Phosphorylation states greatly regulate the activity and gating properties of Cav3.1 T-type Ca2+ channels

Ca_v3.1 T-type Ca²⁺ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Ca_v3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Ca_v3.1 channel has been poorly investigated. In this work, we analyzed rat Ca_v3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Ca_v3.1 phosphorylation map which includes the reported mouse Ca_v3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Ca_v3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Ca_v3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Ca_v3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.     

Population genetic analysis and origin discrimination of snow crab (Chionoecetes opilio) using microsatellite markers

Major habitats for the snow crab Chionoecetes opilio are mostly found within the northwest Atlantic and North Pacific Oceans. However, the East Sea populations of C. opilio, along with its relative the red snow crab (C. japonicas), are two of the most important commercial crustacean species for fisheries on the east coast of the Korean Peninsula. The East Sea populations of C. opilio are facing declining resources due to overfishing and global climate change. Thus, an analysis of population structure is necessary for future management. Five Korean and one Russian group of C. opilio were analyzed using nine microsatellite markers that were recently developed using next-generation sequencing. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The number of alleles per locus varied from 4 to 18 with a mean of 12, and allelic richness per locus ranged from 4.0 to 17.1 across all populations with a mean of 9.7. The Hardy–Weinberg equilibrium test revealed significant deviation in three out of nine loci in some populations after sequential Bonferroni correction and all of them had higher expected heterozygosity than observed heterozygosity. Null alleles were presumed in four loci, which explained the homozygosity in three loci. The pairwise fixation index (F _ST ) values among the five Korean snow crab populations did not differ significantly, but all of the pairwise F _ST values between each of the Korean snow crab populations and the Russian snow crab population differed significantly. An UPGMA dendrogram revealed clear separation of the Russian snow crab population from the Korean snow crab populations. Assignment tests based on the allele distribution discriminated between Korean and Russian origins with 93 % accuracy. Therefore, the snow crab populations around the Korean Peninsula need to be managed separately from the populations in Bering Sea in global scale resource management. Also, this information can be used for identification of snow crab origin which is problematic in worldwide crab trade.     

Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca²⁺ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca²⁺ selective filters of all high-voltage-activated Ca²⁺ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca²⁺ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca_v3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca_v3.2, including relative permeability between Ba²⁺ and Ca²⁺, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd²⁺ blocking sensitivity, and pH effects, in distinctive ways.     

Spontaneous and stimulated interleukin-6 and tumor necrosis factor-alpha production at delivery and three months after birth

OBJECTIVE: To study the production and interrelations of maternal and neonatal cytokines (IL-6 and TNF-alpha) during labor, after vaginal delivery and at three months after delivery. METHOD: The unstimulated concentrations of cytokines in the supernatants of whole-blood cultures and concentrations after PMA (phorbol 12-myristate 13-acetate) and concanavalin (conA) stimulation were determined by enzyme-linked immunosorbent assays (ELISAs). The blood samples were from the peripheral veins of 27 healthy women during term labor and immediately after delivery and three months after delivery. Neonatal samples were taken at birth (cord blood) and three months after delivery. RESULTS: IL-6 responses to stimulation were increased in the parturients and in umbilical cord blood at delivery compared with maternal and neonatal samples obtained 3 months postpartum. In contrast, the production of maternal TNF-alpha in peripheral blood was down-regulated at delivery compared with values 3 months postpartum. After an IL-6 and TNF-alpha burst in umbilical cord samples, neonatal cytokine production was at a low level three months after delivery. IL-6 production tended to be higher in both umbilical cord blood as well as in maternal samples after delivery in women who were younger. In addition, TNF-alpha production in umbilical cord blood was significantly higher in those women who were younger. CONCLUSIONS: The production of IL-6 was up-regulated in both the maternal and in umbilical cord blood at delivery. The production of TNF-alpha was up-regulated in umbilical cord blood compared with neonatal values 3 months after birth. Maternal age had effects on IL-6 and TNF-alpha production at delivery.     

Modulation of Ca(v)3.2 T-type Ca2+ channels by protein kinase C

Although T-type Ca2+ channels have been implicated in numerous physiological functions, their regulations by protein kinases have been obscured by conflicting reports. We investigated the effects of protein kinase C (PKC) on Ca(v)3.2 T-type channels reconstituted in Xenopus oocytes. Phorbol-12-myristate-13-acetate (PMA) strongly enhanced the amplitude of Ca(v)3.2 channel currents (approximately 3-fold). The augmentation effects were not mimicked by 4alpha-PMA, an inactive stereoisomer of PMA, and abolished by preincubation with PKC inhibitors. Our findings suggest that PMA upregulates Ca(v)3.2 channel activity via activation of oocyte PKC.     

Cloning and expression of the human T-type channel Ca(v)3.3: insights into prepulse facilitation

The full-length human Ca(v)3.3 (alpha(1I)) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3' untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5 mM Ca(2+) as charge carrier. From a holding potential of -100 mV, step depolarizations elicited inward currents with an apparent threshold of -70 mV, a peak of -30 mV, and reversed at +40 mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Ca(v)3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Ca(v)3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Ca(v)3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100 mV, 200 ms) increased the fraction of channels that gated at low voltages. In contrast, human Ca(v)3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Ca(v)3.3 is similar to high voltage-activated Ca(2+) channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity.     

3D pharmacophore based virtual screening of T-type calcium channel blockers

Virtual screening of the commercial databases was done by using a three dimensional pharmacophore previously developed for T-type calcium channel blockers using CATALYSTtrade mark program. Biological evaluation of 25 selected virtual hits resulted in the discovery of a highly potent compound VH04 with IC(50) value of 0.10 microM, eight times as potent as the known selective T-type calcium channel blocker, mibefradil. Search for similar compounds yielded several hits with micro-molar IC(50) values and high T-type calcium channel selectivity. Based on the structure of the virtual hits, small molecule libraries with novel scaffolds were designed, synthesis and biological evaluation of which are currently in progress. This result shows a successful example of ligand based drug discovery of potent T-type calcium channel blockers.     

Multiple structural elements contribute to the slow kinetics of the Cav3.3 T-type channel

Molecular cloning and expression studies established the existence of three T-type Ca(2+) channel (Ca(v)3) alpha(1) subunits: Ca(v)3.1 (alpha(1G)), Ca(v)3.2 (alpha(1H)), and Ca(v)3.3 (alpha(1I)). Although all three channels are low voltage-activated, they display considerable differences in their kinetics, with Ca(v)3.1 and Ca(v)3.2 channels activating and inactivating much faster than Ca(v)3.3 channels. The goal of the present study was to determine the structural elements that confer the distinctively slow kinetics of Ca(v)3.3 channels. To address this question, a series of chimeric channels between Ca(v)3.1 and Ca(v)3.3 channels were constructed and expressed in Xenopus oocytes. Kinetic analysis showed that the slow activation and inactivation kinetics of the Ca(v)3.3 channel were not completely abolished by substitution with any one portion of the Ca(v)3.1 channel. Likewise, the Ca(v)3.1 channel failed to acquire the slow kinetics by simply adopting one portion of the Ca(v)3.3 channel. These findings suggest that multiple structural elements contribute to the slow kinetics of Ca(v)3.3 channels.     

Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

Ca_v3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca_v3.2 and modulation by redox agents. His¹⁹¹ is found on the extracellular face of the Ca_v3.2 channel on the IS3-S4 linker and is not conserved in other Ca_v3 channels. Mutation of the corresponding residue in Ca_v3.1 to histidine, Gln¹⁷², significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca_v3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3–S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.     

A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels

Molecular cloning studies have revealed that heterogeneity of T-type Ca2+ currents in native tissues arises from the three isoforms of Ca(v)3 channels: Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3. From pharmacological analysis of the recombinant T-type channels, low concentrations (<50 microM) of nickel were found to selectively block the Ca(v)3.2 over the other isoforms. To date, however, the structural element(s) responsible for the nickel block on the Ca(v)3.2 T-type Ca2+ channel remain unknown. Thus, we constructed chimeric channels between the nickel-sensitive Ca(v)3.2 and the nickel-insensitive Ca(v)3.1 to localize the region interacting with nickel. Systematic assaying of serial chimeras suggests that the region preceding domain I S4 of Ca(v)3.2 contributes to nickel block. Point mutations of potential nickel-interacting sites revealed that H191Q in the S3-S4 loop of domain I significantly attenuated the nickel block of Ca(v)3.2, mimicking the nickel-insensitive blocking potency of Ca(v)3.1. These findings indicate that His-191 in the S3-S4 loop is a critical residue conferring nickel block to Ca(v)3.2 and reveal a novel role for the S3-S4 loop to control ion permeation through T-type Ca2+ channels.