The proximal C-terminus of alpha(1C) subunits is necessary for junctional membrane targeting of cardiac L-type calcium channels

In cardiac myocytes, LTCCs (L-type calcium channels) form a functional signalling complex with ryanodine receptors at the JM (junctional membrane). Although the specific localization of LTCCs to the JM is critical for excitation-contraction coupling. their targeting mechanism is unclear. Transient transfection of GFP (green fluorescent protein)-alpha(1S) or GFP-alpha(1C) but not P/Q-type calcium channel alpha(1A), in dysgenic (alpha(1S)-null) GLT myotubes results in correct targeting of these LTCCs to the JMs and restoration of action-potential-induced Ca2+ transients. To identify the sequences of alpha(1C) responsible for JM targeting, we generated a range of alpha(1C)-alpha(1A) chimaeras, deletion mutants and alanine substitution mutants and studied their targeting properties in GLT myotubes. The results revealed that amino acids L-1681 QAGLRTL(1688) and P(1693)EIRRAIS(1700), predicted to form two adjacent alpha-helices in the proximal C-terminus, are necessary for the JM targeting of alpha(1C). The efficiency of restoration of action-potential-induced Ca2+ transients in GLT myotubes was significantly decreased by mutations in the targeting motif. JM targeting was not disrupted by the distal C-terminus of alpha(1C) which binds to the second alpha-helix. Therefore we have identified a new structural motif in the C-terminus of alpha(1C) that mediates the targeting of cardiac LTCCs to JMs independently of the interaction between proximal and distal C-termini of alpha(1C).ArticleBIOCHEMICAL JOURNAL. 448:221-231 (2012)journal articl

Two mechanistically distinct effects of dihydropyridine nifedipine on Ca(V)1.2 L-type Ca2+ channels revealed by Timothy syndrome mutation

Dihydropyridine Ca2+ channel antagonists (DHPs) block Ca(V)1.2 L-type Ca2+ channels (LTCCs) by stabilizing their voltage-dependent inactivation (VDI); however, it is still not clear how DHPs allosterically interact with the kinetically distinct (fast and slow) VDI. Thus, we analyzed the effect of a prototypical DHP, nifedipine on LTCCs with or without the Timothy syndrome mutation that resides in the I-II linker (LI-II) of Ca(V)1.2 subunits and impairs VDI. Whole-cell Ba2+ currents mediated by rabbit Ca(V)1.2 with or without the Timothy mutation (G436R) (analogous to the human G406R mutation) were analyzed in the presence and absence of nifedipine. In the absence of nifedipine, the mutation significantly impaired fast closed-and open-state VDI (CSI and OSI) at -40 and 0 mV, respectively, but did not affect channels' kinetics at -100 mV. Nifedipine equipotently blocked these channels at -80 mV. In wild-type LTCCs, nifedipine promoted fast CSI and OSI at -40 and 0 mV and promoted or stabilized slow CSI at -40 and -100 mV, respectively. In LTCCs with the mutation, nifedipine resumed the impaired fast CSI and OSI at -40 and 0 mV, respectively, and had the same effect on slow CSI as in wild-type LTCCs. Therefore, nifedipine has two mechanistically distinct effects on LTCCs: the promotion of fast CSI/OSI caused by LI-II at potentials positive to the sub-threshold potential and the promotion or stabilization of slow CSI caused by different mechanisms at potentials negative to the subthreshold potential.ArticleEUROPEAN JOURNAL OF PHARMACOLOGY. 685(1-3):15-23 (2012)journal articl

NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG

<div><p>The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression.</p></div

NQO1 expression and 17-AAG sensitivity in melanoma and NSCLC cell lines.

<p>NQO1 expression and 17-AAG sensitivity in melanoma and NSCLC cell lines.</p

Combination treatment with 17-AAG and CDDP or DTIC.

<p>Combination treatment with 17-AAG and CDDP or DTIC.</p

NRF2-dependent NQO1 expression in melanocytes and melanoma cells harboring wild-type KEAP1.

<p>(A) Immortalized melanocytes (LP/TERT) were transfected with siRNA against NRF2 or control siRNA (Cont), or treated with transfection medium alone (Mock, −). Total RNA was extracted on day 3 and subjected to RT-PCR to quantify NRF2, NQO1 and GAPDH cDNAs. The data were expressed as mean±S.D. of three independent experiments (n = 3), and statistical significance was expressed as *, <i>p</i><0.05; **, <i>p</i><0.01; and ***, <i>p</i><0.001 compared to control siRNA samples. Whole extracts were prepared on day 4 and subjected to immunoblotting for detection of NRF2, NQO1 and TUBA. (B) HMV-II cells were treated as described in (A), and RT-PCR and immunoblotting were carried out on day 3 and day 6, respectively. (C) C32 cells were treated and subjected to RT-PCR and immunoblotting as described in (B).</p

Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper amine oxidase, which contains a prosthetic copper ion and a protein-derived redox cofactor, topa quinone. We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a non-planar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the copper. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions