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G Yellen

Publications and source records attributed to G Yellen.

At least 19 recordsLinked to original sources
PubMed1998

The activation gate of a voltage-gated K+ channel can be trapped in the open state by an intersubunit metal bridge.

Voltage-activated K+ channels are integral membrane proteins containing a potassium-selective transmembrane pore gated by changes in the membrane potential. This activation gating (opening) occurs in milliseconds and involves a gate at the cytoplasmic side of the pore. We found that substituting cysteine at a particular position in the last transmembrane region (S6) of the homotetrameric Shaker K+ channel creates metal binding sites at which Cd2+ ions can bind with high affinity. The bound Cd2+ ions form a bridge between the introduced cysteine in one channel subunit and a native histidine in another subunit, and the bridge traps the gate in the open state. These results suggest that gating involves a rearrangement of the intersubunit contacts at the intracellular end of S6. The recently solved structure of a bacterial K+ channel shows that the S6 homologs cross in a bundle, leaving an aperture at the bundle crossing. In the context of this structure, the metal ions form a bridge between a cysteine above the bundle crossing and a histidine below the bundle crossing in a neighboring subunit. Our results suggest that gating occurs at the bundle crossing, possibly through a change in the conformation of the bundle itself.

Amino Acid Substitution↗
PubMed1997

Defective "pacemaker" current (Ih) in a zebrafish mutant with a slow heart rate.

At a cellular level, cardiac pacemaking, which sets the rate and rhythm of the heartbeat, is produced by the slow membrane depolarization that occurs between action potentials. Several ionic currents could account for this pacemaker potential, but their relative prominence is controversial, and it is not known which ones actually play a pacemaking role in vivo. To correlate currents in individual heart cells with the rhythmic properties of the intact heart, we have examined slow mo (smo), a recessive mutation we discovered in the zebrafish Danio rerio. This mutation causes a reduced heart rate in the embryo, a property we can quantitate because the embryo is transparent. We developed methods for culture of cardiocytes from zebrafish embryos and found that, even in culture, cells from smo continue to beat relatively slowly. By patch-clamp analysis, we discovered that a large repertoire of cardiac currents noted in other species are present in these cultured cells, including sodium, T-type, and L-type calcium and several potassium currents, all of which appear normal in the mutant. The only abnormality appears to be in a hyperpolarization-activated inward current with the properties of Ih, a current described previously in the nervous system, pacemaker, and other cardiac tissue. smo cardiomyocytes have a reduction in Ih that appears to result from severe diminution of one kinetic component of the Ih current. This provides strong evidence that Ih is an important contributor to the pacemaking behavior of the intact heart.

Animals↗
PubMed1997

Gated access to the pore of a voltage-dependent K+ channel.

Voltage-activated K+ channels are integral membrane proteins that open or close a K(+)-selective pore in response to changes in transmembrane voltage. Although the S4 region of these channels has been implicated as the voltage sensor, little is known about how opening and closing of the pore is accomplished. We explored the gating process by introducing cysteines at various positions thought to lie in or near the pore of the Shaker K+ channel, and by testing their ability to be chemically modified. We found a series of positions in the S6 transmembrane region that react rapidly with water-soluble thiol reagents in the open state but not the closed state. An open-channel blocker can protect several of these cysteines, showing that they lie in the ion-conducting pore. At two of these sites, Cd2+ ions bind to the cysteines without affecting the energetics of gating; at a third site, Cd2+ binding holds the channel open. The results suggest that these channels open and close by the movement of an intracellular gate, distinct from the selectivity filter, that regulates access to the pore.

Animals↗
PubMed1997

Trapping of organic blockers by closing of voltage-dependent K+ channels: evidence for a trap door mechanism of activation gating.

Small organic molecules, like quaternary ammonium compounds, have long been used to probe both the permeation and gating of voltage-dependent K+ channels. For most K+ channels, intracellularly applied quaternary ammonium (QA) compounds such as tetraethylammonium (TEA) and decyltriethylammonium (C10) behave primarily as open channel blockers: they can enter the channel only when it is open, and they must dissociate before the channel can close. In some cases, it is possible to force the channel to close with a QA blocker still bound, with the result that the blocker is "trapped." Armstrong (J. Gen. Physiol. 58:413-437) found that at very negative voltages, squid axon K+ channels exhibited a slow phase of recovery from QA blockade consistent with such trapping. In our studies on the cloned Shaker channel, we find that wild-type channels can trap neither TEA nor C10, but channels with a point mutation in S6 can trap either compound very efficiently. The trapping occurs with very little change in the energetics of channel gating, suggesting that in these channels the gate may function as a trap door or hinged lid that occludes access from the intracellular solution to the blocker site and to the narrow ion-selective pore.

Animals↗
PubMed1996

Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K+ channels.

Many blockers of Na+ and K+ channels act by blocking the pore from the intracellular side. For Shaker K+ channels, such intracellular blockers vary in their functional effect on slow (C-type) inactivation: Some blockers interfere with C-type inactivation, whereas others do not. These functional differences can be explained by supposing that there are two overlapping "subsites" for blocker binding, only one of which inhibits C-type inactivation through an allosteric effect. We find that the ability to bind to these subsites depends on specific structural characteristics of the blockers, and correlates with the effect of mutations in two distinct regions of the channel protein. These interactions are important because they affect the ability of blockers to produce use-dependent inhibition.

Allosteric Regulation↗
PubMed1996

The inward rectification mechanism of the HERG cardiac potassium channel.

A human genetic defect associated with 'long Q-T syndrome', an abnormality of cardiac rhythm involving the repolarization of the action potential, was recently found to lie in the HERG gene, which codes for a potassium channel. The HERG K+ channel is unusual in that it seems to have the architectural plan of the depolarization-activated K+ channel family (six putative transmembrane segments), yet it exhibits rectification like that of the inward-rectifying K+ channels, a family with different molecular structure (two transmembrane segments). We have studied HERG channels expressed in mammalian cells and find that this inward rectification arises from a rapid and voltage-dependent inactivation process that reduces conductance at positive voltages. The inactivation gating mechanism resembles that of C-type inactivation, often considered to be the 'slow inactivation' mechanism of other K+ channels. The characteristics of this gating suggest a specific role for this channel in the normal suppression of arrhythmias.

Animals↗
PubMed1996

Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K+ channel.

Quaternary ammonium blockers inhibit many voltage-activated potassium (K+) channels from the intracellular side. When applied to Drosophila Shaker potassium channels expressed in mammalian cells, these rapidly reversible blockers produced use-dependent inhibition through an unusual mechanism--they promoted an intrinsic conformational change known as C-type inactivation, from which recovery is slow. The blockers did so by cutting off potassium ion flow to a site in the pore, which then emptied at a rate of 10(5) ions per second. This slow rate probably reflected the departure of the last ion from the multi-ion pore: Permeation of ions (at 10(7) per second) occurs rapidly because of ion-ion repulsion, but the last ion to leave would experience no such repulsion.

Binding Sites↗
PubMed1996

On the use of thiol-modifying agents to determine channel topology.

A powerful tool in the study of cloned ion channels is the combined use of site-directed mutagenesis and chemical modification. Site-directed mutagenesis is used to introduce new cysteine residues at specific positions in a channel protein, and chemical modification by thiol-specific reagents is then used to assess the exposure of the introduced cysteins. This method has been used to assess secondary structure, membrane topology and conformational changes. We report that one commonly used, charged reagent (MTSEA; aminoethyl methanethiosulfonate) can cross the membrane quite readily. We also find that other reagents that are quite membrane-impermeant can cross the membrane when patches are electrically leaky. Both of these undesired effects can be controlled by the use of a thiol scavenger. These findings argue for caution in the use of modifying reagents to determine the membrane topology of channels and other membrane proteins.

Animals↗
PubMed1996

Dynamic rearrangement of the outer mouth of a K+ channel during gating.

With prolonged stimulation, voltage-activated K+ channels close by a gating process called inactivation. This inactivation gating can occur by two distinct molecular mechanisms: N-type, in which a tethered particle blocks the intracellular mouth of the pore, and C-type, which involves a closure of the external mouth. The functional motion involved in C-type inactivation was studied by introducing cysteine residues at the outer mouth of Shaker K+ channels through mutagenesis, and by measuring state-dependent changes in accessibility to chemical modification. Modification of three adjacent residues in the outer mouth was 130-10,000-fold faster in the C-type inactivated state than in the closed state. At one position, state-dependent bridging or crosslinking between subunits was also possible. These results give a consistent picture in which C-type inactivation promotes a local rearrangement and constriction of the channel at the outer mouth.

Cell Line↗
PubMed1996

N-type inactivation and the S4-S5 region of the Shaker K+ channel.

The intracellular segment of the Shaker K+ channel between transmembrane domains S4 and S5 has been proposed to form at least part of the receptor for the tethered N-type inactivation "ball." We used the approach of cysteine substitution mutagenesis and chemical modification to test the importance of this region in N-type inactivation. We studied N-type inactivation or the block by a soluble inactivation peptide ("ball peptide") before and after chemical modification by methanethiosulfonate reagents. Particularly at position 391, chemical modification altered specifically the kinetics of ball peptide binding without altering other biophysical properties of the channel. Results with reagents that attach different charged groups at 391 C suggested that there are both electrostatic and steric interactions between this site and the ball peptide. These findings identify this site to be in or near the receptor site for the inactivation ball. At many of the other positions studied, modification noticeably inhibited channel current. The accessible cysteines varied in the state-dependence of their modification, with five- to tenfold changes in reactions rate depending on the gating state of the channel.

Amino Acid Sequence↗
PubMed1995

Covalent modification of engineered cysteines in the nicotinic acetylcholine receptor agonist-binding domain inhibits receptor activation.

We constructed and characterized a series of nicotinic receptor mutants with a cysteine substituted for one of the amino acid residues in the alpha-subunit between positions 183 and 198. This region of the receptor is known to participate in agonist binding and channel activation. The goal of this 'cysteine scanning mutagenesis' is to introduce the reactivity of a free thiol group into functionally important protein domains; modification of the introduced cysteines can then be used to probe the structure and function of the targeted region. Mutants were examined by coexpression with the beta-, gamma- and delta-subunits in Xenopus oocytes using two-microelectrode voltage clamp recording. Twelve of fourteen mutants expressed receptors with properties comparable with the wild-type, including sensitivity to reduction by dithiothreitol (DTT). This indicates that introduction of an additional cysteine within this region of the receptor did not interfere with formation of the native disulphide between alpha Cys-192 and alpha Cys-193. Only one mutation, alpha Y198C, caused dramatic changes in the EC50 for acetylcholine (ACh) and in the sensitivity to DTT. We then examined the effects of the thiol modification and found two mutants, alpha H186C and alpha V188C, that showed significant decreases in responsiveness to ACh after exposure to methylmethanethiosulphonate (MMTS). Dose-response measurements show that exposure of alpha H186C mutants to MMTS causes a shift in apparent agonist affinity without changing the peak response, and this is not reversible by DTT. In contrast, the MMTS-treated alpha V188C mutants show changes in both apparent affinity and peak response which are readily reversed by DTT. Together, our data show that these two nearby residues occupy markedly different environments relative to the contact points for ACh. They also demonstrate that cysteine-substitution mutagenesis can be successfully applied to protein domains that include functionally important disulphides.

Acetylcholine↗
PubMed1995

Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms.

Voltage-activated K+ currents and their inactivation properties are important for controlling frequency-dependent signaling in neurons and other excitable cells. Two distinct molecular mechanisms for K+ channel inactivation have been described: N-type, which involves rapid occlusion of the open channel by an intracellular tethered blocker, and C-type, which involves a slower change at the extracellular mouth of the pore. We find that frequency-dependent cumulative inactivation of Shaker channels is very sensitive to changes of extracellular [K+] in the physiological range, with much more inactivation at low [K+]out, and that it results from the interaction of N- and C-type inactivation. N-type inactivation enhances C-type inactivation by two mechanisms. First, it inhibits outward K+ flux, which normally fills an external ion site and thus prevents C-type inactivation. Second, it keeps the channel's activation gate open even after repolarization, allowing C-type inactivation to occur for a prolonged period.

Cell Line↗
PubMed1995

A discrete site for general anesthetics on a postsynaptic receptor.

General anesthetics depress central nervous system excitability via a mechanism that probably involves effects on synaptic ion channels, but the fundamental molecular nature of the site where they act is unknown. Although the importance of hydrophobicity for general anesthetic drug potency has long been established, it remains uncertain whether these "nonspecific" drugs act on membrane proteins directly or by modification of the physical properties of the lipid membrane or the lipid-protein interface. We find that specific mutations in the acetylcholine receptor pore-forming M2 domains enhance the sensitivity of the receptor to the general anesthetics isoflurane, hexanol, and octanol, suggesting that these agents act by binding directly to a discrete protein site at or near these residues. The sensitivity of the receptor to block by general anesthetics increases with increased hydrophobicity of these residues, demonstrating that hydrophobic forces dominate the interaction of drugs with their protein site. Furthermore, octanol inhibits both wild-type and mutant nicotinic acetylcholine receptors preferentially after channel opening, which is consistent with a mechanism where drugs bind within the receptor's pore. Similar sites on postsynaptic ion channels in brain may represent general anesthetic targets for modulating consciousness.

Amino Acid Sequence↗
PubMed1994

Conductance mutations of the nicotinic acetylcholine receptor do not act by a simple electrostatic mechanism.

Fixed negative charges in many cation channels raise the single-channel conductance, apparently by an electrostatic mechanism: their effects are accentuated in solutions of low ionic strength and attenuated at high ionic strength. The charges of specific amino acids near the ends of the proposed pore-lining M2 segment of the nicotinic acetylcholine receptor, termed the extracellular and cytoplasmic rings, have recently been shown to influence the single-channel K+ conductance (Imoto, K., C. Busch, B. Sakmann, M. Mishina, T. Konno, J. Nakai, H. Bujo, Y. Mori, K. Fukuda and S. Numa. 1988. Nature 335:645-648). We examined whether these charges might act by a direct electrostatic effect on the energy of ions in the pore, rather than indirectly by inducing a structural change. To this end, we measured the conductances of charge mutants over a range of K+ concentrations (ionic strengths). As expected, we found that negative charge mutations raise the conductance, and positive charge mutations lower it. The effects of cytoplasmic-ring mutations are accentuated at low ionic strength, but they are not completely attenuated at high ionic strength. The effects of extracellular-ring mutations are independent of ionic strength. These results are inconsistent with the simplest electrostatic model. We suggest a modified model that qualitatively accounts for the data.

Amino Acid Sequence↗
PubMed1994

Cysteines in the Shaker K+ channel are not essential for channel activity or zinc modulation.

We investigated whether the cysteine residues in Shaker potassium (K+) channels are essential for activation and inactivation gating or for modulation of activation gating by external zinc (Zn2+). Mutants of the Shaker K+ channel were prepared in which all seven cysteine residues were replaced (C-less). These changes were made in both wild-type Shaker H4 channels and in a deletion mutant (delta 6-46) lacking N-type ("fast") inactivation. Replacement of all cysteines left most functional properties of the K+ currents unaltered. The most noticeable difference between the C-less and wild-type currents was the faster C-type inactivation of the C-less channel which could be attributed largely to the mutation of Cys462. This is consistent with the effects of previously reported mutations of nearby residues in the S6 region. There were also small changes in the activation gating of C-less currents. Modulation by external Zn2+ of the voltage dependence and rate of activation gating is preserved in the C-less channels, indicating that none of the cysteines in the Shaker K+ channel plays an important role in Zn2+ modulation.

Animals↗