Patch-clamp technique, mechanism studies

Variations of the patch-clamp technique have been used for studying neurotransmitter transduction mechanisms (Smith 1995). 

Figure 3 Possible mechanisms of MS channel activation by bilayer deformation forces. Hydrophobic mismatch and bilayer curvature are considered as deformation forces of pressure-induced changes in the lipid bilayer causing conformational changes in MS channels as indicated by the example of MscL (13). These changes were studied experimentally by reconstituting purified MscL proteins in liposome bilayers prepared from synthetic phosphatidylcholine lipids of well-defined composition. The changes in functional properties were examined by the patch-clamp technique, whereas the structural changes were determined by EPR and FRET spectroscopy. (Reproduced from Reference 12, with permission).

The development of in vitro brain slice and isolated neuron techniques has greatly facilitated detailed studies of the electrophysiology of a wide range of neuronal types in the adult and neonatal vertebrate central nervous system (CNS). Particularly advantageous are the greater mechanical stability that these preparations provide over in vivo models and the control allowed over the composition of the extracellular environment. In addition, the development of the patch-clamp technique has opened up the possibility of direct access to the intracellular environment via internal patch pipet solutions. In combination, these approaches have enabled detailed investigations of neuronal membrane properties, the cellular actions of neurotransmitters, and synaptic mechanisms. 

Polar Cell Systems for Membrane Transport Studies Direct current electrical measurement in epithelia steady-state and transient analysis, 171, 607 impedance analysis in tight epithelia, 171, 628 electrical impedance analysis of leaky epithelia theory, techniques, and leak artifact problems, 171, 642 patch-clamp experiments in epithelia activation by hormones or neurotransmitters, 171, 663 ionic permeation mechanisms in epithelia biionic potentials, dilution potentials, conductances, and streaming potentials, 171, 678 use of ionophores in epithelia characterizing membrane properties, 171, 715 cultures as epithelial models porous-bottom culture dishes for studying transport and differentiation, 171, 736 volume regulation in epithelia experimental approaches, 171, 744 scanning electrode localization of transport pathways in epithelial tissues, 171, 792. 

Approaches to artificial ion channels have, for instance, made use of macrocyclic units [6.72,6.74] (see also below), of peptide [8.183-8.185] and cyclic peptide [8.186] components, of non-peptidic polymers [8.187] and of various amphiphilic molecules [6.11, 8.188, 8.189]. The properties of such molecules incorporated in bilayer membranes may be studied by techniques such as ion conductance [6.69], patch-clamp [8.190] or NMR [8.191, 8.192] measurements. However, the nature of the superstructure formed and the mechanism of ion passage (carrier, channel, pore, defect) are difficult to determine and often remain a matter of conjecture. 

Ions and small molecules may be transported across cell membranes or lipid bilayers by artificial methods that employ either a carrier or channel mechanism. The former mechanism is worthy of brief investigation as it has several ramifications in the design of selectivity filters in artificial transmembrane channels. To date there are few examples where transmembrane studies have been carried out on artificial transporters. The channel mechanism is much more amenable to analysis by traditional biological techniques, such as planar bilayer and patch clamp methods, so perhaps it is not surprising that more work has been done to model transmembrane channels. 

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