| The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ATP-gated Cl- channel that when mutated leads to the disease cystic fibrosis. Data obtained with the excised, patch-clamp technique indicate that CFTR opens in bursts of activity comprised of four kinetically distinguishable states; an open state that undergoes rapid block by the pH buffer MOPS (01), an open state not blocked by MOPS (02), a closed state due to MOPS block (Cblocked) and an interburst closed state (C). By developing a conditional probability analysis, we were able to define whether CFTR bursts began or ended via the 01 or the 02 state. By studying the propensity of CFTR to open and close via either 01 or 02, we noted several novel gating behaviors of CFTR. First, because ATP stimulated opening into both 01 and 02 but to differing degrees, this suggests that ATP can bind to two different sites to open CFTR. When bursts were defined based on if they began and ended via 01 or 02, we found that ATP had widely different effects on the occurrence of the burst types. This indicated that ATP is involved in more than just channel opening. Also, because ATP increased how long the channel remained open, this also indicates that ATP interacts with the open CFTR channel. Additional findings include; gating in ADP and saturating ATP (5 mM) was similar to that found in 1 mM ATP, impairing ATP binding to NBD1 (K464A) increased the likelihood of the 02 state, decreasing ATP concentration caused burst durations to be more varied, CFTR channels often go through the same burst type repeatedly, and CFTR possesses at least two different C states. To explain the novel data, we developed a parsimonious model of CFTR gating where ATP binds to either NBD to open the CFTR channel. Once open, an additional ATP may bind to the free NBD to stabilize the open state. Support for the new gating model is argued based on its ability to describe ours and other published data as well as by comparing the model to other possible models of gating. |
