The development of an ion-sensitive solid-state device is described. The device combines the principles of an MOS transistor and a glass electrode and can be used for measurements of ion activities in electrochemical and biological environments. Some preliminary results are given.

Development of an Ion-Sensitive Solid-State Device for Neurophysiological Measurements

1 The ion-selective and ion transport properties of glycine receptor (GlyR) and gamma-aminobutyric acid receptor (GABAR) channels in the soma membrane of mouse spinal cord neurones were investigated using the whole-cell, cell-attached and outside-out patch versions of the patch-clamp technique 2 Current-voltage (I-V) relations of transmitter-activated currents obtained from whole-cell measurements with 145 mM-Cl- intracellularly and extracellularly, showed outward rectification In voltage-jump experiments, the instantaneous I-V relations were linear, and the steady-state I-V relations were rectifying outwardly indicating that the gating of GlyR and GABAR channels is voltage sensitive 3 The reversal potential of whole-cell currents shifted 56 mV per tenfold change in internal Cl- activity indicating activation of Cl(-)-selective channels The permeability ratio of K+ to Cl- (PK/PCl) was smaller than 005 for both channels 4 The permeability sequence for large polyatomic anions was formate greater than bicarbonate greater than acetate greater than phosphate greater than propionate for GABAR channels; phosphate and propionate were not measurably permeant in GlyR channels This indicates that open GlyR and GABAR channels have effective pore diameters of 52 and 56 A, respectively The sequence of relative permeabilities for small anions was SCN- greater than I- greater than Br- greater than Cl- greater than F- for both channels 5 GlyR and GABAR channels are multi-conductance-state channels In cell-attached patches the single-channel slope conductances close to 0 mV membrane potential were 29, 18 and 10 pS for glycine, and 28, 17 and 10 pS for GABA-activated channels The most frequently observed (main) conductance states were 29 and 17 pS for the GlyR and GABAR channel, respectively 6 In outside-out patches with equal extracellular and intracellular concentrations of 145 mM-Cl-, the conductance states were 46, 30, 20 and 12 pS for GlyR channels and 44, 30, 19 and 12 pS for GABAR channels The most frequently occurring main state was 46 pS for the GlyR and 30 pS for the GABAR channel 7 Single-channel conductances measured in equal 140 mM concentrations of small anions on both membrane faces revealed a conductance sequence of Cl- greater than Br- greater than I- greater than SCN- greater than F- for both channels This is nearly the inverse sequence of that found for the permeability of these ions indicating the presence of binding sites for ions in the channel(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones.

Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. This review summarizes our current knowledge of this large protein family and discusses recent advances in our understanding of their structure and physiological functions.

Claudins and the Modulation of Tight Junction Permeability

We discuss the view that the folding of many, perhaps most, integral membrane proteins can be considered as a two-stage process. In stage I, hydrophobic alpha-helices are established across the lipid bilayer. In stage II, they interact to form functional transmembrane structures. This model is suggested by the nature of transmembrane segments in known structures, refolding experiments, the assembly of integral membrane protein from fragments, and the existence of very small integral membrane protein subunits. It may extend to proteins with a variety of functions, including the formation of transmembrane aqueous channels. The model is discussed in the context of the forces involved in membrane protein folding and the interpretation of sequence data.

Membrane protein folding and oligomerization: the two-stage model.

An epithelial cell line (MDCK) was used to prepare monolayers which, in vitro, develop properties of transporting epithelia. Monolayers were formed by plating cells at high densities (10(6) cells/cm2) on collagen-coated nylon cloth disks to saturate the area available for attachment, thus avoiding the need for cell division. An electrical resistance developed within 4-6 h after plating and achieved a steady-state value of 104 +/- 1.8 omega-cm2 after 24 h. Mature monolayers were morphologically and functionally polarized. They contained junctional complexes composed of desmosomes and tight junctions with properties similar to those of "leaky" epithelia. Monolayers were capable of maintaining a spontaneous electrical potential sensitive to amiloride, produced a net water flux from the apical to basal side, and discriminated between Na+ and Cl- ions. The MDCK permeability barrier behaves as a "thin" membrane with negatively charged sites. It has: (a) a linear conductance/concentration relationship; (b) an asymmetric instantaneous current/voltage relationship; (c) a reduced ability to discriminate between Na+ and Cl- caused by lowering the pH; and (d) a characteristic pattern of ionic selectivity which suggests that the negatively charged sites are highly hydrates and of medium field strength. Measurements of Na+ permeability of electrical and tracer methods ruled out exchange diffusion as a mechanism for ion permeation and the lack of current saturation in the I/deltapsi curves does not support the involvement of carriers. The discrimination between Na+ and Cl- was severely but reversibly decreased at low pH, suggesting that Na+-specific channels which exclude Cl- contain acidic groups dissociated at neutral pH. Bound Ca++ ions are involved in maintaining the integrity of the junctions in MDCK monolayers as was shown by a reversible drop of resistance and opening of the junctions in Ca++-free medium containing EGTA. Several other epithelial cell lines are capable of developing a significant resistance under the conditions used to obtain MDCK monolayers.

/pdf/polarized-monolayers-formed-by-epithelial-cells-on-a-4au5swem16.pdf

Polarized monolayers formed by epithelial cells on a permeable and translucent support.

Cation Selective Glass Electrodes and their Mode of Operation

GENERAL PROPERTIES OF OPEN CHANNELS 207 What are Ion Channels and Why are They Needed? 207 The Energy Profile and Ion Permeation 208 Ionic Selectivity 209 Fluctuations of Channels 211

An introduction to molecular architecture and permeability of ion channels.

�Single channel patch-clamp techniques were used to study nicotinic acetylcholine receptors in cultured rat myotubes . The single channel conductance in pure cesium and sodium levels offat high concentrations, as if a binding site within the channel were saturating . The conductances at very low concentrations, however, are larger than predictedby thesimplest one-site transport model fitted to the high-concentration data . At low concentrations, the current-voltage relations are inwardly rectifying, but they become more ohmic ifa smallamount of divalent cations is added externally . Magnesium and barium are good permeants that have rather high affinities for the channel. Upon adding low millimolar concentrations ofthese divalent cations externally to a membrane bathed in pure cesium, the inward current carried by cesium is decreased . As more divalent cations are added, the inward-going currents continued to decrease and the divalent cation replaces cesium as the main current carrier . The ion transport data are described by considering the size, shape, and possible net charge of the channel . In that way, even the complex features of transportare explained inarealistic physical framework. Theresults are consistent with the channel having long, wide, multiply occupied vestibules that serve as transition zones to the short, selective, singly occupied narrow region of the channel. A small amount of net negative charge within the pore couldproduce concentration-dependent potentials that provide a simple explanation for the more complicatedaspects of the permeation properties .

in Acetylcholine Receptor Channels Ion Transport Related to Structure

Single channel patch-clamp techniques were used to study nicotinic acetylcholine receptors in cultured rat myotubes. The single channel conductance in pure cesium and sodium levels off at high concentrations, as if a binding site within the channel were saturating. The conductances at very low concentrations, however, are larger than predicted by the simplest one-site transport model fitted to the high-concentration data. At low concentrations, the current-voltage relations are inwardly rectifying, but they become more ohmic if a small amount of divalent cations is added externally. Magnesium and barium are good permeants that have rather high affinities for the channel. Upon adding low millimolar concentrations of these divalent cations externally to a membrane bathed in pure cesium, the inward current carried by cesium is decreased. As more divalent cations are added, the inward-going currents continued to decrease and the divalent cation replaces cesium as the main current carrier. The ion transport data are described by considering the size, shape, and possible net charge of the channel. In that way, even the complex features of transport are explained in a realistic physical framework. The results are consistent with the channel having long, wide, multiply occupied vestibules that serve as transition zones to the short, selective, singly occupied narrow region of the channel. A small amount of net negative charge within the pore could produce concentration-dependent potentials that provide a simple explanation for the more complicated aspects of the permeation properties.

/pdf/monovalent-and-divalent-cation-permeation-in-acetylcholine-17l0wx0kk8.pdf

George Eisenman

Papers

Cation Selective Glass Electrodes and their Mode of Operation

An introduction to molecular architecture and permeability of ion channels.

in Acetylcholine Receptor Channels Ion Transport Related to Structure

Monovalent and divalent cation permeation in acetylcholine receptor channels. Ion transport related to structure.

Acetylcholine-activated channel current-voltage relations in symmetrical na solutions.