Yoshiro Sohma

G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects.

Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). CFTR is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis at its nucleotide binding domains (NBDs). G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Patients carrying the G551D mutation present a severe clinical phenotype. On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Residues G551 and G1349 are located at equivalent positions in the highly conserved signature sequence of each NBD. The physiological importance of these residues lies in the fact that the signature sequence of one NBD and the Walker A and B motifs from the other NBD form the ATP-binding pocket (ABP1 and ABP2, named after the location of the Walker A motif) once the two NBDs dimerize. Our studies show distinct gating characteristics for these mutants. The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is ∼100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg2+]. The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. G1349D-CFTR maintains ATP dependence, albeit with a Po ∼10-fold lower than WT. Interestingly, compared to WT results, the ATP dose–response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Thus, our data provide a quantitative explanation at the single-channel level for different phenotypes presented by patients carrying these two mutations. In addition, these results support the idea that CFTRs two ABPs play distinct functional roles in gating.

Glucose-induced electrical activities and insulin secretion in pancreatic islet β-cells are modulated by CFTR

The cause of insulin insufficiency remains unknown in many diabetic cases. Up to 50% adult patients with cystic fibrosis (CF), a disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), develop CF-related diabetes (CFRD) with most patients exhibiting insulin insufficiency. Here we show that CFTR is a regulator of glucose-dependent electrical acitivities and insulin secretion in β-cells. We demonstrate that glucose elicited whole-cell currents, membrane depolarization, electrical bursts or action potentials, Ca2+ oscillations and insulin secretion are abolished or reduced by inhibitors or knockdown of CFTR in primary mouse β-cells or RINm5F β-cell line, or significantly attenuated in CFTR mutant (DF508) mice compared with wild-type mice. VX-809, a newly discovered corrector of DF508 mutation, successfully rescues the defects in DF508 β-cells. Our results reveal a role of CFTR in glucose-induced electrical activities and insulin secretion in β-cells, shed light on the pathogenesis of CFRD and possibly other idiopathic diabetes, and present a potential treatment strategy.

Membrane potential and bicarbonate secretion in isolated interlobular ducts from guinea-pig pancreas

The interlobular duct cells of the guinea-pig pancreas secrete HCO3 − across their luminal membrane into a HCO3 −-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO3 − transport cannot be achieved by luminal Cl−/HCO3 − exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO3 − efflux, we have measured the intracellular potential (Vm) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl−-25 mM HCO3 − solution, a condition similar to the basal state, the resting potential was approximately −60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (∼5 mV) due to activation of electrogenic Na+-HCO3 − cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately −50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO3 − concentration to 125 mM caused a hyperpolarization (∼10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl− efflux across the luminal membrane is minimized by the depletion of intracellular Cl− and offset by the hyperpolarizing effects of Na+-HCO3 − cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO3 − that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO3 − to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO3 −.

A local GABAergic system within rat trigeminal ganglion cells

We investigated the GABAergic system within the Sprague–Dawley rat (2–3‐weeks old) trigeminal ganglion (TG). Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis revealed expression of glutamate decarboxylase (GAD) 65 and GAD67 mRNAs and mRNAs encoding GABAA receptor subunits α1–6, β1–3, γ1–3, and δ. In situ hybridization revealed that GAD65 and GAD67 mRNAs were expressed in neuronal cell bodies but not satellite cells. Immunohistochemical analysis showed that only GAD65 was expressed in all neuronal cell bodies, and approximately 70% of all neurons exhibited GABA immunoreactivity. Satellite cells were strongly immunopositive for GABA. GABAA receptor α1, α5, β2/3 and γ1/2/3 subunit immunoreactivities were observed in the majority of neurons, but no immunoreactivity for α2 was observed. Two types of cells were identified in TG based on cell size and morphology, type A and B. The percentage of cells expressing α3, α4, α6, and δ subunits appeared to be dependent on cell size, as δ and α6 expression were only observed in small (B‐type) neurons. In whole‐cell patch clamp experiments, GABA application induced inward Cl– currents in all neurons examined. The EC50 for GABA varied from 5.3 to 240 µm, and the Hill Coefficient (nH) varied between 0.98 and 2.6 at −60 mV. We found that GABA was released from TG cells by increasing extracellular K+ concentration to 100 mm. We speculate that GABA acts as a nonsynaptically released diffusible neurotransmitter, which may modulate somatic inhibition of neurons within the TG.

CFTR Gating I: Characterization of the ATP-dependent Gating of a Phosphorylation-independent CFTR Channel (ΔR-CFTR)

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed ΔR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the ΔR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal ΔR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of ΔR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and ΔR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the ΔR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.

A Mathematical Model of the Pancreatic Ductal Epithelium

Abstract. A mathematical model of the HCO−3-secreting pancreatic ductal epithelium was developed using network thermodynamics. With a minimal set of assumptions, the model accurately reproduced the experimentally measured membrane potentials, voltage divider ratio, transepithelial resistance and short-circuit current of nonstimulated ducts that were microperfused and bathed with a CO2/HCO−3-free, HEPES-buffered solution, and also the intracellular pH of duct cells bathed in a CO2/HCO−3-buffered solution. The model also accurately simulated: (i) the effect of step changes in basolateral K+ concentration, and the effect of K+ channel blockers on basolateral membrane potential; (ii) the intracellular acidification caused by a Na+-free extracellular solution and the effect of amiloride on this acidification; and (iii) the intracellular alkalinization caused by a Cl−-free extracellular solution and the effect of DIDS on this alkalinization. In addition, the model predicted that the luminal Cl− conductance plays a key role in controlling both the HCO−3 secretory rate and intracellular pH during HCO−3 secretion. We believe that the model will be helpful in the analysis of experimental data and improve our understanding of HCO−3-transporting mechanisms in pancreatic duct cells.

Potentiation of large conductance, Ca2+-activated K+ (BK) channels by α5β1 integrin activation in arteriolar smooth muscle

Injury/degradation of the extracellular matrix (ECM) is associated with vascular wall remodelling and impaired reactivity, a process in which altered ECM–integrin interactions play key roles. Previously, we found that peptides containing the RGD integrin‐binding sequence produce sustained vasodilatation of rat skeletal muscle arterioles. Here, we tested the hypothesis that RGD ligands work through α5β1 integrin to modulate the activity of large conductance, Ca2+‐activated K+ (BK) channels in arteriolar smooth muscle. K+ currents were recorded in single arteriolar myocytes using whole‐cell and single‐channel patch clamp methods. Activation of α5β1 integrin by an appropriate, insoluble α5β1 antibody resulted in a 30–50% increase in the amplitude of iberiotoxin (IBTX)‐sensitive, whole‐cell K+ current. Current potentiation occurred 1–8 min after bead–antibody application to the cell surface. Similarly, the endogenous α5β1 integrin ligand fibronectin (FN) potentiated IBTX‐sensitive K+ current by 26%. Current potentiation was blocked by the c‐Src inhibitor PP2 but not by PP3 (0.1–1 μm). In cell‐attached patches, number of open channels × open probability (NPo) of a 230–250 pS K+ channel was significantly increased after FN application locally to the external surface of cell‐attached patches through the recording pipette. In excised, inside‐out patches, the same method of FN application led to large, significant increases in NPo and caused a leftward shift in the NPo–voltage relationship at constant [Ca2+]. PP2 (but not PP3) nearly abolished the effect of FN on channel activity, suggesting that signalling between the integrin and channel involved an increase in Ca2+sensitivity of the channel via a membrane‐delimited pathway. The effects of α5β1 integrin activation on both whole‐cell and single‐channel BK currents could be reproduced in HEK 293 cells expressing the BK channel α‐subunit. This is the first demonstration at the single‐channel level that integrin signalling can regulate an ion channel. Our results show that α5β1 integrin activation potentiates BK channel activity in vascular smooth muscle through both Ca2+‐ and c‐Src‐dependent mechanisms. This mechanism is likely to play a role in the arteriolar dilatation and impaired vascular reactivity associated with ECM degradation.

CFTR gating II: Effects of nucleotide binding on the stability of open states.

Previously, we demonstrated that ADP inhibits cystic fibrosis transmembrane conductance regulator (CFTR) opening by competing with ATP for a binding site presumably in the COOH-terminal nucleotide binding domain (NBD2). We also found that the open time of the channel is shortened in the presence of ADP. To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Single-channel kinetic analysis of ΔR/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. ΔR/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of ∼100 s, estimated from current relaxation upon nucleotide removal. ADP induces a shorter locked-open state, suggesting that binding of ADP at a second site decreases the locked-open time. To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Two locked-open time constants can be discerned and the relative distribution of each component is altered by changing [ATP] so that increasing [ATP] shifts the relative distribution to the longer locked-open state. Single-channel kinetic analysis for ΔR/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. These results support the idea that occupancy of a second ATP binding site stabilizes the locked-open state. This binding site likely resides in the NH2-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. These results suggest that the binding energy of nucleotide at NBD1 contributes to the overall energetics of the open channel conformation.

On the mechanism of CFTR inhibition by a thiazolidinone derivative

The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172–dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels “inactivate” despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.

State-dependent modulation of CFTR gating by pyrophosphate.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an adenosine triphosphate (ATP)-gated chloride channel. ATP-induced dimerization of CFTRs two nucleotide-binding domains (NBDs) has been shown to reflect the channel open state, whereas hydrolysis of ATP is associated with channel closure. Pyrophosphate (PPi), like nonhydrolytic ATP analogues, is known to lock open the CFTR channel for tens of seconds when applied with ATP. Here, we demonstrate that PPi by itself opens the CFTR channel in a Mg(2+)-dependent manner long after ATP is removed from the cytoplasmic side of excised membrane patches. However, the short-lived open state (tau approximately 1.5 s) induced by MgPPi suggests that MgPPi alone does not support a stable NBD dimer configuration. Surprisingly, MgPPi elicits long-lasting opening events (tau approximately 30 s) when administrated shortly after the closure of ATP-opened channels. These results indicate the presence of two different closed states (C(1) and C(2)) upon channel closure and a state-dependent effect of MgPPi on CFTR gating. The relative amount of channels entering MgPPi-induced long-open bursts during the ATP washout phase decreases over time, indicating a time-dependent dissipation of the closed state (C(2)) that can be locked open by MgPPi. The stability of the C(2) state is enhanced when the channel is initially opened by N(6)-phenylethyl-ATP, a high affinity ATP analogue, but attenuated by W401G mutation, which likely weakens ATP binding to NBD1, suggesting that an ATP molecule remains bound to the NBD1 site in the C(2) state. Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C(2)-equivalent state (C(2)*), which exists before the channel in the C(1) state is opened by ATP. This closed state responds to MgPPi much more inefficiently than the C(2) state. Finally, we show that MgAMP-PNP exerts its effects on CFTR gating via a similar mechanism as MgPPi. The structural and functional significance of our findings is discussed.