Mauricio Montal

Piezo proteins are pore-forming subunits of mechanically activated channels

Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

A missense mutation of the Na+ channel αII subunit gene Nav1.2 in a patient with febrile and afebrile seizures causes channel dysfunction

Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in β1 and αI-subunit genes of voltage-gated Na+ channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R187W) in the gene encoding the α-subunit of neuronal voltage-gated Na+ channel type II (Nav1.2) in a patient with FS associated with afebrile seizures. The mutation R187W occurring on Arg187, a highly conserved residue among voltage-gated Na+ channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNav1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na+ channel conductance was not affected. Prolonged residence in the open state of the R187W mutant channel may augment Na+ influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Nav1.2 in a human disease and propose the R187W mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.

Bcl-2 family proteins as ion-channels.

The Bcl-2 protein family function(s) as important regulators of cellular decisions to heed or ignore death signals. The three-dimensional structure of the Bcl-2 homolog, Bcl-XL, bears a strong resemblance to some pore-forming bacterial toxins. This similarity suggested that the Bcl-2 family proteins may also possess channel-forming capability. This review summarizes the recent initial studies on the in vitro channel activity of Bcl-2, Bcl-XL and Bax and offers some speculation as to the physiological role that these channels may play in the cell death pathway.

Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy

The structures of functional peptides corresponding to the predicted channel-lining M2 segments of the nicotinic acetylcholine receptor (AChR) and of a glutamate receptor of the NMDA subtype (NMDAR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. Both M2 segments form straight transmembrane α-helices with no kinks. The AChR M2 peptide inserts in the lipid bilayer at an angle of 12° relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminal side of the membrane, which is assigned to be intracellular. A model built from these solid-state NMR data, and assuming a symmetric pentameric arrangement of M2 helices, results in a funnel-like architecture for the channel, with the wide opening on the N-terminal intracellular side.

Translocation of botulinum neurotoxin light chain protease through the heavy chain channel

Clostridial botulinum neurotoxins (BoNTs) abort the process of neurotransmitter release at presynaptic motor nerve terminals, causing muscle paralysis. An enigmatic step in the intoxication process is the mechanism by which the neurotoxin heavy chain (HC) forms the conduit for the translocation of the light chain (LC) protease across the endosomal membrane into the cytosol, its site of action. Here we investigate the mechanism of LC translocation by using the combined detection of channel currents and substrate proteolysis, the two hallmark activities of BoNT. Our data are consistent with the translocation of the LC through the HC channel and show that the LC protease activity is retrieved in the trans compartment after translocation. We propose that the BoNT HC–LC complex embedded in the membrane is a transmembrane chaperone, a dynamic structural device that prevents aggregation and achieves translocation of the LC. In this regard, the complex is similar to the protein conducting/translocating channels of the endoplasmic reticulum, mitochondria and chloroplasts.

Botulinum Neurotoxin: A Marvel of Protein Design

Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be the most poisonous protein known. BoNT proteases disable synaptic vesicle exocytosis by cleaving their cytosolic SNARE (soluble NSF attachment protein receptor) substrates. BoNT is a modular nanomachine: an N-terminal Zn(2+)-metalloprotease, which cleaves the SNAREs; a central helical protein-conducting channel, which chaperones the protease across endosomes; and a C-terminal receptor-binding module, consisting of two subdomains that determine target specificity by binding to a ganglioside and a protein receptor on the cell surface and triggering endocytosis. For BoNT, functional complexity emerges from its modular design and the tight interplay between its component modules--a partnership with consequences that surpass the simple sum of the individual components action. BoNTs exploit this design at each step of the intoxication process, thereby achieving an exquisite toxicity. This review summarizes current knowledge on the structure of individual modules and presents mechanistic insights into how this protein machine evolved to this level of sophistication. Understanding the design principles underpinning the function of such a dynamic modular protein remains a challenging task.

Activation of Store-Operated Ca2+ Current in Xenopus Oocytes Requires SNAP-25 but Not a Diffusible Messenger

Depletion of Ca2+ stores in Xenopus oocytes activated entry of Ca2+ across the plasma membrane, which was measured as a current I(soc) in subsequently formed cell-attached patches. I(soc) survived excision into inside-out configuration. If cell-attached patches were formed before store depletion, I(soc) was activated outside but not inside the patches. I(soc) was potentiated by microinjection of Clostridium C3 transferase, which inhibits Rho GTPase, whereas I(soc) was inhibited by expression of wild-type or constitutively active Rho. Activation of I(soc) was also inhibited by botulinum neurotoxin A and dominant-negative mutants of SNAP-25 but was unaffected by brefeldin A. These results suggest that oocyte I(soc) is dependent not on aqueous diffusible messengers but on SNAP-25, probably via exocytosis of membrane channels or regulatory molecules.

Identification of an ion channel activity of the Vpu transmembrane domain and its involvement in the regulation of virus release from HIV‐1‐infected cells

HIV‐1 Vpu catalyzes two independent functions, degradation of the virus receptor CD4 in the endoplasmic reticulum and enhancement of virus release from the cell surface. These activities are confined to distinct structural domains of Vpu, the cytoplasmic tail and the transmembrane (TM) anchor, respectively. It was recently reported that Vpu forms cationselective ion channels in lipid bilayers. Here we report that this property of Vpu is a characteristic of its TM anchor. Expression of full‐length Vpu in Xenopus oocytes increases membrane conductance. The Vpu‐induced conductance is selective to monovalent cations over anions, does not discriminate Na+ over K+ and shows marginal permeability to divalent cations. Notably, introduction of the scrambled TM sequence into full‐length Vpu abrogates its capacity to increase membrane conductance in oocytes and to promote virus release from infected cells. Reconstitution of synthetic Vpu fragments in lipid bilayers identified an ion channel activity for a sequence corresponding to the TM domain of Vpu. In contrast, a peptide with the same amino acid composition but with a scrambled sequence does not form ion channels. Our findings therefore suggest that the ability of Vpu to increase virus release from infected cells may be correlated with an ion channel activity of the TM domain, thereby providing a potential target for drug intervention based on the development of Vpu‐specific channel blockers.

A nonsense mutation of the sodium channel gene SCN2A in a patient with intractable epilepsy and mental decline

Mutations, exclusively missense, of voltage-gated sodium channel α subunit type 1 (SCN1A) and type 2 (SCN2A) genes were reported in patients with idiopathic epilepsy: generalized epilepsy with febrile seizures plus. Nonsense and frameshift mutations of SCN1A, by contrast, were identified in intractable epilepsy: severe myoclonic epilepsy in infancy (SMEI). Here we describe a first nonsense mutation of SCN2A in a patient with intractable epilepsy and severe mental decline. The phenotype is similar to SMEI but distinct because of partial epilepsy, delayed onset (1 year 7 months), and absence of temperature sensitivity. A mutational analysis revealed that the patient had a heterozygous de novo nonsense mutation R102X of SCN2A. Patch-clamp analysis of Nav1.2 wild-type channels and the R102X mutant protein coexpressed in human embryonic kidney 293 cells showed that the truncated mutant protein shifted the voltage dependence of inactivation of wild-type channels in the hyperpolarizing direction. Analysis of the subcellular localization of R102X truncated protein suggested that its dominant negative effect could arise from direct or indirect cytoskeletal interactions of the mutant protein. Haploinsufficiency of Nav1.2 protein is one plausible explanation for the pathology of this patient; however, our biophysical findings suggest that the R102X truncated protein exerts a dominant negative effect leading to the patients intractable epilepsy.

Orientations of amphipathic helical peptides in membrane bilayers determined by solid-state NMR spectroscopy

SummarySolid-state NMR spectroscopy was used to determine the orientations of two amphipathic helical peptides associated with lipid bilayers. A single spectral parameter provides sufficient orientational information for these peptides, which are known, from other methods, to be helical. The orientations of the peptides were determined using the15N chemical shift observed for specifically labeled peptide sites. Magainin, an antibiotic peptide from frog skin, was found to lie in the plane of the bilayer. M2δ, a helical segment of the nicotinic acetylcholine receptor, was found to span the membrane, perpendicular to the plane of the bilayer. These findings have important implications for the mechanisms of biological functions of these peptides.