The ryanodine receptor (RyR) is one of the key proteins involved in excitation-contraction (E-C) coupling in skeletal muscle, where it functions as a Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. RyR consists of a single polypeptide of approximately 560 kDa normally arranged in a homotetrameric structure, which contains a carboxyl (C)-terminal transmembrane domain and a large amino (N)-terminal cytoplasmic domain. To test whether the carboxyl-terminal portion of RyR is sufficient to form a Ca2+ release channel, we expressed the full-length (RyR-wt) and C-terminal (RyR-C, approximately 130 kDa) RyR proteins in a Chinese hamster ovary (CHO) cell line, and measured their Ca2+ release channel functions in planar lipid bilayer membranes. The single-channel properties of RyR-wt were found to be similar to those of RyR from skeletal muscle SR. The RyR-C protein forms a cation-selective channel that shares some of the channel properties with RyR-wt, including activation by cytoplasmic Ca2+ and regulation by ryanodine. Unlike RyR-wt, which exhibits a linear current-voltage relationship and inactivates at millimolar Ca2+, the channels formed by RyR-C display significant inward rectification and fail to close at high cytoplasmic Ca2+. Our results show that the C-terminal portion of RyR contains structures sufficient to form a functional Ca2+ release channel, but the N-terminal portion of RyR also affects the ion-conduction and calcium-dependent regulation of the Ca2+ release channel.

For a cystic fibrosis transmembrane conductance regulator (CFTR) channel to enter its open state, serine residues in the R domain must be phosphorylated by cAMP-dependent protein kinase, and intracellular ATP must bind to the nucleotide-binding folds and subsequently be hydrolyzed. CFTR with its R domain partially removed, ⌬R(708 -835)-CFTR, forms a chloride channel that opens independently of protein kinase A phosphorylation, with open probability approximately one-third that of the wild type CFTR channel. Deletion of this portion of the R domain from CFTR alters the response of the channel to 5-adenylylimidodiphosphate, pyrophosphate, and vanadate, compounds that prolong burst duration of the wild type CFTR channel but fail to do so in the ⌬R-CFTR. In addition, the addition of exogenous unphosphorylated R domain protein, which blocks the wild type CFTR channel, has no effect on the ⌬R-CFTR channel. However, when the exogenous R domain is phosphorylated, significant stimulation of the ⌬R-CFTR channel results; Rich et al. (9) demonstrated that deletion of 128 amino acids from the R domain of CFTR, ⌬R(708 -835), leads to a chloride channel that opens without PKA phosphorylation. This portion of the R domain corresponds to sequences that are not conserved in related proteins, i.e. MDR1 and STE6 (9 -11). The portion of the R domain that remains in ⌬R-CFTR corresponds to amino acids that are present in other ATP-binding cassette transporters between the predicted first nucleotide binding fold and the predicted second membrane-spanning domain (9, 11). Our studies show that unphosphorylated R domain protein synthesized in vitro (amino acids 590 -858) interacts specifically with CFTR to inhibit chloride conductance in a phosphorylationdependent manner (12). These studies were interpreted to be consistent with the hypothesis that the putative "gating particle" of the CFTR channel resides within the R domain.The first and second NBF of CFTR share sequence similarity in certain conserved regions such as Walker A and Walker B motifs, but the overall amino acid homology between the two NBFs of CFTR is only ϳ30%. Functional studies of CFTR containing site-directed mutations in NBF1 and NBF2 suggest that the two NBFs have different roles in the gating of the CFTR channel (13)(14)(15). Mutations predicted to interfere with nucleotide hydrolysis in the first NBF reduce the channel opening rate, but the corresponding mutations in the second NBF result in prolonged channel openings (15). The use of compounds that alter the ATP hydrolysis cycle of CFTR, such as 5Ј-adenylylimidodiphosphate (AMP-PNP), PP i , and vanadate (VO 4 ), provided evidence that hydrolysis of ATP is not only required for channel opening but also is involved in channel closure from the bursting state (16 -19). Based on these studies, it was proposed that ATP hydrolysis at NBF1 initiates a burst of activity and that hydrolysis at NBF2 terminates a burst of open events (15).This dual regulatory mechanism of the CFTR channel (PKA phosphorylation of the R domain ...

We describe an integrated workflow for proteogenomic analysis and global profiling of posttranslational modifications (PTMs) in prokaryotes and use the model cyanobacterium Synechococcus sp. PCC 7002 (hereafter Synechococcus 7002) as a test case. We found more than 20 different kinds of PTMs, and a holistic view of PTM events in this organism grown under different conditions was obtained without specific enrichment strategies. Among 3,186 predicted protein-coding genes, 2,938 gene products (>92%) were identified. We also identified 118 previously unidentified proteins and corrected 38 predicted gene-coding regions in the Synechococcus 7002 genome. This systematic analysis not only provides comprehensive information on protein profiles and the diversity of PTMs in Synechococcus 7002 but also provides some insights into photosynthetic pathways in cyanobacteria. The entire proteogenomics pipeline is applicable to any sequenced prokaryotic organism, and we suggest that it should become a standard part of genome annotation projects.proteogenomics | post-translational modifications | cyanobacteria | photosynthesis | Synechococcus