TRPM4 Channels Mediate Hypertonicity-induced, Ca2+-impermeable, Non-selective Cation Currents in a Cervical Cancer Cell Line, HeLa Cells

Article信州医学雑誌 62(1):33-44(2014)journal articl

β(2)-Adrenergic and M(2)-muscarinic receptors decrease basal t-tubular L-type Ca2+ channel activity and suppress ventricular contractility in heart failure

L-Lype Ca2+ channels (LTCC) play a crucial role in cardiac excitation-contraction coupling. We previously found that in failing ventricular myocytes of mice chronically treated with isoproterenol, basal t-tubular (TT) LTCC activity was halved by activation of protein phosphatase (PP)2A whereas basal surface sarcolemmal (SS) LTCC activity was doubled by inhibition of PP1. Interestingly, chronic treatment of these mice with pertussis toxin almost completely normalized TT and SS LTCC densities and cardiac contractility. In the present study, we therefore sought to identify the G(i/o) protein coupled receptors in cardiac myocytes (i.e. beta(2)-adrenergic, M-2-muscarinic and A(1)-adenosine receptors) that are responsible for these abnormalities in heart failure by chronically administrating mice a selective antagonist of each receptor (ICI118,551, atropine and 8-cyclopentyl-1,3-dipropilxanthine (DPCPX), respectively) with isoproterenol. Compared with mice treated with isoproterenol alone, mice treated with isoproterenol plus ICI118,551 or atropine, but not DPCPX showed significantly lower lung weight/tibial length, higher fractional shortening, lower left ventricular end-diastolic pressure and higher dP/dt(max) and dP/dt(min). In addition, ventricular myocytes of mice treated with isoproterenol plus ICI118,551 or atropine, but not DPCPX exhibited significantly higher TT and lower SS LTCC current densities than those of mice treated with isoproterenol alone due to normalization of the PP activities. These results indicate that beta(2)-adrenergic, M-2-muscarinic, but not A(1)-adenosine receptors contribute to reduced ventricular contractility at least partially by decreasing basal TT LTCC activity in heart failure. Therefore, antagonists of beta(2)-alrenergic and/or M-2-muscarinic receptors can be good adjuncts to beta(1)-adrenergic receptor antagonists in the treatment of heart failure.ArticleEUROPEAN JOURNAL OF PHARMACOLOGY. 724:122-131 (2014)journal articl

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

Accuracy of short-term insomnia onset and composite clinical score in the differential diagnosis of common cold and influenza : A cross-sectional study\n

　Influenza (IA) and the common cold (CC) may present with similar symptoms. However, rapid IA diagnostic tests have a low sensitivity during the early stages of the disease. Therefore, various diagnostic tools are needed. Mild short-term insomnia (MSTI) occurs in patients with CC, but the incidence in patients with IA remains unknown. Hence, we hypothesized the difference between the day of onset of MSTI and the date of fever occurrence in IA to be shorter than that in CC. This study aimed to investigate the incidence of MSTI in patients with CC and IA with fever ( 37.5℃) and the risk of developing acute-onset MSTI (alone or as part of a composite clinical score) to aid in the differential diagnosis during the early disease stage. The relationship between MSTI and other symptoms was analyzed using quadratic logistic regression to model the number of IA cases with composite score day progression from the onset of fever, namely origin day 0. The results revealed a day 0 axis- symmetrical and inverted U-shaped relationship between the onset days of the composite scores and estimated probability of IA. The coefficient of the quadratic term is negative and significantly different from zero, supporting the hypothesis that the onset of IA-related MSTIs is more acute than that of CC-related MSTIs. Principal component analysis was also applied to the onset days for five IA- and CC-related symptoms, in addition to fever (MSTI, cough, rhinorrhea, sore throat, and headache). The maximum day difference in fever onset and at least one additional symptom was established as the cutoff day between IA and CC using receiver operating characteristic analysis. The cutoff value was confirmed based on the MSTI-onset- derived variables using logistic regression. This cross-sectional survey included 100 and 106 CC and IA patients, respectively. The optimal cutoff day were one day before and one day after the onset of fever. For those with CC and MSTI beyond the cutoff day, the diagnostic sensitivity, specificity, and positive likelihood ratio were 7%, 99%, and 7.42, respectively. For patients with CC and any composite clinical score symptoms beyond the cutoff day, the sensitivity, specificity, and positive likelihood ratios were 36%, 92%, and 4.77, respectively. A composite clinical score and MSTI onset with a cutoff day of one day before and after fever onset may help distinguish between early stage CC and IA

The proximal C-terminus of α1C 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