Abstract-The HCN family of ion channel subunits underlies the currents I f in heart and I h and I q in the nervous system.In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a  subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a  subunit for the cardiac delayed rectifier I Kr , these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2001;88:e84-e87.)Key Words: HCN family Ⅲ MiRP1 Ⅲ KCNE family Ⅲ  subunit T he HCN (hyperpolarization-activated cyclic nucleotidegated) family of ion channel subunits has been identified as the molecular correlate of the currents I f in heart and I h and I q in neurons. [1][2][3] However, several ion channels are heteromultimers of a large ␣ subunit (like the HCN family members) and smaller  subunits. The cardiac delayed rectifiers I Kr 4 and I Ks 5 are examples of this basic principle. Their ␣ subunits derive from the ERG and KCNQ families, respectively, but both also contain  subunits from the KCNE family of single transmembranespanning proteins called minK and minK-related peptides (MiRPs). In this study, we report that MiRP1 enhances the expression and speeds the kinetics of activation of the HCN family of channel subunits. From immunoprecipitation experiments, we show that it most probably forms a complex with HCN1. Using RNase protection assays (RPAs), we demonstrate that MiRP1 mRNA is prevalent in the primary cardiac pacemaking region, the sinoatrial (SA) node, and barely detectable in ventricle. Cardiac pacemaker activity is generated by a narrow balance of inward (I f ) and outward (I Kr ) currents. Our results demonstrate for the first time the potential importance of a single  subunit in simultaneously regulating both the expression and gating of both inward and outward cardiac pacemaker channels. Materials and Methods Heterologous Expression in Xenopus OocytescRNA encoding mouse HCN1 or HCN2, rat MiRP1 with or without an HA tag at the carboxy-terminal, and rat minK were transcribed using the mMessage mMachine kit (Ambion). Xenopus laevis oocytes were isolated, injected with 2 to 5 ng (50 to 100 nL) of cRNA, and maintained in Barth medium at 18°C for 1 to 3 days. For experiments using both HCN1 or HCN2 and MiRP1 or minK, the respective cRNAs were injected in a 1:0.04 to 1 ratio. Electrophysiological studies on oocytes used the 2-microelectrode voltage clamp. The extracellular recording solution (OR2) contained, in mmol/L, NaCl 80, KCl 2, MgCl 2 1, and Na-HEPES 5 (pH 7.6). Group data are presented as meanϮSEM. Tests of statistical significance for midpoint and slope of activation curves were performed using unpaired Student's t tests. PϽ0.05 is considered significant. RNase Protection AssaysThe procedures for the preparation of total RNA from rabbit he...
Seventeen compounds of the type R3SnAA, where R = methyl (Me) or cyclohexyl (Cyh), and AA is the anion of glycine (gly), DL-a-alanine (ala), DL-a-amino-re-butyric acid (but), DL-a-valine (val), DL-a-leucine (leu), L-a-isoleucine (isoleu), ßalanine ((3-ala), and glycylglycine (glygly), including tri-n-butyltin glycinate (n-Bu3Sn(gly)), have been prepared by azeotropic distillation of water from benzene solutions of the corresponding stannol or bis(trialkyltin) oxide and the acids; DMF is added as a catalyst for R = Me and AA = gly and ala and for n-Bu3Sn(gly). From lowered infrared amino group stretching frequencies and enhanced intensities, twelve of the compounds are identified as amino coordinated to tin, including all the Me3Sn derivatives, n-Bu3Sn(gly), Cyh3Sn(gly), (3-ala, glygly. These compounds, except the last, exhibit Mossbauer QS values in the range 3.2 ± 0.1 mm/sec and ratios of QS to IS generally greater than 2.1, consistent with higher than fourcoordination and pronounced line intensity asymmetry (Goldanskii-Karyagin effect) suggesting associated lattices. Me3Sn-(gly), ala, glygly, and n-Bu3Sn(gly) give ambient-temperature Mossbauer spectra confirming polymeric structures. High v(0-C=0) stretching frequencies, assigned with the aid of deuterated amino group isotopomers, rule out carboxylate carbonyl oxygen coordination to tin in these cases, and infrared and Raman data in the tin-carbon stretching region [v-(Sn-C) is assigned at 490 and 420 cm"1 for the cyclohexyltin group] rule out precisely planar SnC3 skeletons. The aminobridged compounds are depicted as one-dimensional associated lattices of trigonal-bipyramidal units of the axially most electronegative kind. Cyh3Sn(glygly), whose larger QS value and lack of line asymmetry suggest a different structural type, is from lowered infrared stretching frequency evidence coordinated by the amide carbonyl to give six-coordination at tin which from the large QS value must be in the meridian configuration of which no examples are known. Of the five Cyh3Sn derivatives lacking amino-coordination, two (but and val) are, from high v(0-C=0) and reduced QS values, simple, fourcoordinated monomers.The remaining three are higher than four-coordinated from lowered carboxylate stretching frequencies, but QS data rule out axially most electronegative structures. These results are rationalized on the steric effect of the groups at tin and the ce-carbon substituent in the ligand; when both are small, the one-dimensional polymer formed by bridging amino groups is the preferred structure; in intermediate cases carboxylate group coordination, utilizing equatorial or equatorial-axial positions presumably through chelation in monomeric units, becomes preferred; when both are large, only simple, four-coordinated monomers are found. Cyh3SnOH, used for comparison, is shown to be polymeric on the basis of the ambient-temperature Mossbauer spectrum, pronounced line intensity asymmetry, large QS value, and the observation of fragments higher than the molecular ion in the mass spec...
Ein verbesserter Syntheseweg fiir Pentaphenyicyciopentadienol (I), Pentaphenylcyclopentadienyibromid (Z), Pentaphenylcyclopentadien (3), und Pentaphenylcyclopentadienyllithium (4a), die DarsteUung von Decaphenylgermanocen (S), -stannocen (6) und -plumbocen (7) sowie von Pentaphenylstannocen (8) wird beschrieben. Die vollstiindigen analytischen Daten OR-, Raman-, Riintgenpulver-, NMR-, Massen-und "9"Sn-MiiBbauer-Spektren) werden angegeben. Es werden Il9Sn-und 207Pb-CPMAS-NMR-Messungen von verschiedenen Stannoamen und dem Plumbocen 7 mitgeteilt.
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