Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis

Abstract

Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Initially, Cl- conductance in the sweat duct was discovered to be impaired in CF, a finding that has been extended to all CFTR-expressing cells. Subsequent cloning of the gene showed that CFTR functions as a cyclic-AMP-regulated Cl- channel; and some CF-causing mutations inhibit CFTR Cl- channel activity. The identification of additional CF-causing mutants with normal Cl- channel activity indicates, however, that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters, including Cl(-)-coupled HCO3- transport. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues, indicating the importance of this activity. HCO3- and pH affect mucin viscosity and bacterial binding. We have examined Cl(-)-coupled HCO3- transport by CFTR mutants that retain substantial or normal Cl- channel activity. Here we show that mutants reported to be associated with CF with pancreatic insufficiency do not support HCO3- transport, and those associated with pancreatic sufficiency show reduced HCO3- transport. Our findings demonstrate the importance of HCO3- transport in the function of secretory epithelia and in CF.

Figure 1

cAMP-stimulated Cl⁻ and ${\text{HCO}}_3^{-}$ transport by wild-type (WT) CFTR and the CFTR mutants I148T and R117H. The whole-cell Cl⁻ current of HEK293 cells transfected with the indicated constructs was determined (a–c). For, forskolin. The current was normalized with respect to membrane capacitance and GFP fluorescence before averaging (d). Transfected cells were also loaded with MQAE (e–h) or BCECF (i–l) for measurements of [Cl⁻]_i and pH_i, respectively. Cells loaded with MQAE were exposed to a solution in which Cl⁻ was replaced with ${\text{NO}}_3^{-}$ and then stimulated with 5 μM forskolin. For pH measurements, Cl⁻ was replaced with gluconate. After calibration, initial rates of changes in [Cl⁻]_i (h) and pH_i (l) were averaged.

Figure 2

cAMP-stimulated Cl⁻ and ${\text{HCO}}_3^{-}$ transport by CFTR mutants associated with a severe or a mild form of CF. [Cl⁻]_i (a, b, e, f) and ${\text{HCO}}_3^{-}$ (c, d, g, h) transport were measured as in Fig. 1. Summaries of averaged rates are given for Cl⁻ (i) and for ${\text{HCO}}_3^{-}$ (j).

Figure 3

The ${\text{HCO}}_3^{-}:{\text{Cl}}^{-}$ transport ratio of CFTR mutants associated with CF. The ${\text{HCO}}_3^{-}:{\text{Cl}}^{-}$ transport ratios were calculated from the averaged rates summarized in Table 1 of the Supplementary Information. The ratio measured in wild-type CFTR was set to 1. Inset illustrates the different cytoplasmic domains of CFTR. CL1, cytoplasmic loop 1; CL2, cytoplasmic loop 2; NBD1, nucleotide-binding domain 1; RD, regulatory domain; CL3, cytoplasmic loop 3; CL4, cytoplasmic loop 4; NBD2, nucleotide-binding domain 2.