Chimeric Constructs Confer the Human CFTR Chloride Channel with the Properties of Mouse CFTR

Mouse cystic fibrosis transmembrane conductance regulator (CFTR) differs from human CFTR in several important respects: altered gating pattern distinguished by prolonged residence in a sub-conductance state, reduced single-channel conductance and strong inward rectification. Transfer of the regulatory (R) domain of mouse CFTR to human CFTR was without effect, whereas transfer of both nucleotide-binding domains (NBDs) endowed human CFTR with greatly prolonged channel openings without conferring human CFTR with the sub-conductance state, single-channel conductance and inward rectification of mouse CFTR. We therefore hypothesised that the membrane-spanning domains (MSDs) determine these differences between human and mouse CFTR. To test this idea, we constructed the human-mouse CFTR (hmCFTR) chimeras hmTM1-6, hmTM7-12 and hmTM1-6:TM7-12 containing MSD1, MSD2 and MSD1+2 of mouse CFTR on a human CFTR background and studied them in excised inside-out membrane patches from transiently transfected CHO cells. For single-channel studies, we used a large Cl- concentration gradient ([Cl-]i, 147 mM; [Cl-]e, 10 mM) and clamped voltage at –50 mV, whereas for macroscopic studies, we used symmetrical Cl--rich solutions. All intracellular solutions contained PKA (75 nM) and ATP (1 mM) to activate and sustain CFTR activity; temperature was 37 °C. The three hmCFTR chimeras possessed a gating pattern intermediate between human and mouse CFTR. Both the single-channel conductance and open probability of the full open-state decreased in the rank order: human CFTR > hmTM7-12 > hmTM1-6 ≥ hmTM1-6:TM7-12 (n = 5-10). Like human CFTR, hmTM7-12 rarely transitioned to sub-conductance states, whereas hmTM1-6 sojourned to multiple sub-conductance states and hmTM1-6:TM7-12 resided in a tiny sub-conductance state resembling that of mouse CFTR. Human CFTR Cl- currents exhibited weak inward rectification, whereas those of mouse CFTR strongly inwardly rectified. Transfer of both MSDs of mouse CFTR to human CFTR in the chimeras hmTM1-6:TM7-12 reproduced the strong inward rectification of mouse CFTR (n = 4). Interestingly, the inward rectification of hmTM1-6 closely resembled that of mouse CFTR, whereas that of hmTM7-12 was intermediate between human and mouse CFTR (n = 6-9). Taken together, our data demonstrate that sequences from both MSDs specify the gating, conduction and rectification properties of CFTR with sequences from MSD1 playing a dominant role in determining the different rectification behaviour of human and mouse CFTR. Supported by the Biotechnology and Biological Sciences Research Council and Cystic Fibrosis Trust. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.