Interaction of Na+ and Mg2+ ions in acetylcholine receptor channels of frog skeletal muscle changes in character with an increase in agonist concentration

Abstract

Measurements of agonist-induced single-channel currents of nicotinic acetylcholine receptor channels (nAChR) have shown that addition of divalent metal cations (Ca2+, Mg2+, Ba2+) in millimolar amounts causes a decrease of 40% or more in currents carried by monovalent cations (Na+, Cs+). Conventional voltage-clamp measurements were made at frog (Rana pipiens) sartorius endplates to determine if this occlusive interaction between mono- and divalent cations is preserved in macroscopic currents produced by higher concentrations of nAChR agonists, as would be expected on the basis of simple summation of unitary currents. Single ion nAChR currents of Na+ (INa+ ) and Mg2+(IMg2+ ) and the composite current when both cations were present (INa+, Mg2+ ) were measured in high-K+, low ionic strength bathing media during local superfusion with various concentrations of carbamylcholine. It was found that, at lower test carbamylcholine levels of 27 and 54 microM, peak values of INa+, Mg2+ were 40% and 53% of the respective INa+ , in agreement with the effects observed from single-channel measurements. At all higher carbamylcholine levels, however, peak INa+, Mg2+ exceeded INa+ and increased instead with the sum of INa+ and IMg2+ as if there was an independent movement of these ions through the channels rather than the occlusive interaction found at lower carbamylcholine levels. This suggests that with exposure to increasing agonist concentrations above those commonly used in single-channel measurements there are changes in the open state of nAChR affecting cation selectivity possibly in the narrow pore region of the channels.

Modulation of fetal and adult acetylcholine receptors by Ca2+ and Mg2+ at developing mouse end-plates

“…Conversely, channel activation was lengthened with 0.1 µM ACh, but not with 100 µM. Thus we found no single-channel correlate of the reported conversion of Mg 2+ action from inhibition to facilitation with increasing agonist concentration [19]. This discrepancy might be due to the different experimental preparations used (developing mouse or adult frog), or to the facilitation being specific for the conditions (high [Mg 2+ ] e and carbamylcholine as an agonist used in [19]).…”

“…It is also unclear whether the single-channel properties of AChR channels are differently affected by Mg 2+ at various [ACh]. In fact, at variance with "classical" studies, a conversion of Mg 2+ action from block to facilitation of carbamylcholine-evoked responses is observed at the frog neuromuscular junction with high agonist concentrations [19]. Furthermore, there is no general consensus on the effects of divalent cations on the kinetics of AChR channels.…”

Weakening of ion-channel interactions of Na + and Li + in acetylcholine-receptor channels of frog skeletal muscle with an increase in agonist concentration

“…Means of 45 digitized samples of both clamping current and voltage (sampling rate = 100 Hz) were computed at consecutive 0.5-s intervals throughout each experimental trial and stored (64 K Apple II series desktop computer) for later analysis using specialized programs. Previous publications [39,40] should be consulted for further details about the methods used in voltage clamping and agonist superfusion.…”

“…Since the open-state configuration of channel proteins, particularly in the narrow pore region of nAChR, shapes and limits ion traffic through the channel [11,14,28,32,57,60], a possible approach to the question of agonist-dependent secondary open states might be to examine the "mix" of ion species making up nAChR currents at various increasing concentrations of agonists and to see if there is any shift in the preference or interaction among permeating cations. In a preliminary study, in which something of this kind was tried using Na + and Mg 2+ as convenient current carriers with readily distinguishable characteristics [40], it was found that above a certain submaximal "transition" level of agonist (lying between 50 and 100 µM carbamylcholine) the peak Mg 2+ current began to increase disproportionately to the rise of Na + currents and with changes in the composite bi-ionic currents that were most easily explained in terms of altered open states characterized by decreased ion-channel interactions in the pore region and greater independence of the Na + and Mg 2+ fluxes. This proposal is complicated, however, by some other possible actions involving Mg 2+ outside the pore region, including displacement of agonist molecules from outer receptor sites [53,56], and Mg 2+ -specific effects on intracellular phosphorylation pathways [2] which could lead to the opening of other ion channels such as L-type Ca 2+ channels [21,50].…”

“…It would be desirable, therefore, to expand these observations using other test ions as current carriers, particularly the alkali metal cations which would either not have these extra-channel actions or exhibit them to a much lesser degree. As a step in this direction, whole-cell single-ion nAChR currents of Na + and Li + in frog muscle fibers were measured in the present study along the lines reported previously [40] and compared, both singly and in combination, at selected agonist concentrations above and below the putative "transition" level and with respect to a number of features that depend on interactions of the permeating cations with intra-channel protein subunits.…”