a B S TRAC T A literature review reveals many lines of evidence that both delayed rectifier and inward rectifier potassium channels are multi-ion pores. These include unidirectional flux ratios given by the 2-2.5 power of the electrochemical activity ratio, very steeply voltage-dependent block with monovalent blocking ions, relief of block by permeant ions added to the side opposite from the blocking ion, rectification depending on E -EK, and a minimum in the reversal potential or conductance as external K + ions are replaced by an equivalent concentration of TI + ions. We consider a channel with a linear sequence of energy barriers and binding sites. The channel can be occupied by more than one ion at a time, and ions hop in single file into vacant sites with rate constants that depend on barrier heights, membrane potential, and interionic repulsion. Such multi-ion models reproduce qualitatively the special flux properties of potassium channels when the barriers fi~r hopping out of the pore are larger than for hopping between sites within the pore and when there is repulsion between ions. These conditions also produce multiple maxima in the conductance-ion activity relationship. In agreement with Armstrong's hypothesis (1969.J. Gen. Physiol. 54:553-575), inward rectification may be understood in terms of block by an internal blocking cation. Potassium channels must have at least three sites and often contain at least two ions at a time.Evidence has accumulated, for the sodium channel and for several types of potassium channels of electrically excitable cells, that ions interact with the channel and with other ions in it while diffusing across the membrane (French and Adelman, 1976). An earlier paper of Hille (1975 b) discussed a model of the sodium channel in which the permeating ion must pass across a sequence of four energy barriers to cross the membrane. Inasmuch as the model assumed that no more than one ion could be in the channel at a time, it was called a oneion pore. In this paper we consider a similar type of linear, multibarrier model for a multi-ion pore where more than one ion may be in a channel at a time, and the ions are not permitted to pass by each other as they move through the channel. These assumptions lead to phenomena commonly referred to as "single-file diffusion" or the "long pore effect" which have been reported in measurements of the passive movement of ions in potassium channels of nerve, muscle, and other cell membranes. Our goal is to show that the major transport properties of potassium channels may be accounted for by this class of multi-ion channel models. For practical reasons, only a qualitative agreement is demonstrated here. An attempt to make more realistic models would need many more J. GEN. PHYSIOL. 9 The Rockefeller University Press .
Fourteen ORFs have been identified in the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome. ORF 3a of SARS-CoV codes for a recently identified transmembrane protein, but its function remains unknown. In this study we confirmed the 3a protein expression and investigated its localization at the surface of SARS-CoV-infected or 3a-cDNA-transfected cells. Our experiments showed that recombinant 3a protein can form a homotetramer complex through interprotein disulfide bridges in 3a-cDNA-transfected cells, providing a clue to ion channel function. The putative ion channel activity of this protein was assessed in 3a-complement RNA-injected Xenopus oocytes by two-electrode voltage clamp. The results suggest that 3a protein forms a potassium sensitive channel, which can be efficiently inhibited by barium. After FRhK-4 cells were transfected with an siRNA, which is known to suppress 3a expression, followed by infection with SARS-CoV, the released virus was significantly decreased, whereas the replication of the virus in the infected cells was not changed. Our observation suggests that SARS-CoV ORF 3a functions as an ion channel that may promote virus release. This finding will help to explain the highly pathogenic nature of SARS-CoV and to develop new strategies for treatment of SARS infection. caused alarm all over the world. The newly discovered human coronavirus named SARS-associated coronavirus (SARS-CoV) was identified as the causative agent for this disease (1, 2). SARSCoV has a large single-positive-strand RNA genome that contains 14 ORFs. Some of these ORFs encode viral structural proteins, such as spike protein, membrane protein, small envelope protein, and nucleocapsid protein, as well as viral replicase and protease (3). Those proteins play important roles in viral infection and replication. However, functions for other ORFs are not clear. Therefore, identification and characterization of new functional proteins from the ORFs will be helpful for understanding the pathogenesis of SARS-CoV. Up to now there are still no effective drugs or vaccines against SARS-CoV. The identification of new viral proteins and the elucidation of their functions will provide potential targets for design of drugs or vaccines against SARS.Our previous work has revealed that ORF 3a of SARS-CoV is such a viral protein (4). Since then, other publications have concurred in this observation and have shown that it is a structural protein (5-8). ORF 3a is located between the S and E protein loci and encodes a protein of 274 aa. The only available information based on proteomics and immunoblotting suggests that 3a protein is structural in nature, but its localization, topology, and biological function have not been identified.A computed biology analysis of the amino acid sequence of the 3a protein revealed that it has low similarity with any other known protein. Its C-terminal region shares Ϸ50% similarity to Plasmodium calcium pump protein and to the Shewanella outer-membrane porin. Interestingly, comparison of ORFs ...
Recurrent or chronic bronchial infection and poor dental status, mainly resulting from chronic dental infection, may be associated with an increased risk for cerebrovascular ischemia.
Sodium currents were studied under voltage clamp in the presence of neutral, amine, and quaternary local anesthetic compounds. Use-dependent block was observed as a cumulative depression of INa seen with repetitive depolarizing test pulses applied at frequencies of 2-10s-1. With quaternary QX-314, the time constant of use dependence was long, and with neutral benzocaine, very short. With lidocaine and procaine, increasing external pH (pHo) changed the time constant from long to short, but alterations of internal pH have no effect. Inactivation in Na channels was measured by the influence of prepulses on peak INa during test pulses. Single-stimulus inactivation curves were shifted more with lidocaine at high pHo than at low pHo, but inactivation curves measured during pulse trains with any of the drugs and at any pHo were strongly shifted. All measurements show that the drug-receptor reaction was slow for amine drugs at low pHo, as for quaternary drugs at any pHo, and fast for amine drugs at high pHo, as for neutral drugs at any pHo. The major effect of low pHo on amine drugs was to reduce the concentration of drugs in the fiber and to protonate drug molecules on the receptor, thus trapping them in the blocking position for a longer time. Direct effects of pH on the receptor seemed minimal.
To investigate the voltage dependence of the Na+/K+ pump, current-voltage relations were determined in prophase-arrested oocytes of Xenopus laevis. All solutions contained 5 mM Ba2+ and 20 mM tetraethylammonium (TEA) to block K+ channels. If, in addition, the Na+/K+ pump is blocked by ouabain, K(+)-sensitive currents no larger than 50 nA/cm2 remain. Reductions in steady-state current (on the order of 700 nA/cm2) produced by 50 microM ouabain or dihydro-ouabain or by K+ removal, therefore, primarily represent current generated by the Na+/K+ pump. In Na(+)-free solution containing 5 mM K+, Na+/K+ pump current is relatively voltage independent over the potential range from -160 to +40 mV. If external [K+] is reduced below 0.5 mM, negative slopes are observed over this entire voltage range. Similar results are seen in Na(+)- and Ca(2+)-free solutions in the presence of 2 mM Ni2+, an experimental condition designed to prevent Na+/Ca2+ exchange. The occurrence of a negative slope can be explained by the voltage dependence of the apparent affinity for activation of the Na+/K+ pump by external K+, consistent with the existence of an external ion well for K+ binding. In 90 mM Na+, 5 mM K+ solution, Na+/K+ pump current-voltage curves at negative membrane potentials have a positive slope and can be described by a monotonically increasing sigmoidal function. At an extracellular [K+] of 1.3 mM, a negative slope was observed at positive potentials. These findings suggest that in addition to a voltage-dependent step associated with Na+ translocation, a second voltage-dependent step that is dependent on external [K+], possibly external K+ binding, participates in the overall reaction mechanism of the Na+/K+ pump.
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