ANTAGONISM OF SAXITOXIN AND TETRODOTOXIN BLOCK BY INTERNAL MONOVALENT CATIONS IN SQUID AXON

The block of voltage-dependent sodium channels by saxitoxin (STX) and tetrodotoxin (TTX) was investigated in voltage-clamped squid giant axons internally perfused with a variety of permeant monovalent cations. Substitution of internal Na+ by either NH4+ or N2H5+ resulted in a reduction of outward current through sodium channels under control conditions. In contrast, anomalous increases in both inward and outward currents were seen for the same ions if some of the channels were blocked by STX or TTX, suggesting a relief of block by these internal cations. External NH4+ was without effect on the apparent magnitude of toxin block. Likewise, internal inorganic monovalent cations were without effect, suggesting that proton donation by NH4+ might be involved in reducing toxin block. Consistent with this hypothesis, decreases in internal pH mimicked internal perfusion with NH4+ in reducing toxin block. The interaction between internally applied protons and externally applied toxin molecules appears to be competitive, as transient increases in sodium channel current were observed during step increases in intracellular pH in the presence of a fixed STX concentration. In addition to these effects on toxin block, low internal pH produced a voltage-dependent block of sodium channels and enhanced steady-state inactivation. Elevation of external buffer capacity only marginally diminished the modulation of STX block by internal NH4+, suggesting that alkalinization of the periaxonal space and a resultant decrease in the cationic STX concentration during NH4+ perfusion may play only a minor role in the effect. These observations indicate that internal monovalent cations can exert trans-channel influences on external toxin binding sites on sodium channels.

Differential ion dehydration energetics explains selectivity in the non-canonical lysosomal K+ channel TMEM175

Structures of the human lysosomal K+ channel TMEM175 in open and closed states revealed a novel architecture lacking the canonical K+ selectivity filter motif present in previously known K+ channel structures. A hydrophobic constriction composed of four isoleucine residues was resolved in the pore and proposed to serve as the gate in the closed state, and to confer ion selectivity in the open state. Here, we achieve higher-resolution structures of the open and closed states and employ molecular dynamics simulations to analyze the conducting properties of the putative open state, demonstrating that it is capable of permeating K+ ions at the expected rate. Monovalent cations must dehydrate significantly to penetrate the narrow hydrophobic constriction, but ion flow is assisted by a favorable electrostatic field generated by the protein that spans the length of the pore. The balance of these opposing energetic factors explains why permeation is feasible, and why TMEM175 is selective for K+ over Na+, despite the absence of the canonical selectivity filter. Accordingly, mutagenesis experiments reveal an exquisite sensitivity of the channel to perturbations that mitigate the constriction. Together, these data reveal a novel mechanism for selective permeation of ions by TMEM175 that is unlike that of other K+ channels.

Pore mutation N617D in the skeletal muscle DHPR blocks Ca2+ influx due to atypical high-affinity Ca2+ binding

Skeletal muscle excitation-contraction (EC) coupling roots in Ca2+-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region.

Can Channel-Forming Antibiotics In Complex with Carriers Provide Enhanced Muscle Activity?

The presented review and experimental work provides the data regarding the selective permeability of lipid and cell membranes for ions and organic compounds under the influence of channel-forming polyene compounds with a known molecule structure. It has been shown that the polyene antibiotic levorin А2 with an aromatic structure affects a number of physicochemical parameters of lipid membranes. It was established that the permeability of lipid and cellular membranes for monovalent cations, as well as for monosugar and other neutral molecules increases under the influence of a levorin of А2. The biological activity of levorin А2 and the rate of delivery of molecules to the membranes depend on the surface tension and substrate environment of the membranes. It has been shown that in combination with levorin, dimethyl sulfoxide, and citral, the surface tension of the aqueous solutions surrounding the membrane decreases by half. Comparative data on levorin А2 effects on lipid membranes and muscle cell membranes are presented. It is assumed that levorin А2, being a channel-forming compound, can induce the formation of additional permeability channels in the membranes of muscle cells and, with intense muscle activity, enhance the transfer of cation and energy-dependent substrates through the membranes.

Analysis of entrapped and free liquor to gain new insights into kraft pulping

Most of our knowledge on kraft pulping comes from studies on dissolved lignin in the freely drainable black liquor and isolated residual lignin in pulp. However, entrapped liquor in the delignified chips has been shown to differ significantly from the free liquor. The present study has compared three liquor fractions: free, lumen and fiber wall liquor. The free liquor was obtained by draining the delignified chips, the lumen liquor was separated by centrifugation and the fiber wall liquor by subsequent leaching. The liquor in the fiber wall had the lowest concentration of lignin and hydrosulfide ions and the highest concentration of monovalent cations. The dissolved lignin in the fiber wall liquor had the highest molar mass and the highest content of xylan. The highest concentration of dissolved lignin was in the liquor filling the lumen cavities. The lignin in the free liquor had the lowest molar mass and the lowest content of lignin structures containing β-O-4 linkages and aliphatic hydroxyl groups. The lowest mass transfer rate of dissolved lignin was from the lumen liquor to the free liquor probably restricted by the tortuosity of the chip.