[Osvaldo Alvarez remembers his professor].
Explore the source record for details and available documents.
Chile↗
Biomedical subjects
Osvaldo Alvarez
Publications and source records attributed to Osvaldo Alvarez.
S3b amino acid residues do not shuttle across the bilayer in voltage-dependent Shaker K+ channels.
In voltage-dependent channels, positive charges contained within the S4 domain are the voltage-sensing elements. The "voltage-sensor paddle" gating mechanism proposed for the KvAP K+ channel has been the subject of intense discussion regarding its general applicability to the family of voltage-gated channels. In this model, the voltage sensor composed of the S3b and the S4 segment shuttles across the lipid bilayer during channel activation. Guided by this mechanism, we assessed here the accessibility of residues in the S3 segment of the Shaker K+ channel by using cysteine-scanning mutagenesis. Mutants expressed robust K+ currents in Xenopus oocytes and reacted with methanethiosulfonate ethyltrimethylammonium in both closed and open conformations of the channel. Because Shaker has a long S3-S4 linker segment, we generated a deletion mutant with only three residues to emulate the KvAP structure. In this short linker mutant, all of the tested residues in the S3b were accessible to methanethiosulfonate ethyltrimethylammonium in both closed and open conformations. Because the S3b moves together with the S4 domain in the paddle model, we tested the effects of deleting two negative charges or adding a positive charge to this region of the channel. We found that altering the S3b net charge does not modify the total gating charge involved in channel activation. We conclude that the S3b segment is always exposed to the external milieu of the Shaker K+ channel. Our results are incompatible with any model involving a large membrane displacement of segment S3b.
Amino Acid Sequence↗
SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP.
We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca2+ concentration ([Ca2+]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 1263-1277, 1998), with low, moderate, and high maximal fractional open times (Po). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 microM cytoplasmic [Ca2+]. At 10 microM [Ca2+], low-Po channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation (KaATP) = 422 microM] than moderate-Po channels (KaATP = 82 microM). Oxidation of low-Po channels with thimerosal or 2,2'-dithiodipyridine (DTDP) gave rise to moderate-Po channels and decreased KaATP from 422 to 82 microM. At 0.1 microM cytoplasmic [Ca2+], ATP induced an almost negligible activation of low-Po channels. After oxidation to high-Po behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low-Po channels at 10 microM [Ca2+] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode.
2,2'-Dipyridyl↗
Anion permeation in human ClC-4 channels.
ClC-4 and ClC-5 are mammalian ClC isoforms with unique ion conduction and gating properties. Macroscopic current recordings in heterologous expression systems revealed very small currents at negative potentials, whereas a substantially larger instantaneous current amplitude and a subsequent activation were observed upon depolarization. Neither the functional basis nor the physiological impact of these channel features are currently understood. Here, we used whole-cell recordings to study pore properties of human ClC-4 channels heterologously expressed in tsA201 or HEK293 cells. Variance analysis demonstrated that the prominent rectification of the instantaneous macroscopic current amplitude is due to a voltage-dependent unitary current conductance. The single channel amplitudes are very small, i.e., 0.10 +/- 0.02 pA at +140 mV for external Cl(-) and internal I(-). Conductivity and permeability sequences were determined for various external and internal anions, and both values increase for anions with lower dehydration energies. ClC-4 exhibits pore properties that are distinct from other ClC isoforms. These differences can be explained by assuming differences in the size of the pore narrowing and the electrostatic potentials within the ion conduction pathways.
Anions↗
Tonic and phasic receptor neurons in the vertebrate olfactory epithelium.
Olfactory receptor neurons (ORNs) respond to odorants with characteristic patterns of action potentials that are relevant for odor coding. Prolonged odorant exposures revealed three populations of dissociated toad ORNs, which were mimicked by depolarizing currents: tonic (TN, displaying sustained firing, 49% of 102 cells), phasic (PN, exhibiting brief action potential trains, 36%) and intermediate neurons (IN, generating trains longer than PN, 15%). We studied the biophysical properties underlying the differences between TNs and PNs, the most extreme cases among ORNs. TNs and PNs possessed similar membrane capacitances (approximately 4 pF), but they differed in resting potential (-82 versus -64 mV), input resistance (4.2 versus 2.9 G(Omega)) and unspecific current, I(u) (TNs: 0 < I(u) 1 pA/pF). Firing behavior did not correlate with differences in voltage-gated conductances. We developed a mathematical model that accurately simulates tonic and phasic patterns. Whole cell recordings from rat ORNs in fragments (approximately 4 mm(2)) of olfactory epithelium showed that such a tissue normally contains tonic and phasic receptor neurons, suggesting that this feature is common across a wide range of vertebrates. Our findings show that the individual passive electrical properties can govern the firing patterns of ORNs.
Action Potentials↗
Molecular coupling between voltage sensor and pore opening in the Arabidopsis inward rectifier K+ channel KAT1.
Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open.
Algorithms↗
Counting channels: a tutorial guide on ion channel fluctuation analysis.
Ion channels open and close in a stochastic fashion, following the laws of probability. However, distinct from tossing a coin or a die, the probability of finding the channel closed or open is not a fixed number but can be modified (i.e., we can cheat) by some external stimulus, such as the voltage. Single-channel records can be obtained using the appropriate electrophysiological technique (e.g., patch clamp), and from these records the open probability and the channel conductance can be calculated. Gathering these parameters from a membrane containing many channels is not straightforward, as the macroscopic current I = iNP(o), where i is the single-channel current, N the number of channels, and P(o) the probability of finding the channel open, cannot be split into its individual components. In this tutorial, using the probabilistic nature of ion channels, we discuss in detail how i, N, and P(o max) (the maximum open probability) can be obtained using fluctuation (nonstationary noise) analysis (Sigworth FJ. G Gen Physiol 307: 97-129, 1980). We also analyze the sources of possible artifacts in the determination of i and N, such as channel rundown, inadequate filtering, and limited resolution of digital data acquisition by use of a simulation computer program (available at www.cecs.cl).
Animals↗