, low-voltage activated T-type channel (Fan et al., PNAS, 2010). In this study, we investigated the cause of this insensitivity. Since the IIS1-S3 region of P/Q alpha-1 has too few cytosolic amino acids to be a Gem binding site, and since we had identified a different alpha-1 region that binds Gem, we focused on altered biophysical properties of the chimera. We observed that the chimeric channel inactivated much slower than WT P/Q channel did; we therefore examined whether slow inactivation was responsible for Gem insensitivity. Mutating residues known to increase channel inactivation enhanced Gem inhibition, whereas mutating residues known to slow channel inactivation weakened or abolished Gem inhibition. Replacing beta3 with beta2a, an auxiliary subunit known to slow channel inactivation, also weakened Gem inhibition. Furthermore, recovery from inactivation of WT P/Q channels in the presence of Gem was markedly slower than without Gem. Thus, Gem appeared to stabilize the channel in the inactivated state. These results shed light on a new factor - altered regulation by RGK proteins - that may contribute to HVA calcium channel-related channelopathies. Indeed, we found that familial hemiplegic migraine mutations that speed P/Q channel inactivation strengthened Gem inhibition, whereas Timothy syndrome mutations that slow channel inactivation greatly weakened Gem inhibition.

Voltage-Gated Calcium Channel α2δ Subunits in Lipid Rafts: The Importance of Proteolytic Cleavage Into α2 and δ

The High Voltage-Activated (HVA) subgroup of voltage-gated calcium channels contain an α1 subunit, which forms the selective pore and determines the main functional properties of the channel. The α1 subunit is associated with auxiliary subunits including β and α2δ, which modulate trafficking and functional properties of the channels.There are four known genes encoding α2δ subunits, which are believed to have similar structure. They consist of two peptides: the highly glycosylated α2 which is entirely extracellular is disulfide-bonded to a δ subunit that links the protein into the plasma membrane. The α2 and δ peptides are encoded by a single gene as an uninterrupted α2δ pre-protein, which is further processed post-translationally. We have recently shown that α2δ subunits are glycosyl phophatidyl inositol (GPI)-achored proteins (Davies et al., 2010, PNAS 107:1654-1659), and this is essential for their function, and explains their localization in lipid raft fractions (Davies et al, 2006, J. Neurosci. 26: 8748-8757).We further studied the mechnism and consequences of the proteolytic cleavage of the α2δ subunits (α2δ-1, -2 and -3)and identified the cleavage sites in α2δ-2 and α2δ-3. Western blots from brain tissue revealed only the mature form of the protein strongly associated with lipid rafts. However, transfecting mammalian cells with cDNA for α2δ-1,-2,and -3 resulted in incomplete cleavage (∼ 40-60% processing) most probably due to limitations of the cleavage-mediating protease(s) in heterologous expression systems. The mature form is localized in lipid rafts, suggesting that maturation of the protein might occur in localized membrane domains. Moreover, electrophysiological recordings demonstrated that proteolytic cleavage is key to the function of these subunits.We are currently examining the nature of the protease(s) involved in this proteolytic processing.

Modulation of Calcium-Dependent Inactivation of L-Type Ca2+ Channels via β-Adrenergic Signaling in Thalamocortical Relay Neurons

Neuronal high-voltage-activated (HVA) Ca2+ channels are rapidly inactivated by a mechanism that is termed Ca2+-dependent inactivation (CDI). In this study we have shown that β-adrenergic receptor (βAR) stimulation inhibits CDI in rat thalamocortical (TC) relay neurons. This effect can be blocked by inhibition of cAMP-dependent protein kinase (PKA) with a cell-permeable inhibitor (myristoylated protein kinase inhibitor-(14–22)-amide) or A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide, suggesting a critical role of these molecules downstream of the receptor. Moreover, inhibition of protein phosphatases (PP) with okadaic acid revealed the involvement of phosphorylation events in modulation of CDI after βAR stimulation. Double fluorescence immunocytochemistry and pull down experiments further support the idea that modulation of CDI in TC neurons via βAR stimulation requires a protein complex consisting of CaV1.2, PKA and proteins from the AKAP family. All together our data suggest that AKAPs mediate targeting of PKA to L-type Ca2+ channels allowing their phosphorylation and thereby modulation of CDI.

Amitriptyline modulates calcium currents and intracellular calcium concentration in mouse trigeminal ganglion neurons

Migraine is increasingly recognized as a channelopathy, and abnormalities of voltage-activated ionic channels could represent the molecular basis for the altered neuronal functioning. The high-voltage-activated (HVA) Ca2+ channels in the trigeminovascular system play a role in the pathophysiology of migraine. In the present study, effects of amitriptyline (AMT), a commonly used migraine prophylactic drug, on the HVA calcium currents (ICa) were examined in mouse trigeminal ganglion neurons using whole-cell patch clamp technique. AMT produced concentration- and use-dependent inhibition of HVA ICa. Bath application of GÖ-6983 (a selective protein kinase C inhibitor) or H89 (a protein kinase A inhibitor) did not reduce the AMT-induced inhibition of HVA ICa. A similar inhibition was observed when calcium imaging was used to directly monitor the effects of AMT on KCl-induced increments of intracellular Ca2+ concentration ([Ca2+]i). By blocking HVA Ca2+ channels and Ca2+ entry into cells, AMT could prevent the release of neurotransmitters and help restore the neuronal threshold for excitation. Our findings suggest interesting therapeutic mechanisms for AMT in migraine prevention.

Identification of a disulfide bridge essential for structure and function of the voltage-gated Ca2+ channel α2δ-1 auxiliary subunit

Voltage-gated calcium (CaV) channels are transmembrane proteins that form Ca2+-selective pores gated by depolarization and are essential regulators of the intracellular Ca2+ concentration. By providing a pathway for rapid Ca2+ influx, CaV channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. CaV channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α1 subunit, which typically contains associated β and α2δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α2δ-1 is post-translationally cleaved into an extracellular α2 polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α2δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α2) and Cys1047 (in the extracellular domain of δ) prevented the association of the α2 and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α2δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (CaV2.2α1/β3) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α2δ-1 function.

Nerve Terminal Nicotinic Acetylcholine Receptors Initiate Quantal GABA Release from Perisomatic Interneurons by Activating Axonal T-Type (Cav3) Ca2+Channels and Ca2+Release from Stores

Release of conventional neurotransmitters is mainly controlled by calcium (Ca2+) influx via high-voltage-activated (HVA), Cav2, channels (“N-, P/Q-, or R-types”) that are opened by action potentials. Regulation of transmission by subthreshold depolarizations does occur, but there is little evidence that low-voltage-activated, Cav3 (“T-type”), channels take part. GABA release from cortical perisomatic-targeting interneurons affects numerous physiological processes, and yet its underlying control mechanisms are not fully understood. We investigated whether T-type Ca2+channels are involved in regulating GABA transmission from these cells in rat hippocampal CA1 using a combination of whole-cell voltage-clamp, multiple-fluorescence confocal microscopy, dual-immunolabeling electron-microscopy, and optogenetic methods. We show that Cav3.1, T-type Ca2+channels can be activated by α3β4 nicotinic acetylcholine receptors (nAChRs) that are located on the synaptic regions of the GABAergic perisomatic-targeting interneuronal axons, including the parvalbumin-expressing cells. Asynchronous, quantal GABA release can be triggered by Ca2+influx through presynaptic T-type Ca2+channels, augmented by Ca2+from internal stores, following focal microiontophoretic activation of the α3β4 nAChRs. The resulting GABA release can inhibit pyramidal cells. The T-type Ca2+channel-dependent mechanism is not dependent on, or accompanied by, HVA channel Ca2+influx, and is insensitive to agonists of cannabinoid, μ-opioid, or GABABreceptors. It may therefore operate in parallel with the normal HVA-dependent processes. The results reveal new aspects of the regulation of GABA transmission and contribute to a deeper understanding of ACh and nicotine actions in CNS.

Differential regulation of low and high voltage-activated calcium channels in neonatal rat myocytes following chronic PKA modulation

The biophysical properties of voltage-dependent cardiac calcium channels (VDCC) can be modulated by protein kinases. In this study, we investigate whether long-term treatment with protein kinase A (PKA) modulators alters the VDCC activity in neonatal ventricular myocytes. Using whole-cell patch-clamp recordings, we found an increase in high-voltage activated (HVA) current density and a corresponding decrease in low-voltage activated (LVA) current density in neonatal rat ventricular myocytes up to 6 days in culture. Long-term exposure to 8Br-cAMP, a PKA stimulator, increased the HVA current density at 7 and 24 hours. In contrast, H89, a PKA inhibitor, caused a biphasic change in the HVA, an initial reduction at 7 hours exposure followed by an increase up to 4 days. In addition, H89 caused a sustained increase in LVA currents from 7 hours to 4 days. These findings suggest that chronic exposure to H89 changes LVA and HVA calcium current activities in cardiac myocytes. PKA is a key target of beta-adrenoceptor activiation, thus, our findings suggest long-term repeated use of beta-adrenergic drugs may induce unexpected functional alteration of VDCCs.

Ethanol alters calcium signaling in axonal growth cones

Calcium (Ca2+) channels are sensitive to ethanol and Ca2+ signaling is a critical regulator of axonal growth and guidance. Effects of acute and chronic exposure to ethanol (22, 43, or 87 mM) on voltage-gated Ca2+ channels (VGCCs) in whole cells, and KCl-induced Ca2+ transients in axonal growth cones, were examined using dissociated hippocampal cultures. Whole-cell patch-clamp analysis in neurons with newly-formed axons (Stage 3) revealed that rapidly inactivating, low-voltage activated (LVA) and non-inactivating, high-voltage activated (HVA) currents were both inhibited in a dose-dependent manner by acute ethanol, with relatively greater inhibition of HVA currents. When assessed by Fluo-4-AM imaging, baseline fluorescence and Ca2+ response to ethanol in Stage 3 neurons was similar compared to neurons without axons, but peak Ca2+ transient amplitudes in response to bath-applied KCl were greater in Stage 3 neurons and were decreased by acute ethanol. The amplitude of Ca2+ transients elicited specifically in axonal growth cones by focal application of KCl was also inhibited by acute exposure to moderate-to-high concentrations of ethanol (43 or 87 mM), whereas a lower concentration (22 mM) had no effect. When 43 or 87 mM ethanol was present continuously in the medium, KCl-evoked Ca2+ transient amplitudes were also reduced in growth cones. In contrast, Ca2+ transients were increased by continuous exposure to 22 mM ethanol. Visualization using a fluorescent dihydropyridine analog revealed that neurons continuously exposed to ethanol expressed increased amounts of L-type Ca2+ channels, with greater increases in axonal growth cones than cell bodies. Thus, acute ethanol reduces Ca2+ current and KCl-induced Ca2+ responses in whole cells and axonal growth cones, respectively, and chronic exposure is also generally inhibitory despite apparent up-regulation of L-type channel expression. These results are consistent with a role for altered growth cone Ca2+ signaling in abnormal neuromorphogenesis associated with fetal alcohol spectrum disorders.

Forced‐exercise delays neuropathic pain in experimental diabetes: effects on voltage‐activated calcium channels

Physical exercise produces a variety of psychophysical effects, including altered pain perception. Elevated levels of centrally produced endorphins or endocannabinoids are implicated as mediators of exercise-induced analgesia. The effect of exercise on the development and persistence of disease-associated acute/chronic pain remains unclear. In this study, we quantified the physiological consequence of forced-exercise on the development of diabetes-associated neuropathic pain. Euglycemic control or streptozotocin (STZ)-induced diabetic adult male rats were subdivided into sedentary or forced-exercised (2-10 weeks, treadmill) subgroups and assessed for changes in tactile responsiveness. Two weeks following STZ-treatment, sedentary rats developed a marked and sustained hypersensitivity to von Frey tactile stimulation. By comparison, STZ-treated diabetic rats undergoing forced-exercise exhibited a 4-week delay in the onset of tactile hypersensitivity that was independent of glucose control. Exercise-facilitated analgesia in diabetic rats was reversed, in a dose-dependent manner, by naloxone. Small-diameter (< 30 μm) DRG neurons harvested from STZ-treated tactile hypersensitive diabetic rats exhibited an enhanced (2.5-fold) rightward (depolarizing) shift in peak high-voltage activated (HVA) Ca(2+) current density with a concomitant appearance of a low-voltage activated (LVA) Ca(2+) current component. LVA Ca(2+) currents present in DRG neurons from hypersensitive diabetic rats exhibited a marked depolarizing shift in steady-state inactivation. Forced-exercise attenuated diabetes-associated changes in HVA Ca(2+) current density while preventing the depolarizing shift in steady-state inactivation of LVA Ca(2+) currents. Forced-exercise markedly delays the onset of diabetes-associated neuropathic pain, in part, by attenuating associated changes in HVA and LVA Ca(2+) channel function within small-diameter DRG neurons possibly by altering opioidergic tone.

Homodimerization of the Src Homology 3 Domain of the Calcium Channel β-Subunit Drives Dynamin-dependent Endocytosis

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory β-subunit (Ca(V)β), a membrane associated guanylate kinase containing one guanylate kinase (β-GK) and one Src homology 3 (β-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although β-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, β-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that β-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the β-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)β form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)β with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that β-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)β. We propose that a change in the oligomeric state of Ca(V)β is the functional switch between channel activator and channel internalizer.

Ca V2.3 Channels Are Critical for Oscillatory Burst Discharges in the Reticular Thalamus and Absence Epilepsy

Neurons of the reticular thalamus (RT) display oscillatory burst discharges that are believed to be critical for thalamocortical network oscillations related to absence epilepsy. Ca²+-dependent mechanisms underlie such oscillatory discharges. However, involvement of high-voltage activated (HVA) Ca²+ channels in this process has been discounted. We examined this issue closely using mice deficient for the HVA Ca(v)2.3 channels. In brain slices of Ca(v)2.3⁻/⁻, a hyperpolarizing current injection initiated a low-threshold burst of spikes in RT neurons; however, subsequent oscillatory burst discharges were severely suppressed, with a significantly reduced slow afterhyperpolarization (AHP). Consequently, the lack of Ca(v)2.3 resulted in a marked decrease in the sensitivity of the animal to γ-butyrolactone-induced absence epilepsy. Local blockade of Ca(v)2.3 channels in the RT mimicked the results of Ca(v)2.3⁻/⁻ mice. These results provide strong evidence that Ca(v)2.3 channels are critical for oscillatory burst discharges in RT neurons and for the expression of absence epilepsy.

Molecular Determinants of Voltage-Gated Calcium Channel Inhibition by Gem

High voltage-activated (HVA) Ca2+ channels, including L-, N- and P/Q-type channels, are potently inhibited by the Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins. This inhibition was widely thought to depend on the direct association between RGK proteins and the β subunit (Cavβ) of HVA Ca2+ channels, but we recently found that P/Q channel inhibition by Gem protein in inside-out membrane patches required Cavβ but not the Gem-Cavβ interaction, and that Gem coimmunoprecipitated with the P/Q channel α1 subunit in a Cavβ-independent manner. Thus, we proposed that direct interactions between Gem and the α1 subunit (Cavα1) of HVA Ca2+ channels are critical for Gem inhibition. In this study, we investigate the molecular determinants on Cavα1 that are important for Gem inhibition. We construct chimeras between the α1 subunit of P/Q channels and the Gem-insensitive low voltage-activated T-type channels, which do not bind or require Cavβ. We find that grafting the α-interaction domain (AID), the high-affinity Cavβ binding site on Cavα1, or grafting the entire AID-containing I-II loop from P/Q channel into T-channels (TPQ I-II loop), is sufficient for Cavβ binding and gating regulation but does not bestow RGK inhibition. However, adding the first two transmembrane segments (IIS1-IIS2) of the second homology repeat onto the TPQ I-II loop construct, produced a Gem-sensitive T-channel. This inhibition persists with non-interacting Gem and Cavβ mutants, indicating that the Cavβ-Gem interaction is not necessary, just as in the case of Gem inhibition of P/Q channels. In complementary experiments, substituting only IIS1-IIS2 of P/Q channel α1 with that of T-channel's severely attenuates RGK inhibition. This supports a paradigm in which Gem directly binds and inhibits Cavβ-primed Cavα1 on the plasma membrane. We are currently investigating the role of other Cavα1 regions in Gem inhibition.

Altered profile and D2-dopamine receptor modulation of high voltage-activated calcium current in striatal medium spiny neurons from animal models of Parkinson's disease

In the present work we analyzed the profile of high voltage-activated (HVA) calcium (Ca2+) currents in freshly isolated striatal medium spiny neurons (MSNs) from rodent models of both idiopathic and familial forms of Parkinson's disease (PD). MSNs were recorded from reserpine-treated and 6-hydroxydopamine (6-OHDA)-lesioned rats, and from DJ-1 and PINK1 (PTEN induced kinase 1) knockout (−/−) mice. Our analysis showed no significant changes in total HVA Ca2+ current. However, we recorded a net increase in the L-type fraction of HVA Ca2+ current in dopamine-depleted rats, and of both N- and P-type components in DJ-1−/− mice, whereas no significant change in Ca2+ current profile was observed in PINK1−/− mice. Dopamine modulates HVA Ca2+ channels in MSNs, thus we also analyzed the effect of D1 and D2 receptor activation. The effect of the D1 receptor agonist SKF 83822 on Ca2+ current was not significantly different among MSNs from control animals or PD models. However, in both dopamine-depleted rats and DJ-1−/− mice the D2 receptor agonist quinpirole inhibited a greater fraction of HVA Ca2+ current than in the respective controls. Conversely, in MSNs from PINK1−/− mice we did not observe alterations in the effect of D2 receptor activation. Additionally, in both reserpine-treated and 6-OHDA-lesioned rats, the effect of quinpirole was occluded by the selective L-type Ca2+ channel blocker nifedipine, while in DJ-1−/− mice it was mostly occluded by ω-conotoxin GVIA, blocker of N-type channels. These results demonstrate that both dopamine depletion and DJ-1 deletion induce a rearrangement in the HVA Ca2+ channel profile, specifically involving those channels that are selectively modulated by D2 receptors.

Changes in high voltage-activated calcium current in dorsal root ganglion neurons isolated from rats with neuropathic pain

Objective To investigate the changes in high voltage-activated （HVA） calcium current in dorsal root ganglion （DRG） neurons isolated from rats with neuropathic pain. Methods Pathogen-free male SD rats aged 4-6 weeks weighing 180-220 g were used in this study. The animals were anesthetized with intraperitoneal pentobarbital sodium 50 mg/kg. Neuropathic pain was induced by ligation of L5 spinal nerve between DRG and sciatic nerve. The nerve was transected distal to the ligature. The animals which showed positive signs of neuropathic pain were decapitated on the 14th postoperative day. L5 and L4 DRGs were isolated and the neurons in the ganglia were enzymatically dissociated （group L5 and L4）. The control group received no surgery （group C）. The HVA Ca2＋ current was recorded using whole-cell patch clamp technique. Results Peak calcium current density was significantly lower in group L5 and L4 than in group C, and was significantly lower in group L5 than in group L4 . Halfactivation value （Va 1/2） was also significantly lower in group L5 than in group C and L4 （P 〈 0.05）. The relative contribution of N-type to the total HVA Ca2＋ current was significantly greater in group L5 than in group C and L4（P 〈 0.05）. There was no significant difference in the steady-state inactivation curves among the 3 groups. Conclusion In rats with neuropathic pain, the HVA Ca2＋ current in the injured DRG neurons may play a key role in the induction of neuropathic pain. Key words: Calcium channels; Neuralgia; Ganglia,spinal; Neurons

Characteristics of calcium currents in rat geniculate ganglion neurons.

Geniculate ganglion (GG) cell bodies of chorda tympani (CT), greater superficial petrosal (GSP), and posterior auricular (PA) nerves transmit orofacial sensory information to the rostral nucleus of the solitary tract (rNST). We used whole cell recording to study the characteristics of the Ca(2+) channels in isolated Fluorogold-labeled GG neurons that innervate different peripheral receptive fields. PA neurons were significantly larger than CT and GSP neurons, and CT neurons could be further subdivided based on soma diameter. Although all GG neurons possess both low voltage-activated (LVA) "T-type" and high voltage-activated (HVA) Ca(2+) currents, CT, GSP, and PA neurons have distinctly different Ca(2+) current expression patterns. Of GG neurons that express T-type currents, the CT and GSP neurons had moderate and PA neurons had larger amplitude T-type currents. HVA Ca(2+) currents in the GG neurons were separated into several groups using specific Ca(2+) channel blockers. Sequential applications of L, N, and P/Q-type channel antagonists inhibited portions of Ca(2+) current in all CT, GSP, and PA neurons to a different extent in each neuron group. No difference was observed in the percentage of L- and N-type Ca(2+) currents reduced by the antagonists in CT, GSP, and PA neurons. Action potentials in GG neurons are followed by a Ca(2+) current initiated after depolarization (ADP) that may influence intrinsic firing patterns. These results show that based on Ca(2+) channel expression the GG contains a heterogeneous population of sensory neurons possibly related to the type of sensory information they relay to the rNST.

Characterization of voltage-activated ionic currents in the GnRH-containing terminalis nerve in transgenic zebrafish

The terminalis nerve (TN) is in a class of cranial nerves that plays important roles in animal development, physiology and behavior. Here, we report a study on the characterization of voltage-activated ionic currents in GnRH-containing TN cells in zebrafish. The experiments were performed using acutely dissociated TN cells from the transgenic zebrafish Tg (GnRH-3::GFP). In the transgenic zebrafish, the TN cells express GFP under the transcriptional control of the zebrafish GnRH-3 promoter. In all of the GnRH-containing TN cells examined, we recorded both low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium current (ICa). The characteristics of the ICa were similar to those described in other zebrafish cell types. However, the distribution patterns of the currents in the GnRH-containing TN cells were different in comparison to the distribution of the currents in other cell types. In addition, we characterized TTX-sensitive sodium current (INa) and 4AP-sensitive and TEA-resistant potassium current (IK). The characteristics of voltage-activated INa and IK in the GnRH-containing TN cells were similar to those described in other zebrafish cell types. Together, the data from this study revealed the electrophysiological properties of the GnRH-containing TN cells, thereby providing insight on the regulatory mechanisms of TN-signaling in animal physiology.

The β Subunit of Voltage-Gated Ca2+Channels

Calcium regulates a wide spectrum of physiological processes such as heartbeat, muscle contraction, neuronal communication, hormone release, cell division, and gene transcription. Major entryways for Ca(2+) in excitable cells are high-voltage activated (HVA) Ca(2+) channels. These are plasma membrane proteins composed of several subunits, including α(1), α(2)δ, β, and γ. Although the principal α(1) subunit (Ca(v)α(1)) contains the channel pore, gating machinery and most drug binding sites, the cytosolic auxiliary β subunit (Ca(v)β) plays an essential role in regulating the surface expression and gating properties of HVA Ca(2+) channels. Ca(v)β is also crucial for the modulation of HVA Ca(2+) channels by G proteins, kinases, and the Ras-related RGK GTPases. New proteins have emerged in recent years that modulate HVA Ca(2+) channels by binding to Ca(v)β. There are also indications that Ca(v)β may carry out Ca(2+) channel-independent functions, including directly regulating gene transcription. All four subtypes of Ca(v)β, encoded by different genes, have a modular organization, consisting of three variable regions, a conserved guanylate kinase (GK) domain, and a conserved Src-homology 3 (SH3) domain, placing them into the membrane-associated guanylate kinase (MAGUK) protein family. Crystal structures of Ca(v)βs reveal how they interact with Ca(v)α(1), open new research avenues, and prompt new inquiries. In this article, we review the structure and various biological functions of Ca(v)β, with both a historical perspective as well as an emphasis on recent advances.

Interaction of T-type calcium channel Ca(V)3.3 with the β-subunit.

The β-subunit of high-voltage-activated (HVA) calcium channels is essential for the regulation of expression and gating. On the other hand, various reports have suggested that β subunits play no role in the regulation of low-voltage-activated T-type channels. In addition there has been no clear demonstration of a physical interaction between the α-subunit of T-type channel with β-subunit. In this study, we systematically investigated the interaction between Ca(V)α and Ca(V)β. The four Ca(V)β isoforms were expressed in a bacterial system and purified into homogeneity, whereas the ten types of Ca(V)α alpha interaction domain (AID) peptides were chemically synthesized. All possible combinations of Ca(V)α and Ca(V)β were then tested for by in vitro immunoassays. We describe here the identification of a new interaction between Ca(V)3.3 and Ca(V)β proteins. This interaction is of low affinity compared to that between the AID of the HVA α-subunit and the alpha-binding pocket (ABP) site of the β-subunit. The AID peptide of HVA channel exerted no effect on the Ca(V)3.3-Ca(V)β interaction, thus demonstrating that another site not in the ABP of Ca(V)β protein played a role in binding with Ca(V)3.3. This is the first demonstration of an α-β subunit interaction in a T-type calcium channel.

Direct inhibition of P/Q-type voltage-gated Ca 2+ channels by Gem does not require a direct Gem/Ca v β interaction

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca(2+) channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca(2+) channel beta subunits (Ca(v)beta), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca(2+) channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca(v)beta but not the Gem/Ca(v)beta interaction, and that Gem coimmunoprecipitates with the P/Q channel alpha(1) subunit (Ca(v)alpha(1)) in a Ca(v)beta-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca(v)alpha(1) critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca(v)alpha(1) abolishes Gem inhibition of P/Q channels and confers Ca(v)beta-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca(v)alpha(1) that binds Ca(v)beta). Our results challenge the prevailing view regarding the role of Ca(v)beta in RGK inhibition of high voltage-activated Ca(2+) channels and prompt a paradigm in which Gem directly binds and inhibits Ca(v)beta-primed Ca(v)alpha(1) on the plasma membrane.