Strategies for studying permeation at voltage-gated ion channels

Voltage-dependent ion channels are presently thought to consist of several distinct functional regions: (a) activation gates, (b) inactivation gates, and permeation pathways. This chapter focuses on permeation pathways and may spur new ideas about experiments that use site-directed mutagenesis to probe the ion conduction pathway. Some hubris is required to attempt a survey of this field since individual families -- K^+, Na^+, or Ca^(2+) -- have been reviewed in detail (15, 68, 115, 127). My unified treatment is motivated by the structural similarity suggested by recent cDNA sequencing data on this group (see, for instance, 24). There have been many excellent previous treatments of ion channel permeation (6, 15, 34, 35, 51, 53, 68, 73, 74, 115, 127)

The N-Word: Lessons Taught and Lessons Learned

In the fall of 2008, I dared to teach a fifteen-week course that focused on a single word, a word arguably like no other, a word adorned with these emotionally colorful descriptors: “the most explosive of racial epithets,” “our cruelest word,” “the most toxic in the English language,” “the most troubling word in our language,” “almost magical in its negative power,” “six simple letters that convey centuries of pain, evil and contempt,” “an almost universally known word of contempt,” “occupies a place in the soul where logic and reason never go,” and “the filthiest, dirtiest, nastiest word in the English language.” I have since taught the course three more times. Because of the overwhelming success of my multimedia and multi-genre undergraduate course, “The N-word: Lessons Taught and Lessons Learned,” both for my students and for me, and because of the peculiar and alleged post-racial American historical moment in which we now are living with the first African American U.S. President, this reflective pedagogical piece, “The N-Word: Lessons taught and Lessons Learned,” is particularly relevant and timely. Indeed, although the use and history of the “nigger” with its various interracial, intraracial, and intracultural associations have garnered public attention in American classrooms, in the American media, and in American popular culture, deeper implications surrounding this word, the word “nigger” has not had the kind of sustained classroom exploration my semester -long course afforded. Putting this single word under a critical microscope underscored for me and my students the fact that ideas about language and identity, about language and public performance, and about language and American race relations inextricably connect youths and elders, blacks and whites, males and females, children and adults, the international and the domestic, past and present, public and private, and the personal and the political. Specifically, this pedagogical reflection offers a social and political context for the course, an intellectual rationale for the course, specific and detailed course content, students’ responses to the course, students\u27 and teacher\u27s overarching lessons gleaned from the course, and bibliographic suggestions for classroom practitioners and critically curious others navigating the ocean of materials on the word that journalist Farai Chideya has called “the all-American trump card, the nuclear bomb of racial epithets.”

New Views of Multi-Ion Channels

Thus, most site-directed mutagenesis data render it untenable to consider that two or more roughly equivalent high affinity sites govern selectivity in multi-ion pores. The papers by Dang and McCleskey and Kiss et al. respond to this challenge by showing that a model with a single high affinity site, flanked by two binding sites of lower affinity close to the pore entrances, can generate much of the classical multi-ion behavior. The sites need not interact, and the two flanking sites could arise from one of several mechanisms: a featureless charged vestibule, a dehydration step, or a specific weak binding site. The multi-ion pore remains a cornerstone of permeation theory, but the new theory features only a single high affinity site and no mutual repulsion. The high flux rate occurs because ions pause at the flanking sites and reequilibrate thermally, gaining enough energy to move over the next barrier

Nonrefundable Retainers: Impermissible Under Fiduciary, Statutory and Contract Law

Since the New York Court of Appeals banned nonrefundable retainers, numerous other courts have joined in prohibiting this widespread practice of lawyers charging a fee for services in advance and keeping the fee even if the services are not performed. This may reflect increased judicial recognition of the effect of egregious fee practices on the image of the bar and the role such practices play in the declining esteem in which the legal profession is held. Among the more provocative contributors to this ongoing debate, Professor Steven Lubet recently reviewed our work advocating the ban against nonrefundable retainers and posed a number of questions about the per se prohibition against them. In this Article, we respond to Professor Lubet\u27s questions as well as to those posed by other scholars. In Part I, we discuss the client discharge right, the cornerstone upon which the absolute ban on nonrefundable retainers rests, and respond to a series of arguments concerning its meaning, how it may be impaired, and whether it may be waived. In Part II, we show that legitimate purposes that may be served by nonrefundable retainers cannot avoid impairing the client discharge right, but that other fee arrangements that do not impair that right can easily be designed to serve some of those ends. In Part III, we extend an invitation to address the issues we have identified to all those who believe that a per se ban against nonrefundable retainers is unnecessary to prevent the abuses they generate. We have previously extended this invitation to devise a less inclusive rule that would in a practical and self-effectuating way prohibit the abuses while permitting the attainment of legitimate purposes. Unfortunately for the debate, however, none of the participants have accepted this invitation. We therefore think it bears repeating

Gain of function mutants: Ion channels and G protein-coupled receptors

Many ion channels and receptors display striking phenotypes for gain-of-function mutations but milder phenotypes for null mutations. Gain of molecular function can have several mechanistic bases: selectivity changes, gating changes including constitutive activation and slowed inactivation, elimination of a subunit that enhances inactivation, decreased drug sensitivity, changes in regulation or trafficking of the channel, or induction of apoptosis. Decreased firing frequency can occur via increased function of K+ or Cl- channels. Channel mutants also cause gain-of-function syndromes at the cellular and circuit level; of these syndromes, the cardiac long-QT syndromes are explained in a more straightforward way than are the epilepsies. G protein-coupled receptors are also affected by activating mutations

Rates and equilibria at the acetylcholine receptor of electrophorus electroplaques. A study of neurally evoked postsynaptic currents and of voltage-jump relaxations

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15°C the rate constant, α, equals 1.2 ms^(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/τ as the sum of the rate constant α for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate α, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to α-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10^(7) to 2x10^(8) M^(-1)s^(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10^(2) to 3x10^(3)s^(-1)

An Intermediate State of the {gamma}-Aminobutyric Acid Transporter GAT1 Revealed by Simultaneous Voltage Clamp and Fluorescence

The rat {gamma}-aminobutyric acid transporter GAT1 expressed in Xenopus oocytes was labeled at Cys74, and at one or more other sites, by tetramethylrhodamine-5-maleimide, without significantly altering GAT1 function. Voltage-jump relaxation analysis showed that fluorescence increased slightly and monotonically with hyperpolarization; the fluorescence at -140 mV was ~0.8% greater than at +60 mV. The time course of the fluorescence relaxations was mostly described by a single exponential with voltage-dependent but history-independent time constants ranging from ~20 ms at +60 mV to ~150 ms at -140 mV. The fluorescence did not saturate at the most negative potentials tested, and the midpoint of the fluorescence–voltage relation was at least 50 mV more negative than the midpoint of the charge–voltage relation previously identified with Na+ binding to GAT1. The presence of {gamma}-aminobutyric acid did not noticeably affect the fluorescence waveforms. The fluorescence signal depended on Na+ concentration with a Hill coefficient approaching 2. Increasing Cl- concentration modestly increased and accelerated the fluorescence relaxations for hyperpolarizing jumps. The fluorescence change was blocked by the GAT1 inhibitor, NO-711. For the W68L mutant of GAT1, the fluorescence relaxations occurred only during jumps to high positive potentials, in agreement with previous suggestions that this mutant is trapped in one conformational state except at these potentials. These observations suggest that the fluorescence signals monitor a novel state of GAT1, intermediate between the E*out and Eout states of Hilgemann, D.W., and C.-C. Lu (1999. J. Gen. Physiol. 114:459–476). Therefore, the study provides verification that conformational changes occur during GAT1 function

Functional Stoichiometry at the Nicotinic Receptor. The Photon Cross Section for Phase 1 Corresponds to Two Bis-Q Molecules per Channel

These experiments examine changes in the agonist-induced conductance that occur when the agonist-receptor complex is perturbed. Voltage-clamped Electrophorus electroplaques are exposed to the photoisomerizable agonist trans-Bis-Q A 1-µs laser flash photoisomerizes some trans-Bis-Q molecules bound to receptors; because the cis configuration is not an agonist, receptor channels close within a few hundred microseconds. This effect is called phase 1. We compare (a) the fraction of channels that close during phase 1 with (b) the fraction of trans-Bis-Q molecules that undergo trans → cis photoisomerization. Parameter a is measured as the fractional diminution in voltage-clamp currents during phase 1. Parameter b is measured by changes in the optical spectra of Bis-Q solutions caused by flashes . At low flash intensities, a is twice b, which shows that the channel can be closed by photoisomerizing either of two bound agonist molecules. Conventional dose-response studies with trans-Bis-Q also give a Hill coefficient of two. As a partial control for changes in the photochemistry caused by binding of Bis-Q to receptors, spectral measurements are performed on the photoisomerizable agonist QBr, covalently bound to solubilized acetylcholine receptors from Torpedo. The bound and free agonist molecules have the same photoisomerization properties. These results verify the concept that the open state of the acetylcholine receptor channel is much more likely to be associated with the presence of two bound agonist molecules than with a single such molecule

Establishing an Ion Pair Interaction in the Homomeric {rho}1 {gamma}-Aminobutyric Acid Type A Receptor That Contributes to the Gating Pathway

{gamma}-Aminobutyric acid type A (GABAA) receptors are members of the Cys-loop superfamily of ligand-gated ion channels. Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state, but the mechanism of gating is not well understood. Here we utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study the gating interface of the human homopentameric {rho}1 GABAA receptor. We have identified an ion pair interaction between two conserved charged residues, Glu92 in loop 2 of the extracellular domain and Arg258 in the pre-M1 region. We hypothesize that the salt bridge exists in the closed state by kinetic measurements and free energy analysis. Several other charged residues at the gating interface are not critical to receptor function, supporting previous conclusions that it is the global charge pattern of the gating interface that controls receptor function in the Cys-loop superfamily