Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) phosphorylates the β 2a subunit of voltagegated Ca 2+ channels at Thr498 to facilitate cardiac L-type Ca 2+ channels. CaMKII colocalizes with β 2a in cardiomyocytes and also binds to a domain in β 2a that contains Thr498 and exhibits amino acid sequence similarity to the CaMKII autoinhibitory domain and to a CaMKII binding domain in the NMDA receptor NR2B subunit (Grueter et al., 2006. Mol. Cell 23:641). Here we explore the selectivity of the actions of CaMKII among Ca 2+ channel β subunit isoforms. CaMKII phosphorylates the β 1b , β 2a , β 3 and β 4 isoforms with similar initial rates and final stoichiometries of 6-12 mole phosphate per mole protein. However, activated/autophosphorylated CaMKII binds to β 1b and β 2a with similar apparent affinity, but does not bind to β 3 or β 4 . Pre-phosphorylation of β 1b and β 2a by CaMKII substantially reduces the binding of autophosphorylated CaMKII. Residues surrounding Thr498 in β 2a are highly conserved in β 1b , but are different in β 3 and β 4 . Site-directed mutagenesis of this domain in β 2a showed that Thr498 phosphorylation promotes dissociation of CaMKII-β 2a complexes in vitro and reduces interactions of CaMKII with β 2a in cells. Mutagenesis of Leu493 to Ala substantially reduces CaMKII binding in vitro and in intact cells but does not interfere with β 2a phosphorylation at Thr498. In combination, these data show that phosphorylation dynamically regulates the interactions of specific isoforms of the VGCC β subunits with CaMKII. † This work was supported by National Institutes of Health (NIH) research grants to R. Figure 1) showing that CaMKII binding to GST-β 1b and GST-β 2a in a glutathione agarose cosedimentation assay is dependent on prior autophosphorylation of CaMKII is available free of charge via the Internet at http://pubs.acs.org. SUPPORTING INFORMATION AVAILABLE Supplementary data (Supplementary NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 February 1. [18][19][20]. In addition to these roles in feedback regulation, CaMKII is involved in crosstalk between Ca 2+ channels: for example, LTCC activation leads to depression of R-type Ca 2+ channels in dendritic spines via a poorly defined CaMKII-dependent mechanism (21).We recently showed that CaMKII-dependent facilitation of cardiac Ca V 1.2 LTCCs is mediated by phosphorylation of the β 2a subunit at Thr498 in cardiomyocytes (22). Moreover, β 2a acts as a CaMKII associated protein (CaMKAP) that directly interacts with CaMKII in vitro and in intact cells. Our findings showed that VGCC regulation may be strongly enhanced or modified by association of CaMKII with the β 2a subunit. In the current study, we demonstrate that CaMKII phosphorylates all of the β subunit isoforms, but interacts with β subunits in an isoform specific manner; this CaMKII-β subunit interaction is negatively modulated by phosphorylation of the β subunit. MATERIALS AND METHODS Generation of plasmid constructsThe open reading f...

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This analysis sets out to specifically discuss the polyfunctionality of 跟 [kai 55 ] in Waxiang (Sinitic), whose lexical source is the verb 'to follow'. Amongst its various uses, we find a preposition 'with, along', a marker of adjuncts and a NP conjunction, thus superficially resembling its Mandarin cognate 跟 gēn 'with'. Curiously, however, it has also evolved into a direct object marker in Waxiang, with a function similar to that of preposition 把 bă < 'hold, take' as found in the S-bă-O-VP or so-called 'disposal' form in standard Mandarin. The pathways of grammaticalization for 跟 [kai 55 ] in Waxiang are thus discussed in order to determine how it has developed this unusual grammatical function in one of the linguistic zones of China where verbs of giving or taking are, in fact, the main source for grammaticalized object markers in 'disposal' constructions.On the basis of 16 th and 17 th century Southern Min literature (Sinitic), a comparison is also made with analogous developments for comitative 共 gòng 'with' to provide support for our hypothesis that the direct object marking use has evolved from the oblique function of a benefactive or dative, and is clearly separate from the crosslinguistically well-attested pathway that leads to its use as a conjunction. We would thus like to propose that these data contribute a new pattern to the stock of 6 The symbol is used for any syllable which has no known corresponding Chinese character.7 Note that the word for 'carpenter' in example (12) is different from that in example (4) due to different informants. 16 The tone mark indicates the tonal category instead of the tonal quality for convenience of comparison: 1. 阴 平 Yīnpíng, 2. 阳 平 Yángpíng, 3. 上 声 Shǎngshēng, 4. 去声 Qùshēng and 5. 入声 Rùshēng respectively. In dialects which distinguish all the 阴 Yīn and 阳 Yáng registers, the one tone mark will stand for both of them. For example, the tone mark 4 stands for both 阴去 Yīnqù and 阳去 Yángqù. 19 Note that the Jishou dialect also has the locative 'along' use of 跟 gēn but its polsyemy does not extend to the object marking use: 20 Note that phonetic transcriptions were not provided in the original data. For the convenience of quotation, we have adopted the practice of romanizing such examples in the pīnyīn system devised for Mandarin but using small capitals to signal the fact it does not indicate the pronunciation of the given Mandarin dialect. 21 We do not wish to suggest here that only geographically isolated languages can undergo the development from comitative > accusative, as one of our reviewers has queried with reason. Rather, the objective here is to provide a possible explanation as to why Waxiang did not adopt or borrow the marker ba that is predominant in surrounding dialects of Southwestern Mandarin. 31 The characters for 有德 yu tec are missing, indicated by '__'. Chappell (2000, 2006, 2007) has carried out research on this topic for contemporary Sinitic languages in order to examine the diversity of lexical sources as well as the evolution of these objec...

In interstellar environment, fullerene species readily react with large molecules (e.g., PAHs and their derivatives) in the gas phase, which may be the formation route of carbon dust grains in space. In this work, the gas-phase ionmolecule collision reaction between fullerene cations (C n + , n=32, 34, ..., 60) and functionalized PAH molecules (9-hydroxyfluorene, C 13 H 10 O) are investigated both experimentally and theoretically. The experimental results show that fullerene/9-hydroxyfluorene cluster cations are efficiently formed, leading to a series of large fullerene/9hydroxyfluorene cluster cations (e.g., [The binding energies and optimized structures of typical fullerene/9-hydroxyfluorene cluster cations were calculated. The bonding ability plays a decisive role in the cluster formation processes. The reaction surfaces, modes and combination reaction sites can result in different binding energies, which represent the relative chemical reactivity. Therefore, the geometry and composition of fullerene/9-hydroxyfluorene cluster cations are complicated. In addition, there is an enhanced chemical reactivity for smaller fullerene cations, which is mainly attributed to the newly formed deformed carbon rings (e.g., 7 C-ring). As part of the coevolution network of interstellar fullerene chemistry, our results suggest that ion-molecule collision reactions contribute to the formation of various fullerene/9-hydroxyfluorene cluster cations in the ISM, providing insights into different chemical reactivity caused by oxygenated functional groups (e.g., hydroxyl, OH, or ether, C-O-C) on the cluster formations.