Liposome reconstitution and modulation of recombinant N -methyl- d -aspartate receptor channels by membrane stretch

Abstract

In this study, the heteromeric N-methyl-D-aspartate (NMDA) receptor channels composed of NR1a and NR2A subunits were expressed, purified, reconstituted into liposomes, and characterized by using the patch clamp technique. The protein exhibited the expected electrophysiological profile of activation by glutamate and glycine and internal Mg 2؉ blockade. We demonstrated that the mechanical energy transmitted to membrane-bound NMDA receptor channels can be exerted directly by tension developed in the lipid bilayer. Membrane stretch and application of arachidonic acid potentiated currents through NMDA receptor channels in the presence of intracellular Mg 2؉ . The correlation of membrane tension induced by either mechanical or chemical stimuli with the physiological Mg 2؉ block of the channel suggests that the synaptic transmission can be altered if NMDA receptor complexes experience local changes in bilayer thickness caused by dynamic targeting to lipid microdomains, electrocompression, or chemical modification of the cell membranes. The ability to study gating properties of NMDA receptor channels in artificial bilayers should prove useful in further study of structure-function relationships and facilitate discoveries of new therapeutic agents for treatment of glutamate-mediated excitotoxicity or analgesic therapies.arachidonic acid ͉ mechanosensation ͉ NMDA receptor ͉ patch clamp ͉ bilayer model T he ionotropic, glutamate-activated N-methyl-D-aspartate (NMDA) receptors are ligand-gated cation channels that play an important role in both physiological and pathological processes, including long-term potentiation and synaptic plasticity (1, 2), neuronal excitotoxicity, and cognitive deficits attributable to aging and pain (3-8). NMDA receptors are formed by heterooligomers of various NR1 and NR2 subunits (9). The secondary structure of the NMDA receptor monomer predicts four transmembrane segments (M1 to M4) with an extracellular N terminus and the C terminus located intracellularly. The M2 domain forms a cytoplasmic reentrant loop that lines the channel pore (9, 10). This region harbors a narrow constriction, forming the selectivity filter that controls voltage-dependent Mg 2ϩ block (10-12). The C-terminal tail binds to cytoskeletal complexes, including kinases and structural proteins, which further modulate the function of the NMDA receptor channel (3, 13).Mechanosensitivity is an important signal transduction mechanism that underlies a number of key biological processes ranging from cellular growth, cell volume and blood pressure regulation, touch, and pain sensation to cardiac arrhythmia, muscular dystrophy, and neuronal degeneration (14,15). In prokaryotes, the mechanosensory transduction is carried out by mechanosensitive (MS) channels of small (MscS-like) and large (MscL-like) conductance (14,16,17). Many eukaryotic ion channels can be gated by mechanical forces. For example, the signaling properties of some membrane proteins, including ion channels, can change dramatically after undergoing a dynamic targeting t...

A neuroscientist's guide to lipidomics

“…As these results were obtained in a minimal system that lacks cellular proteins, they unambiguously demonstrate that mechanical deformation of the lipid bilayer is sufficient to modulate the gating properties of NMDA channels. The data further suggest that administration of exogenous arachidonic acid closely mimics the effects of membrane stretch, probably by bending the liposomal membrane 14 . In fact, a general role for lipid-mediated membrane flexing in mechanosensation has been proposed 15 .…”

“…A similar mechanism probably operates in other instances of lipid regulation of mechanosensitive channels -including transient receptor potential vanilloid-1 (TrPv1) channels 10 , Twik-related K + 1 (TreK1) channels 11 and glutamate N-methyl-d-aspartate (NMDA) receptor channels 12,13 . A recent series of experiments with purified NMDA receptors reconstituted in liposomes has shown that membrane stretch and arachidonic acid application reduce Mg 2+ blockade of NMDA channel activity and concomitantly enhance ion currents through the channels 14 . As these results were obtained in a minimal system that lacks cellular proteins, they unambiguously demonstrate that mechanical deformation of the lipid bilayer is sufficient to modulate the gating properties of NMDA channels.…”

Feeling the hidden mechanical forces in lipid bilayer is an original sense

“…Although there are antecedents (39,40), recent works generate new excitement because of the deep knowledge on the structure and function of K 2p summarized above. Berrier et al (41) have recently reconstituted an enriched fraction of mouse TREK-1 into liposomes and examined patches excised from them.…”

Tuning the mechanosensitivity of a BK channel by changing the linker length

“…These channels transduce mechanical stimuli into intracellular signals, such as elevation in intracellular Ca 2+ concentration and electrical activities [1][2][3][4][5][6], which are important in many physiological functions and pathological conditions, for example, sound detection [4], cell volume regulation [7], touch sensation [8], hypo-osmotic shock protection [9], cell locomotion [10], muscular dystrophy and cardiac arrhythmias [11]. The BK channel is one type of MS channels which is widely expressed in both excitable and non-excitable cells and plays an important role in, for example, vascular smooth muscle tone regulation, neuronal firing and endocrine cell secretion [12][13][14][15][16].…”