Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that apparently has evolved from an ancestral active transporter. Key to the CFTR's switch from pump to channel function may have been the appearance of one or more "lateral portals." Such portals connect the cytoplasm to the transmembrane channel pore, allowing a continuous pathway for the electrodiffusional movement of Cl- ions. However, these portals remain the least well-characterized part of the Cl- transport pathway; even the number of functional portals is uncertain, and if multiple portals do exist, their relative functional contributions are unknown. Here, we used patch-clamp recording to identify the contributions of positively charged amino acid side chains located in CFTR's cytoplasmic transmembrane extensions to portal function. Mutagenesis-mediated neutralization of several charged side chains reduced single-channel Cl- conductance. However, these same mutations differentially affected channel blockade by cytoplasmic suramin and Pt(NO2)42- anions. We considered and tested several models by which the contribution of these positively charged side chains to one or more independent or non-independent portals to the pore could affect Cl- conductance and interactions with blockers. Overall, our results suggest the existence of a single portal that is lined by several positively charged side chains that interact electrostatically with both Cl- and blocking anions. We further propose that mutations at other sites indirectly alter the function of this single portal. Comparison of our functional results with recent structural information on CFTR completes our picture of the overall molecular architecture of the Cl- permeation pathway.
Highlights
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl؊ channel that apparently has evolved from an ancestral active transporter
In this work we have studied the effect of mutating positively charged residues potentially contributing to portal function (Fig. 1, B and E) on both ClϪ conductance and block by cytoplasmic suramin and Pt(NO2)42Ϫ ions
We found that neutralization of another nearby positively charged side chain, Lys[978] (that we could not previously investigate in cysteine-less CFTR for technical reasons (17)), caused a similar, significant reduction in ClϪ conductance (Fig. 2C)
Summary
Members of which function as ATP-dependent pumps (1, 2). It is presumed that some evolutionary change has allowed CFTR to function not as a pump but as an ion channel (3–5). As an ion channel, CFTR must exhibit a continuous aqueous pathway that allows free electrodiffusional movement of ClϪ and other small anions between the cytoplasm and the extracellular solution in the channel open state. Such a continuous open pathway is incompatible with the function of a pump. Positive electrostatic potential around this single large portal supports the idea that it is involved in electrostatic attraction of cytoplasmic ClϪ ions (8) In addition to their functional importance in ClϪ conduction (17, 18), the portals formed by the TMEs may be the site of action of some CFTR inhibitors. Our results suggest that CFTR possesses one dominant lateral portal with a cytoplasmic entrance between TMEs 4 and 6 and that a number of positively charged side chains surrounding this portal act to attract cytoplasmic anions to the pore
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