Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligandgated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization.ATP binding cassette transporter | cystic fibrosis | ligand | constitutive | mutant C ystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily, although it is the only known ion channel in this transporter family (1). Like other ABC transporters, CFTR uses ATP binding to its two nucleotide binding domains (NBDs) to drive conformational rearrangements of its transmembrane domains (2, 3). CFTR channel opening is linked to ATP binding to each of two sites at the interface of an NBD1-NBD2 dimer (2, 3). Subsequent hydrolysis, typically at site 2 (primarily composed of sequences from NBD2), promotes channel closure by clearing ligand from this site (4, 5). The coupling between ATP binding and pore opening is presumably mediated by the cytosolic loops that physically link the NBDs to the transmembrane domains (6, 7).Because CFTR is an enzyme that normally hydrolyzes its ligand as part of the channel gating cycle, the extent to which its properties are similar to those of more conventional ligand-gated channels is an interesting issue. Ligand-gated channels such as acetylcholine receptors obey the principles of protein alloster...

Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO2/HCO3 − solution displayed all the hallmarks of an electrogenic Na+/HCO3 − cotransporter: (a) a DIDS-sensitive pHi recovery following the initial CO2-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na+-dependent outward current under voltage-clamp conditions (−60 mV). All three variants had similar external HCO3 − dependencies (apparent KM of 4–6 mM) and external Na+ dependencies (apparent KM of 21–36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (−60 mV) expressing NBCe1-A exhibited peak HCO3 −-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current–voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (Vm = −60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO3 −-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.

We studied the regulation of intracellular pH (pHi) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pHi. Intrinsic buffering power (βI) was 10.5 mM (pH unit)−1 at pHi 7.0, and decreased linearly with pHi; the best-fit line to the data had a slope of −10.0 mM (pH unit)−2. In the absence of HCO3 −, pHi recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pHi 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pHi. Acid extrusion was inhibited 68% (pHi 6.23) by substituting Li+ for Na+ in the bath solution. Switching from a CO2/HCO3 −-free to a CO2/HCO3 −-containing bath solution caused mean steady state pHi to increase from 6.82 to 6.90, due to a Na+-driven HCO3 − transporter. The HCO3 −-induced pHi increase was unaffected by amiloride, but was inhibited 75% (pHi 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pHi 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO2/HCO3 −-induced pHi increase was blocked when external Na+ was replaced with N-methyl-d-glucammonium (NMDG+). In the presence of HCO3 −, the Na+-driven HCO3 − transporter contributed to the pHi recovery from an acid load. For example, HCO3 − shifted the plot of acid-extrusion rate vs. pHi by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO3 − increased acid extrusion 3.8-fold (pHi 6.20). When astrocytes were acid loaded in amiloride, with Li+ as the major cation, HCO3 − failed to elicit a substantial increase in pHi. Thus, Li+ does not appear to substitute well for Na+ on the HCO3 − transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na+-driven HCO3 − transporter are the predominant acid extruders in astrocytes.

We screened rat brain cDNA libraries and used 5' rapid amplification of cDNA ends to clone two electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The other clone (rb2NBC) is identical to rb1NBC except for 61 unique COOH-terminal amino acids, the result of a 97-bp deletion near the 3' end of the open-reading frame. Using RT-PCR, we confirmed that mRNA from rat brain contains this 97-bp deletion. Furthermore, we generated rabbit polyclonal antibodies that distinguish between the unique COOH-termini of rb1NBC (alpharb1NBC) and rb2NBC (alpharb2NBC). alpharb1NBC labels an approximately 130-kDa protein predominantly from kidney, and alpharb2NBC labels an approximately 130-kDa protein predominantly from brain. alpharb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; alpharb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO(2)/10 mM HCO(-)(3) to Xenopus oocytes injected with rb2NBC cRNA causes 1) pH(i) to recover from the initial CO(2)-induced acidification and 2) the cell to hyperpolarize. Subsequently, removing external Na(+) reverses the pH(i) increase and elicits a rapid depolarization. In the presence of 450 microM DIDS, removing external Na(+) has no effect on pH(i) and elicits a small hyperpolarization. The rate of the pH(i) decrease elicited by removing Na(+) is insensitive to removing external Cl(-). Thus rb2NBC is a DIDS-sensitive, electrogenic NBC that is predominantly expressed in brain of at least rat.

1. We used the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to study the regulation of intracellular pH (pH1) in single pyramidal neurons freshly isolated from the hippocampal CAI region of immature (2-to 10-day-old) and more mature (21-to 30-day-old) rats.2. Whether isolated from immature or mature rats, neurons had a broad range of initial pHi values (6 3-7 7) when the cells were examined in solutions buffered with Hepes and no CO2/HCO3-. The initial pH, distribution for neurons isolated from immature rats was best fitted with a Gaussian distribution with a mean of 6-95. In contrast, the initial pHi distribution for neurons isolated from mature rats was best fitted with the sum of two Gaussian distributions with means of 6-68 and 7-32.3. When neurons with a relatively low initial pHi in Hepes-buffered solutions were acid loaded, pH, recovered very slowly. Neurons with a relatively high initial pHi recovered rapidly. The rate constant for the exponential pHi recovery increased with initial pHi. All pHi recoveries required Nae. 4. Both for neurons with a relatively high (>7 705) and a relatively low (<7 05) initial pHi, net acid extrusion rates (Jttal = dpHi/dt x buffering power) decreased linearly with increasing pH,. Compared with the line for neurons with a relatively low initial pHi, that for neurons with a relatively high pH1 had a significantly greater slope and was alkaline shifted by 0-6-0-7 pH units.5. Removing external Nae in the absence of C02/HC03-caused pH1 to decrease by -0 3 in neurons with a relatively low initial pH,, and by -0 5 in neurons with a relatively high initial pHi. This initial acidification was followed by a slower, partial pH, recovery in -32% of neurons with a relatively low initial pH,, but only -14 % of neurons with a relatively high pHi.6. When exposed to C02/HC03-, all neurons initially acidified. Neurons with a relatively low initial pHi recovered to a pHi -0-2 pH units greater than the initial value. Among neurons with higher initial pHi values, some did not recover at all, whereas others recovered to a value similar to or above the initial pHi. On average, the final C02/HC03-pH1 for neurons with a relatively high initial pH1 was similar to the pHi in Hepes buffer. Neurons with a relatively high pHi in Hepes buffer continued to be more alkaline (by -0f2 pH units) in C02/HC03.