Boris Martinac

TRPC1 forms the stretch-activated cation channel in vertebrate cells

The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na+, K+, Ca2+ and Mg2+) flux across the cell membrane, and is implicated in cell-volume regulation, cell locomotion, muscle dystrophy and cardiac arrhythmias. However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp. The oocyte was chosen because it expresses the prototypical MscCa (≥107MscCa/oocyte) that is preserved in cytoskeleton-deficient membrane vesicles. We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (Mr 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1). Heterologous expression of the human TRPC1 resulted in a >1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels.

Open channel structure of MscL and the gating mechanism of mechanosensitive channels

Mechanosensitive channels act as membrane-embedded mechano-electrical switches, opening a large water-filled pore in response to lipid bilayer deformations. This process is critical to the response of living organisms to direct physical stimulation, such as in touch, hearing and osmoregulation. Here, we have determined the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using electron paramagnetic resonance spectroscopy and site-directed spin labelling. MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphology. Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1). Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin–spin interactions. The open state is highly dynamic, supporting a water-filled pore of at least 25 Å, lined mostly by TM1. These structures suggest a plausible molecular mechanism of gating in mechanosensitive channels.

Mechanosensitive ion channels: molecules of mechanotransduction

Cells respond to a wide variety of mechanical stimuli, ranging from thermal molecular agitation to potentially destructive cell swelling caused by osmotic pressure gradients. The cell membrane presents a major target of the external mechanical forces that act upon a cell, and mechanosensitive (MS) ion channels play a crucial role in the physiology of mechanotransduction. These detect and transduce external mechanical forces into electrical and/or chemical intracellular signals. Recent work has increased our understanding of their gating mechanism, physiological functions and evolutionary origins. In particular, there has been major progress in research on microbial MS channels. Moreover, cloning and sequencing of MS channels from several species has provided insights into their evolution, their physiological functions in prokaryotes and eukaryotes, and their potential roles in the pathology of disease.

Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution.

Mechanosensitive ion channels (MSCs) which could provide for fast osmoregulatory responses in bacteria, remain unidentified as molecular entities. MSCs from Escherichia coli (strain AW740) were examined using the patch-clamp technique, either (a) in giant spheroplasts, (b) after reconstitution by fusing native membrane vesicles with asolectin liposomes, or (c) by reassembly of octylglucoside-solubilized membrane extract into asolectin liposomes. MSC activities were similar in all three preparations, consisting of a large nonselective MSC of 3-nS conductance (in 200 mM KCl) that was activated by high negative pressures, and a small weakly anion-selective MSC of 1 nS activated by lower negative pressures. Both channels appeared more sensitive to suction in liposomes than in spheroplasts. After gel filtration of the solubilized membrane extract and reconstituting the fractions, both large MSC and small MSC activities were retrieved in liposomes. The positions of the peaks of channel activity in the column eluate, assayed by patch sampling of individual fractions reconstituted in liposomes, showed an apparent molecular mass under nondenaturing conditions of about 60-80 kDa for the large and 200-400 kDa for the small MSC. We conclude that (a) the large MSC and the small MSC are distinct molecular entities, (b) the fact that both MSCs were functional in liposomes following chromatography strongly suggests that these channels are gated by tension transduced via lipid bilayer, and (c) chromatographic fractionation of detergent-solubilized membrane proteins with subsequent patch sampling of reconstituted fractions can be used to identify and isolate these MS channel proteins.

Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels

We have modified the procedure of Criado and Keller (1987) to study ion channels of Escherichia coli reconstituted in liposomes. The modifications include (a) excluding the use of any detergent and (b) inducing blisters from liposomes with Mg2+. These blisters, which appear to be unilamellar, are stable for hours. They could be repeatedly sampled with different patch-clamp pipettes each achieving seal resistance greater than 10 GOhms. Activities of three types of ion channels are often observed by use of this method, including two voltage-sensitive cation channels of different conductances. Even the mechanosensitive channel, previously recorded from live E. coli cells (Martinac et al., 1987), was also detected in these blisters. Apparently the channel protein and any accessory structures, postulated to be needed for mechanotransduction, can be reconstituted together by this method.

Revisiting TRPC1 and TRPC6 mechanosensitivity

This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-activated mechano-sensitive Ca2+ permeable cation channel (MscCa). We have transiently expressed TRPC1 and TRPC6 in African green monkey kidney (COS) or Chinese hamster ovary (CHO) cells and monitored the activity of the stretch-activated channels using a fast pressure clamp system. Although both TRPC1 and TRPC6 are highly expressed at the protein level, the amplitude of the mechano-sensitive current is not significantly altered by overexpression of these subunits. In conclusion, although several TRPC channel members, including TRPC1 and TRPC6, have been recently proposed to form MscCa in vertebrate cells, the functional expression of these TRPC subunits in heterologous systems remains problematic.

Mechanosensitive Channels in Microbes

All cells, including microbes, detect and respond to mechanical forces, of which osmotic pressure is most ancient and universal. Channel proteins have evolved such that they can be directly stretched open when the membrane is under turgor pressure. Osmotic downshock, as in rain, opens bacterial mechanosensitive (MS) channels to jettison osmolytes, relieving pressure and preventing cell lysis. The ion flux through individual channel proteins can be observed directly with a patch clamp. MS channels of large and small conductance (MscL and MscS, respectively) have been cloned, crystallized, and subjected to biophysical and genetic analyses in depth. They are now models to scrutinize how membrane forces direct protein conformational changes. Eukaryotic microbes have homologs from animal sensory channels of the TRP superfamily. The MS channel in yeast is also directly sensitive to membrane stretch. This review examines the key concept that proteins embedded in the lipid bilayer can respond to the changes in the mechanical environment the lipid bilayer provides.

Ion channels in microbes

Studies of ion channels have for long been dominated by the animalcentric, if not anthropocentric, view of physiology. The structures and activities of ion channels had, however, evolved long before the appearance of complex multicellular organisms on earth. The diversity of ion channels existing in cellular membranes of prokaryotes is a good example. Although at first it may appear as a paradox that most of what we know about the structure of eukaryotic ion channels is based on the structure of bacterial channels, this should not be surprising given the evolutionary relatedness of all living organisms and suitability of microbial cells for structural studies of biological macromolecules in a laboratory environment. Genome sequences of the human as well as various microbial, plant, and animal organisms unambiguously established the evolutionary links, whereas crystallographic studies of the structures of major types of ion channels published over the last decade clearly demonstrated the advantage of using microbes as experimental organisms. The purpose of this review is not only to provide an account of acquired knowledge on microbial ion channels but also to show that the study of microbes and their ion channels may also hold a key to solving unresolved molecular mysteries in the future.

The Biological Activity of the Prototypic Cyclotide Kalata B1 Is Modulated by the Formation of Multimeric Pores

The cyclotides are a large family of circular mini-proteins containing a cystine knot motif. They are expressed in plants as defense-related proteins, with insecticidal activity. Here we investigate their role in membrane interaction and disruption. Kalata B1, a prototypic cyclotide, was found to induce leakage of the self-quenching fluorophore, carboxyfluorescein, from phospholipid vesicles. Alanine-scanning mutagenesis of kalata B1 showed that residues essential for lytic activity are clustered, forming a bioactive face. Kalata B1 was sequestered at the membrane surface and showed slow dissociation from vesicles. Electrophysiological experiments showed that conductive pores were induced in liposome patches on incubation with kalata B1. The conductance calculated from the current-voltage relationship indicated that the diameter of the pores formed in the bilayer patches is 41–47 Å. Collectively, the findings provide a mechanistic explanation for the diversity of biological functions ascribed to this fascinating family of ultrastable macrocyclic peptides.

Mechanosensitive channels of bacteria.

Publisher Summary Mechanosensitive (MS) channel activities have been documented in animal, plant, and bacterial cells by patch-clamp techniques. Mechanosensitive channels are thought to be one of the principal molecular devices by which a cell detects and responds to mechanical stimuli, playing a role in the senses of touch, hearing, and balance, as well as in cardiovascular regulation. Despite their importance for much of biological life, the molecular entities and mechanisms responsible for MS channel activities are only now being elucidated. Perhaps the most successful means of identifying molecular players involved in mechanosensation has been the use of genetic techniques. However, the ability to interpret the role of proteins in mechanosensation from a strictly genetic approach is limited, and electrophysiologic evidence definitively demonstrating that these candidate genes do indeed encode MS channel subunits has yet to be reported. The study of bacterial MS channels has involved several diverse approaches. MscL can now be studied using electrophysiologic, biochemical, genetic, and whole-cell physiologic techniques. This chapter describes some of the diverse methodologies that have been and can be used to study the structural and functional properties of bacterial MS channels.