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Abstract: 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 Cav3.2 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 Cav3.2, 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. [Copyright &y& Elsevier]

We study the opto-electrical properties of Natronomonas pharaonis sensory rhodopsin II (NpSRII) by using a near-field microwave microprobe (NFMM) under external light illumination. To investigate the possibility of application of NFMM to biological macromolecules, we used time dependent properties of NPSRII before/after light activation which has three distinct states - ground-state, M-state, and O-state. The diagnostic ability of NFMM is demonstrated by measuring the microwave reflection coefficient ( S11) spectrum of NpSRII under steady-state illumination in the wavelength range of 350-650 nm. Moreover, we present microwave reflection coefficient S11 spectra in the same wavelength range for two fast-photocycling rhodopsins: green light-absorbing proteorhodopsin (GPR) and Gloeobacter rhodopsin (GR). In addition the frequency sweep shift can be detected completely even for tiny amounts of sample (∼10-3 OD of rhodopsin). Based on these results NFMM shows both very high sensitivity for detecting conformational changes and produces a good time-resolved spectrum. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

Redox regulation is one of the ubiquitous mechanisms to modulate ion channels. We here investigated how 5,5'-dithio-bis (2-nitrobenzoic acid), a cysteine specific oxidizing reagent, modulates Cav3.1 and Cav3.2 T-type Ca2 + channels expressed in Xenopus oocytes. Application of the reagent inhibited Cav3.1 and Cav3.2 currents in a dose-dependent manner. The oxidizing reagent (1 mM) reduced the peak amplitude of Cav3.1 and Cav3.2 currents by ~50% over 2-3 minutes and the decreased currents were fully recovered upon washout of it. The reagent slowed the activation and inactivation kinetics of Cav3.1, Cav3.2, and Cav3.3 channel currents. Notably, the reagent positively shifted both activation and steady-state inactivation curves of Cav3.1, while it did not those of Cav3.2. Utilizing chimeric channels from Cav3.1 and Cav3.2, we localized the domains III and IV of Cav3.1 responsible for the positive shifts of channel activation and steady-state inactivation. These findings provide hints relevant to the electrophysiological and molecular mechanisms accounting for the oxidative regulation of T-type channels. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels CaV3.1 and CaV3.2 by nitrous oxide (N2O). To identify functionally important channel structures, chimeras with reciprocal exchange of the N-terminal domains I and II and C-terminal domains III and IV were examined. In whole-cell recordings N2O significantly inhibited CaV3.2, and – less pronounced – CaV3.1. A CaV3.2-prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cell-attached patches containing ≤3 channels N2O reduced average peak current of CaV3.2 by decreasing open probability and open time duration. Effects on CaV3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single CaV3.1 channels were significantly less active than CaV3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within CaV3.2 (H191), are responsible for the subtype-prevalent N2O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N2O on CaV3.2. Such a fingerprint of single channels can help identifying the molecular nature of native channels. This is exemplified by a characterization of single channels expressed in human hMTC cells as functional homologues of recombinant CaV3.1. [Copyright &y& Elsevier]

Abstract: We recently reported that a histidine (H191) in the S3–S4 loop of domain I is critical for nickel inhibition of the Cav3.2 T-type Ca2+ channel. As in Cav3.2, two histidine residues are commonly found in the IS3–IS4 loops of mammalian Cav2.3 Ca2+ channels, which are also blocked by low micromolar concentrations of nickel. We show here by site-directed mutagenesis and electrophysiology that both residues contribute to the nickel sensitivity of Cav2.3, with H183 being more critical than H179. These findings strongly suggest that both H179 and H183 in the IS3–IS4 loop are essential structural determinants required for nickel sensitive inhibition of the Cav2.3. [Copyright &y& Elsevier]

T-type Ca2+ channels play essential roles in numerous cellular processes. Recently, we reported that phorbol-12-myristate-13-acetate (PMA) potently enhanced the current amplitude of Cav3.2 T-type channels reconstituted in Xenopus oocytes. Here, we have compared PMA modulation of the activities of Cav3.1, Cav3.2 and Cav3.3 channels, and have investigated the underlying mechanism. PMA augmented the current amplitudes of the three T-type channel isoforms, but the fold stimulations and time courses differed. The augmentation effects were not mimicked by 4α-PMA, an inactive stereoisomer of PMA, but were abolished by preincubation with protein kinase C (PKC) inhibitors, indicating that PMA augmented T-type channel currents via activation of oocyte PKC. The stimulation effect on Cav3.1 channel activity by PKC was mimicked by endothelin when endothelin receptor type A was coexpressed with Cav3.1 in the Xenopus oocyte system. Pharmacological studies combined with fluorescence imaging revealed that the surface density of Cav3.1 T-type channels was not significantly changed by activation of PKC. The PKC effect on Cav3.1 was localized to the cytoplasmic II–III loop using chimeric channels with individual cytoplasmic loops of Cav3.1 replaced by those of Cav2.1. [ABSTRACT FROM AUTHOR]

Abstract: High voltage-activated Ca2+ channel expression and gating is controlled by their β subunits. Although the sites of interaction are known at the atomic level, how β modulates gating remains to be determined. Using a chimeric approach, β subunit regulation was conferred to a low voltage-activated channel. Regulation was dependent on a rigid linker connecting the α1 interaction domain to IS6. Chimeric channels also revealed a role for IS6 in channel gating. Taken together, these results support a direct coupling model where β subunits alter movements in IS6 that occur as the channel transits between closed, open, and inactivated states. [Copyright &y& Elsevier]

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 Cav3.2 T-type channels reconstituted in Xenopus oocytes. Phorbol-12-myristate-13-acetate (PMA) strongly enhanced the amplitude of Cav3.2 channel currents (∼3-fold). The augmentation effects were not mimicked by 4α-PMA, an inactive stereoisomer of PMA, and abolished by preincubation with PKC inhibitors. Our findings suggest that PMA upregulates Cav3.2 channel activity via activation of oocyte PKC. [Copyright &y& Elsevier]

Molecular diversity in T-type Ca2+ channels is produced by expression of three genes, and alternative splicing of those genes. Prompted by differences noted between rat and human Cav3.3 sequences, we searched for splice variants. We cloned six variants, which are produced by splicing at exon 33 and exon 34. Expression of the variants differed between brain regions. The electrophysiological properties of the variants displayed similar voltage-dependent gating, but differed in their kinetic properties. The functional impact of splicing was inter-related, suggesting an interaction. We conclude that alternative splicing of the Cav3.3 gene produces channels with distinct properties. [Copyright &y& Elsevier]

Ca2+ entry through Cav1.3 Ca2+ channels plays essential roles in diverse physiological events. We employed yeast-two-hybrid (Y2H) assays to mine novel proteins interacting with Cav1.3 and found Snapin2, a synaptic protein, as a partner interacting with the long carboxyl terminus (CTL) of rat Cav1.3L variant. Co-expression of Snapin with Cav1.3L/Cavβ3/α2δ2 subunits increased the peak current density or amplitude by about 2-fold in HEK-293 cells and Xenopus oocytes, without affecting voltage-dependent gating properties and calcium-dependent inactivation. However, the Snapin up-regulation effect was not found for rat Cav1.3S containing a short CT (CTS) in which a Snapin interaction site in the CTL was deficient. Luminometry and electrophysiology studies uncovered that Snapin co-expression did not alter the membrane expression of HA tagged Cav1.3L but increased the slope of tail current amplitudes plotted against ON-gating currents, indicating that Snapin increases the opening probability of Cav1.3L. Taken together, our results strongly suggest that Snapin directly interacts with the CTL of Cav1.3L, leading to up-regulation of Cav1.3L channel activity via facilitating channel opening probability. [ABSTRACT FROM AUTHOR]

Abstract Expression of rat α1G, human α1H and rat α1I subunits of voltage-activated Ca2 + channels in HEK-293 cells yields robust Ca2 + inward currents with 1.25 mm Ca2 + as the charge carrier. Both similarities and marked differences are found between their biophysical properties. Currents induced by expression of α1G show the fastest activation and inactivation kinetics. The α1H and α1I currents activate and inactivate up to 1.5- and 5-fold slower, respectively. No differences in the voltage dependence of steady state inactivation are detected. Currents induced by expression of α1G and α1H deactivate with time constants of up to 6 ms at a test potential of - 80 mV, but currents induced by α1I deactivate about three-fold faster. Recovery from short-term inactivation is more than three-fold slower for currents induced by α1H and α1I in comparison to α1G. In contrast to these characteristics, reactivation after long-term inactivation was fastest for currents arising from expression of α1I and slowest in cells expressing α1H calcium channels. The calcium inward current induced by expression of α1I is increased by positive prepulses while currents induced by α1H and α1G show little ( < 5%) or no facilitation. The data thus provide a characteristic fingerprint of each channel’s activity, which may allow correlation of the α1G, α1H and α1I induced currents with their in vivo counterparts. [ABSTRACT FROM AUTHOR]

Abstract Voltage-dependent activity around the resting potential is determinant in neuronal physiology and participates in the definition of the firing pattern. Low-voltage-activated T-type Ca2 + channels directly affect the membrane potential and control a number of secondary Ca2 + -dependent permeabilities. We have studied the ability of the cloned T-type channels (α1G,H,I) to carry Ca2 + currents in response to mock action potentials. The relationship between the spike duration and the current amplitude is specific for each of the T-type channels, reflecting their individual kinetic properties. Typically the charge transfer increases with spike broadening, but the total Ca2 + entry saturates at different spike durations according to the channel type: 4 ms for α1G; 7 ms for α1H; and > 10 ms for α1I channels. During bursts, currents are inhibited and/or transiently potentiated according to the α1 channel type, with larger effects at higher frequency. The inhibition may be induced by voltage-independent transitions toward inactivated states and/or channel inactivation through intermediate closed states. The potentiation is explained by an acceleration in the channel activation kinetics. Relatively fast inactivation and slow recovery limit the ability of α1G and α1H channels to respond to high frequency stimulation ( > 20 Hz). In contrast, the slow inactivation of α1I subunits allows these channels to continue participating in high frequency bursts (100 Hz). The biophysical properties of α1G, H and I channels will therefore dramatically modulate the effect of neuronal activities on Ca2 + signalling. [ABSTRACT FROM AUTHOR]