A conditional probability analysis of cystic fibrosis transmembrane conductance regulator gating indicates that ATP has multiple effects during the gating cycle.

ATP-binding cassette (ABC) transporters bind and hydrolyze ATP. In the cystic fibrosis transmembrane conductance regulator Cl(-) channel, this interaction with ATP generates a gating cycle between a closed (C) and two open (O1 and O2) conformations. To understand better how ATP controls channel activity, we examined gating transitions from the C to the O1 and O2 states and from these open states to the C conformation. We made three main observations. First, we found that the channel can open into either the O1 or O2 state, that the frequency of transitions to both states was increased by ATP concentration, and that ATP increased the relative proportion of openings into O1 vs. O2. These results indicate that ATP can interact with the closed state to open the channel in at least two ways, which may involve binding to nucleotide-binding domains (NBDs) NBD1 and NBD2. Second, ATP prolonged the burst duration and altered the way in which the channel closed. These data suggest that ATP also interacts with the open channel. Third, the channel showed runs of specific types of open-closed transitions. This finding suggests a mechanism with more than one cycle of gating transitions. These data suggest models to explain how ATP influences conformational transitions in cystic fibrosis transmembrane conductance regulator and perhaps other ABC transporters.

Changes in either cytosolic or nucleoplasmic inositol 1,4,5-trisphosphate levels can control nuclear Ca2+ concentration.

The free nucleoplasmic Ca2+ concentration ([Ca2+]n) may regulate many nuclear events, such as gene transcription. Since the nucleus may possess the enzymes necessary to generate the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), and because the nuclear envelope may enclose an Ins(1,4,5)P3-releasable Ca2+ store, we tested the hypothesis that nuclear and/or cytosolic levels of Ins(1,4,5)P3 can control [Ca2+]n. To assay [Ca2+]n, we measured the fluorescence of the Ca2+ indicator fluo 3 in the nucleus of Xenopus oocytes by confocal microscopy. When we injected Ins(1,4,5)P3 into the cytosol, [Ca2+]n increased. This increase in [Ca2]n still occurred when heparin was present in the nucleus, but was abolished when heparin was present in the cytosol, indicating that cytosolic Ins(1,4,5)P3 levels could control [Ca2+]n. When we injected Ins(1,4,5)P3 directly into the nucleus, [Ca2+]n increased, even when heparin was present in the cytosol, indicating that Ins(1,4,5)P3 could control [Ca2+]n from within the nucleus. These results provide functional evidence for Ins(1,4,5)P3 receptors facing the nucleoplasm and raise the possibility that a phosphoinositide cycle situated at the nuclear membranes can control Ca(2+)-dependent nuclear functions.