Rattlesnake Phospholipase A2 Increases CFTR-Chloride Channel Current and Corrects ∆F508CFTR Dysfunction: Impact in Cystic Fibrosis.

Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmembrane regulator (CFTR; a cyclic AMP-regulated chloride channel) is the most frequent mutation associated with cystic fibrosis. This mutation affects the maturation and gating of CFTR protein. The search for new high-affinity ligands of CFTR acting as dual modulators (correctors/activators) presents a major challenge in the pharmacology of cystic fibrosis. Snake venoms are a rich source of natural multifunctional proteins, potential binders of ion channels. In this study, we identified the CB subunit of crotoxin from Crotalus durissus terrificus as a new ligand and allosteric modulator of CFTR. We showed that CB interacts with NBD1 of both wild type and ∆F508CFTR and increases their chloride channel currents. The potentiating effect of CB on CFTR activity was demonstrated using electrophysiological techniques in Xenopus laevis oocytes, in CFTR-HeLa cells, and ex vivo in mouse colon tissue. The correcting effect of CB was shown by functional rescue of CFTR activity after 24-h ΔF508CFTR treatments with CB. Moreover, the presence of fully glycosylated CFTR was observed. Molecular docking allowed us to propose a model of the complex involving of the ABCß and F1-like ATP-binding subdomains of ΔF508-NBD1. Hydrogen-deuterium exchange analysis confirmed stabilization in these regions, also showing allosteric stabilization in two other distal regions. Surface plasmon resonance competition studies showed that CB disrupts the ∆F508CFTR-cytokeratin 8 complex, allowing for the escape of ∆F508CFTR from degradation. Therefore CB, as a dual modulator of ΔF508CFTR, constitutes a template for the development of new anti-CF agents.

Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1.

Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.

A calcium-permeable non-selective cation channel in the thick ascending limb apical membrane of the mouse kidney.

Non-selective cation channels have been described in the basolateral membrane of the renal tubule, but little is known about functional channels on the apical side. Apical membranes of microdissected fragments of mouse cortical thick ascending limbs were searched for ion channels using the cell-free configuration of the patch-clamp technique. A cation channel with a linear current-voltage relationship (19pS) that was permeable both to monovalent cations [P(NH4)(1.7)>P(Na) (1.0)=P(K) (1.0)] and to Ca(2+) (P(Ca)/P(Na)≈0.3) was detected. Unlike the basolateral TRPM4 Ca(2+)-impermeable non-selective cation channel, this non-selective cation channel was insensitive to internal Ca(2+), pH and ATP. The channel was already active after patch excision, and its activity increased after reduced pressure was applied via the pipette. External gadolinium (10(-5)M) decreased the channel-open probability by 70% in outside-out patches, whereas external amiloride (10(-4)M) had no effect. Internal flufenamic acid (10(-4)M) inhibited the channel in inside-out patches. Its properties suggest that the current might be supported by the TRPM7 protein that is expressed in the loop of Henle. The conduction properties of the channel suggest that it could be involved in Ca(2+) signaling.

Properties of an inwardly rectifying K(+) channel in the basolateral membrane of mouse TAL.

We investigated the properties of K(+) channels in the basolateral membrane of the cortical thick ascending limb (CTAL) using the patch-clamp technique. Approximately 34% of cell-attached patches contained an inwardly rectifying K(+) channel (K(+)-to-Na(+) permeability ratio approximately 22), having an inward conductance (G(in)) of 44 pS and an outward conductance (G(out)) of approximately 10 pS (G(in)/G(out) approximately 4). Channel activity (NP(o)) increased with depolarization. When the cytosolic sides of inside-out patches were exposed to an Mg(2+)-free medium, the channel had a G(in) of 50 pS and was weakly inwardly rectifying (G(in)/G(out) approximately 1). Cytosolic Mg(2+) reduced G(out), yielding a G(in)/G(out) of 3.8 at 1.3 mM Mg(2+). Internal Na(+) also yielded a G(in)/G(out) of 1.6 at 20 mM Na(+). Spermine reduced NP(o) on inside-out membrane patches. Sensitivity to spermine at depolarizing voltages [half-maximal inhibitory concentration (K(i)) = 0.2 microM] was much greater than at hyperpolarizing voltages (K(i) = 26 microM). Half-inactivation by 0.5 microM spermine occurred at a clamp potential of 43 mV, with an effective valence of 1.25. A sigmoid relationship between bath pH and NP(o) of inside-out membrane patches was observed, with a pK of 7.6 and a Hill coefficient of 1.8. Intracellular acidification also reduced the NP(o) of cell-attached patches. This channel is probably a major component of K(+) conductance in the CTAL basolateral membrane.

An inward rectifier K(+) channel at the basolateral membrane of the mouse distal convoluted tubule: similarities with Kir4-Kir5.1 heteromeric channels.

In this study, K(+) channels present in the basolateral membrane of the distal convoluted tubule (DCT) were investigated using patch-clamp methods. In addition, Kir4.1, Kir4.2 and Kir5.1 inward rectifier channels were investigated using RT-PCR and immunohistochemistry (Kir4.1). DCTs were microdissected from collagenase-treated mouse kidneys. One type of K(+) channel was detected in about 50 % of cell-attached patches from the DCT basolateral membrane; this channel was inwardly rectifying and had an inward conductance (g(in)) of approximately 40 pS at an external [K(+)] of 145 mM. The current-voltage relationship was linear when inside-out patches were exposed to a Mg(2+)-free medium. Mg(2+) at a concentration of 1.2 mM considerably reduced the outward conductance (g(out)), yielding a g(in)/g(out) ratio of approximately 4.7. The polycation spermine (5 x 10(-7) M) reduced the open probability (P(o)) by 50 %. Channel activity was dependent upon the intracellular pH, with acid pH decreasing, and basic pH increasing, P(o). Internal ATP (2 mM) and Ca(2+) (up to 10(-3) M) had no effect. Channel activity declined irreversibly when the inner side of the patch was exposed to Mg(2+). Kir4.1, Kir4.2 and Kir5.1 mRNAs were all detected in the DCT. The Kir4.1 protein co-localised with the Na(+)-Cl(-) cotransporter, which is specific to the DCT, and was located on basolateral membranes. The DCT K(+) channel differs from other functionally identified renal K(+) channels with regard to its inhibition by spermine and insensitivity to internal ATP and Ca(2+). At the current state of knowledge, the channel is similar to Kir4.1-Kir5.1 and Kir4.2-Kir5.1 heteromeric channels, but not to Kir4.1 or Kir4.2 homomeric channels.