Flagellated envelopes of Halobacterium salinarium cells were prepared by lysis with taurodeoxycholate. After solubilization of the envelopes with Triton X-100 at high ionic strength, flagella and round patches from which numerous flagella emerged were isolated by gel filtration chromatography. We conclude that the flagellar bundle of H. salinarium is inserted into a differentiated polar cap structure.
Mg2+ ions block N-methyl-D-aspartate (NMDA) channels by entering the pore from either the extracellular or the cytoplasmic side of the membrane in a voltage-dependent manner. We have used these two different block phenomena to probe the structure of the subunits forming NMDA channels. We have made several amino acid substitutions downstream of the Q/R/N site in the TMII region of both NR1 and NR2A subunits. Mutant NR1 subunits were coexpressed with wild-type NR2A subunits and vice versa in Xenopus oocytes. We found that individually mutating the first two amino acid residues downstream to the Q/R/N site affects mostly the block by external Mg2+. Mutations Recently, a series of biochemical studies (14-17) have suggested that, instead of traversing the membrane, the region labeled TMII forms a loop, dipping in and out of the membrane from the internal side. Such pore loops have become a common structural motif in the case of the voltage-gated ion channels since they were first proposed for the Shaker potassium channel by Yellen et al. (18). They used tetraethylammonium (TEA), known to block potassium channels from both sides of the membrane, to probe the structure of a 20 amino acid stretch in the linker between the membrane spanning domains S5 and S6. Mutations on both ends of this "P region" were found to affect only the block by external TEA, whereas a mutation in the center of the P region selectively affected the internal TEA block.In the present study, we have used a similar approach to test whether the mutation of certain amino acid residues downstream of the Q/R/N site affects differentially the blocks produced by internal and external Mg2+. Again, the results suggest that permeation in the channel involves a pore loop.N-methyl-D-aspartate (NMDA) receptors form cation selective ion channels at excitatory synapses and can be distinguished from other glutamate-gated ion channels by their pharmacological and biophysical properties (1). The voltage dependence of the NMDA receptor channel, which is thought to play an important role in many physiological and pathophysiological processes, results from an extracellular Mg2+ block (2, 3), which increases markedly with hyperpolarization due to the binding of Mg2+ deep inside the pore (4, 5). Mg2+ ions can also block the NMDA channel from the cytoplasmic surface (6). Like the external Mg2+ block, the internal Mg2+ block is voltage dependent, but it increases with depolarization and the unblocking rate of internal Mg2+ is two orders of magnitude faster than that of external Mg2+. It has been proposed that external and internal Mg2+ bind to two different blocking sites within the channel pore (6, 7).The genes coding for the subunits of the NMDA receptor have recently been cloned, offering new opportunities for structure function studies (8-10). Functional channels are composed of the NR1 subunit and one or more members of the NR2 family. Site-directed mutagenesis rapidly identified an asparagine residue (N) as playing a key role in the external Mg2+ block (11...
The rate of C-type inactivation of the cloned voltage-gated potassium channel, Kv1.3, measured in membrane patches from Xenopus oocytes, increases when the patch is detached from the cell; the structural basis for this on-cell/off-cell change was examined. First, four serine and threonine residues, that are putative sites for phosphorylation by protein kinases A and C, were mutated to alanines. Mutating any one of these residues, or two or three of them simultaneously, does not eliminate the change in C-type inactivation. However, the basal rate of C-type inactivation in the cell-attached patch is markedly slower in the triple phosphorylation site mutant. Second, a homologous potassium channel, Kv 1.6, does not exhibit the on-cell/off-cell change. When an extracellular histidine at position 401 of Kv1.3 is replaced with tyrosine, the residue at the equivalent position (430) in Kv1.6, the resulting Kv1.3 H401Y mutant channel does not undergo the on-cell/off-cell change. The results indicate that several potentially phosphorylatable intracellular amino acids influence the basal rate of C-type inactivation, but are not essential for the on-cell/off-cell change in inactivation kinetics. In contrast, an extracellular amino acid is critical for this on-cell/off-cell change.
The NMDA receptor channel is blocked by both external and internal Mg2+ ions, which are assumed to bind inside the channel on each side of a central barrier. We have analysed the internal Mg2+ block in recombinant NR1‐NR2A NMDA receptors expressed in Xenopus oocytes. We have determined the effects of mutations of two asparagines that line the selectivity filter of the channel, one located within the NR1 subunit (N598) and the other within the NR2A subunit (N596). The whole‐cell current‐voltage relation of wild‐type NMDA channels shows inward rectification that reflects the voltage‐dependent block produced by the internal Mg2+ of the oocyte. This inward rectification is slightly reduced in the NR2 mutant (N596S) but is abolished in the NR1 mutants (N598Q and N598S). This suggests that the NR1 asparagine plays a larger role than the NR2 asparagine in controlling the internal Mg2+ block. Single‐channel current‐voltage relations confirm that the internal Mg2+ block is reduced in both the NR1 and NR2 mutants. However, the reduction is small and is similar for the two families of mutants. The discrepancy between whole‐cell and single‐channel data is partly due to differential effects of internal Mg2+ on the open probabilities of the two conductance states present in NR1 mutant channels. The results suggest that mutations of NR1 and NR2 asparagines lower the central barrier to Mg2+. An additional contribution of the NR2 asparagine to the external Mg2+ binding site (and possibly to the external barrier that controls access to this site) may account for the marked relief of external Mg2+ block produced by the NR2 mutation.
We transfected cultured hippocampal neurons with the cDNA of the voltage-gated K+ channel Kv1.3 to investigate the mechanisms by which a specific ion channel influences excitability. In transfected neurons under voltage clamp we observed an additional outward current that was blocked selectively by margatoxin. Under current-clamp conditions, Kv1.3-expressing neurons fired tonically over a wide range of stimulation intensity. In non-transfected neurons, or in Kv1.3-expressing cells blocked with margatoxin, only a few action potentials were elicited before a stationary depolarized state was reached. We attribute the specific effect of Kv1.3 to its particularly slow deactivation near the resting potential. A computational model showed that a continuous outwards current arises in Kv1.3-expressing neurons during the interspike intervals. It expands the dynamic range so that these neurons still fire tonically at stimulus current intensities at which non-transfected cells have already been driven into a stationary depolarized state.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.