ALLIN: A tool for annotation of a protein alignment combined with structural visualization.

The physiological, functional, and structural properties of proteins and their pathogenic variants can be summarized using many tools. The information relating to a single protein is often spread among different sources requiring different programs for access. It is not always easy to select, simultaneously visualize, and compare specific properties of different proteins.

Vesicular CLC chloride/proton exchangers in health and diseases.

Chloride is one of the most abundant anions in the human body; it is implicated in several physiological processes such as the transmission of action potentials, transepithelial salt transport, maintenance of cellular homeostasis, regulation of osmotic pressure and intracellular pH, and synaptic transmission. The balance between the extracellular and intracellular chloride concentrations is controlled by the interplay of ion channels and transporters embedded in the cellular membranes. Vesicular members of the CLC chloride protein family (vCLCs) are chloride/proton exchangers expressed in the membrane of the intracellular organelles, where they control vesicular acidification and luminal chloride concentration.

New Insights into the Mechanism of NO Selectivity in the Human Kidney Chloride Channel ClC-Ka and the CLC Protein Family.

Background: The mechanism of anion selectivity in the human kidney chloride channels ClC-Ka and ClC-Kb is unknown. However, it has been thought to be very similar to that of other channels and antiporters of the CLC protein family, and to rely on anions interacting with a conserved Ser residue (Ser) at the center of three anion binding sites in the permeation pathway S. In both CLC channels and antiporters, mutations of Ser alter the anion selectivity.

TMEM16 proteins: unknown structure and confusing functions.

The TMEM16 family of membrane proteins, also known as anoctamins, plays key roles in a variety of physiological functions that range from ion transport to phospholipid scrambling and to regulating other ion channels. The first two family members to be functionally characterized, TMEM16A (ANO1) and TMEM16B (ANO2), form Ca(2+)-activated Cl(-) channels and are important for transepithelial ion transport, olfaction, phototransduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. The roles of other family members, such as TMEM16C (ANO3), TMEM16D (ANO4), TMEM16F (ANO6), TMEM16G (ANO7) and TMEM16J (ANO9), remain poorly understood and controversial.

Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter.

CLC-type exchangers mediate transmembrane Cl(-) transport. Mutations altering their gating properties cause numerous genetic disorders. However, their transport mechanism remains poorly understood.

Purified TMEM16A is sufficient to form Ca2+-activated Cl- channels.

Ca(2+)-activated Cl(-) channels (CaCCs) are key regulators of numerous physiological functions, ranging from electrolyte secretion in airway epithelia to cellular excitability in sensory neurons and muscle fibers. Recently, TMEM16A (ANO1) and -B were shown to be critical components of CaCCs. It is still unknown whether they are also sufficient to form functional CaCCs, or whether association with other subunits is required.

Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel.

Phospholipid (PL) scramblases disrupt the lipid asymmetry of the plasma membrane, externalizing phosphatidylserine to trigger blood coagulation and mark apoptotic cells. Recently, members of the TMEM16 family of Ca(2+)-gated channels have been shown to be involved in Ca(2+)-dependent scrambling. It is however controversial whether they are scramblases or channels regulating scrambling.

Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H(+)/Cl(-) exchanger.

Active exchangers dissipate the gradient of one substrate to accumulate nutrients, export xenobiotics and maintain cellular homeostasis. Mechanistic studies have suggested that two fundamental properties are shared by all exchangers: substrate binding is antagonistic, and coupling is maintained by preventing shuttling of the empty transporter. The CLC H(+)/Cl(-) exchangers control the homeostasis of cellular compartments in most living organisms, but their transport mechanism remains unclear.

PIP2-mediated HCN3 channel gating is crucial for rhythmic burst firing in thalamic intergeniculate leaflet neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate a pacemaking current, I(h), which regulates neuronal excitability and oscillatory activity in the brain. Although all four HCN isoforms are expressed in the brain, the functional contribution of HCN3 is unknown. Using immunohistochemistry, confocal microscopy, and whole-cell patch-clamp recording techniques, we investigated HCN3 function in thalamic intergeniculate leaflet (IGL) neurons, as HCN3 is reportedly preferentially expressed in these cells.

A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels.

The two human CLC Cl(-) channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel beta subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca(2+) and H(+) on ClC-Ka and ClC-Kb in Xenopus oocytes.

Identification of sites responsible for the potentiating effect of niflumic acid on ClC-Ka kidney chloride channels.

Background And Purpose: ClC-K kidney Cl(-) channels are important for renal and inner ear transepithelial Cl(-) transport, and are potentially interesting pharmacological targets. They are modulated by niflumic acid (NFA), a non-steroidal anti-inflammatory drug, in a biphasic way: NFA activates ClC-Ka at low concentrations, but blocks the channel above approximately 1 mM. We attempted to identify the amino acids involved in the activation of ClC-Ka by NFA.

Proton block of the CLC-5 Cl-/H+ exchanger.

CLC-5 is a H(+)/Cl(-) exchanger that is expressed primarily in endosomes but can traffic to the plasma membrane in overexpression systems. Mutations altering the expression or function of CLC-5 lead to Dent's disease. Currents mediated by this transporter show extreme outward rectification and are inhibited by acidic extracellular pH.

CLC channels and transporters: proteins with borderline personalities.

Controlled chloride movement across membranes is essential for a variety of physiological processes ranging from salt homeostasis in the kidneys to acidification of cellular compartments. The CLC family is formed by two, not so distinct, sub-classes of membrane transport proteins: Cl(-) channels and H(+)/Cl(-) exchangers. All CLC's are homodimers with each monomer forming an individual Cl- permeation pathway which appears to be largely unaltered in the two CLC sub-classes.

Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters.

Ion binding to secondary active transporters triggers a cascade of conformational rearrangements resulting in substrate translocation across cellular membranes. Despite the fundamental role of this step, direct measurements of binding to transporters are rare. We investigated ion binding and selectivity in CLC-ec1, a H(+)-Cl(-) exchanger of the CLC family of channels and transporters.

Molecular switch for CLC-K Cl- channel block/activation: optimal pharmacophoric requirements towards high-affinity ligands.

ClC-Ka and ClC-Kb Cl(-) channels are pivotal for renal salt reabsorption and water balance. There is growing interest in identifying ligands that allow pharmacological interventions aimed to modulate their activity. Starting from available ligands, we followed a rational chemical strategy, accompanied by computational modeling and electrophysiological techniques, to identify the molecular requisites for binding to a blocking or to an activating binding site on ClC-Ka.

Mechanism of interaction of niflumic acid with heterologously expressed kidney CLC-K chloride channels.

CLC-K Cl(-) channels belong to the CLC protein family. In kidney and inner ear, they are involved in transepithelial salt transport. Mutations in ClC-Kb lead to Bartter's syndrome, and mutations in the associated subunit barttin produce Bartter's syndrome and deafness.

Niflumic acid inhibits chloride conductance of rat skeletal muscle by directly inhibiting the CLC-1 channel and by increasing intracellular calcium.

Background And Purpose: Given the crucial role of the skeletal muscle chloride conductance (gCl), supported by the voltage-gated chloride channel CLC-1, in controlling muscle excitability, the availability of ligands modulating CLC-1 are of potential medical as well as toxicological importance. Here, we focused our attention on niflumic acid (NFA), a molecule belonging to the fenamates group of non-steroidal anti-inflammatory drugs (NSAID).

Experimental Approach: Rat muscle Cl(-) conductance (gCl) and heterologously expressed CLC-1 currents were evaluated by means of current-clamp (using two-microelectrodes) and patch-clamp techniques, respectively.

Activation and inhibition of kidney CLC-K chloride channels by fenamates.

CLC-K Cl(-) channels are selectively expressed in kidney and ear, where they are pivotal for salt homeostasis, and loss-of-function mutations of CLC-Kb produce Bartter's syndrome type III. The only ligand known for CLC-K channels is a derivative of the 2-p-chlorophenoxypropionic acid (CPP), 3-phenyl-CPP, which blocks CLC-Ka, but not CLC-Kb. Here we show that in addition to this blocking site, CLC-K channels bear an activating binding site that controls channel opening.

Channel or transporter? The CLC saga continues.

It was recently shown that the putative bacterial Cl- channel, ClC-ec1, is in reality a Cl--H+ antiporter. Our group has now shown that this is also the case for two human CLCs, ClC-4 and ClC-5. We found that the flux of Cl- in one direction is stoichiometrically coupled to the movement of protons in the opposite direction, unveiling a behaviour that is typical of a transporter rather than a channel.

Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5.

ClC-4 and ClC-5 are members of the CLC gene family, with ClC-5 mutated in Dent's disease, a nephropathy associated with low-molecular-mass proteinuria and eventual renal failure. ClC-5 has been proposed to be an electrically shunting Cl- channel in early endosomes, facilitating intraluminal acidification. Motivated by the discovery that certain bacterial CLC proteins are secondary active Cl-/H+ antiporters, we hypothesized that mammalian CLC proteins might not be classical Cl- ion channels but might exhibit Cl(-)-coupled proton transport activity.

Molecular determinants of differential pore blocking of kidney CLC-K chloride channels.

The highly homologous Cl(-) channels CLC-Ka and CLC-Kb are important for water and salt conservation in the kidney and for the production of endolymph in the inner ear. Mutations in CLC-Kb lead to Bartter's syndrome and mutations in the small CLC-K subunit barttin lead to Bartter's syndrome and deafness. Here we show that CLC-Ka is blocked by the recently identified blocker 2-(p-chlorophenoxy)-3-phenylpropionic acid of the rat channel CLC-K1 with an apparent K(D) approximately 80 microM.

Investigations of pharmacologic properties of the renal CLC-K1 chloride channel co-expressed with barttin by the use of 2-(p-Chlorophenoxy)propionic acid derivatives and other structurally unrelated chloride channels blockers.

CLC-K chloride channels are expressed in the kidney, where they play a pivotal role in the mechanisms of urine concentration and Na(+) reabsorption. The identification of barttin as an essential beta-subunit of CLC-K channels allowed performance of a pharmacologic characterization of wild-type CLC-K1 expressed in Xenopus oocytes. To this end, a series of 2-(p-chlorophenoxy)propionic acid (CPP) derivatives were screened using the two-microelectrode voltage-clamp technique.