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.

Mutations affecting TEA blockade and ion permeation in voltage-activated K+ channels.

Voltage-dependent ion channels are responsible for electrical signaling in neurons and other cells. The main classes of voltage-dependent channels (sodium-, calcium-, and potassium-selective channels) have closely related molecular structures. For one member of this superfamily, the transiently voltage-activated Shaker H4 potassium channel, specific amino acid residues have now been identified that affect channel blockade by the small ion tetraethylammonium, as well as the conduction of ions through the pore. Furthermore, variation at one of these amino acid positions among naturally occurring potassium channels may account for most of their differences in sensitivity to tetraethylammonium.

Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel.

The active site of voltage-activated potassium channels is a transmembrane aqueous pore that permits ions to permeate the cell membrane in a rapid yet highly selective manner. A useful probe for the pore of potassium-selective channels is the organic ion tetraethylammonium (TEA), which binds with millimolar affinity to the intracellular opening of the pore and blocks potassium current. In the potassium channel encoded by the Drosophila Shaker gene, an amino acid residue that specifically affects the affinity for intracellular TEA has now been identified by site-directed mutagenesis. This residue is in the middle of a conserved stretch of 18 amino acids that separates two locations that are both near the external opening of the pore. These findings suggest that this conserved region is intimately involved in the formation of the ion conduction pore of voltage-activated potassium channels. Further, a stretch of only eight amino acid residues must traverse 80 percent of the transmembrane electric potential difference.

Tight steric closure at the intracellular activation gate of a voltage-gated K(+) channel.

In voltage-gated K(+) channels (Kv), an intracellular gate regulates access from the cytoplasm to the pore by organic channel blockers and by chemical modifiers. But is ion flow itself controlled instead by constriction of the narrow selectivity filter near the extracellular surface? We find that the intracellular gate of Kv channels is capable of regulating access even by the small cations Cd(2+) and Ag(+). It can also exclude small neutral or negatively charged molecules, indicating that the gate operates by steric exclusion rather than electrostatically. Just intracellular to the gated region, channel closure does not restrict access even to very large reagents. Either these Kv channels have a broader inner entrance than seen in the KcsA crystal, even in the closed state, or the region is highly flexible (but nevertheless remains very securely closed nearby).

The inactivation gate of the Shaker K+ channel behaves like an open-channel blocker.

Following voltage-dependent activation, Drosophila Shaker K+ channels enter a nonconducting, inactivated state. This process has been proposed to occur by a "ball-and-chain" mechanism, in which the N-terminus of the protein behaves like a blocker tethered to the cytoplasmic side of the channel and directly occludes the pore to cause inactivation. To complement the ample evidence for the involvement of the N-terminus, we sought evidence that it blocks the pore directly. We found that inactivation exhibits several distinctive properties of pore blockade. First, recovery was speeded by increased external K+ concentrations, just as blockade can be relieved by trans-permeant ions. Second, single-channel experiments show that the channel reopens from the inactivated state upon repolarization. These openings were usually required for recovery, as though the blocking particle must exit the pore before the channel can close.

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.

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.

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.

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.

The internal quaternary ammonium receptor site of Shaker potassium channels.

Quaternary ammonium (QA) compounds inhibit K+ conductance by entering and occluding the open pore of voltage-activated K+ channels. We characterized the effects of a series of alkyl-triethylammonium blockers on the Shaker K+ channel and tested them on a series of site-directed mutants of the channel protein in order to define the structural features of the binding sites. We found that mutations in two regions of the channel protein, the pore (P) region and the last transmembrane sequence (S6), appear to alter QA binding, not through their effects on gating but perhaps through direct effects on the binding site. Several mutations in the P region affect tetraethylammonium binding but have minimal effects on longer blockers, suggesting that the hydrophobic tail contributes to binding in a nonadditive fashion. Binding of the longer blockers can be affected by varying the hydrophobicity of 1 residue within S6 by site-specific substitution, in a manner consistent with a direct hydrophobic interaction between the side chain at this site and the alkyl chains of the blocker.

A novel K+ channel with unique localizations in mammalian brain: molecular cloning and characterization.

Using a cDNA library prepared from circumvallate papillae of rat tongue, we have identified, cloned, and sequenced a novel K+ channel, designated cdrk. The cdrk channel appears to be a member of the Shab subfamily, most closely resembling drk1. Electrophysiologic analysis of expressed cdrk channels reveals delayed rectifier properties similar to those of drk1 channels. Localizations of cdrk mRNA in rat brain and peripheral tissues, assessed by in situ hybridization and Northern blot analysis, differ from any other reported K+ channels. In the brain cdrk mRNA is most concentrated in granule cells of the olfactory bulb and cerebellum. In peripheral tissues, mRNAs for cdrk and drk1 are reciprocally localized, indicating that the K+ channel properties contributed by mammalian Shab homologs may be important in a variety of excitable tissues.

Sodium channels from human brain RNA expressed in Xenopus oocytes. Basic electrophysiologic characteristics and their modification by diphenylhydantoin.

We describe the expression and characterization of sodium channels from human brain RNA in the Xenopus oocyte. The expressed channel, studied by whole-cell voltage clamp, reveals characteristic selectivity for sodium as the permeant ion, voltage-dependent gating, and block by nanomolar concentrations of tetrodotoxin. Such channels are not seen in control oocytes injected with solvent only. The anticonvulsant diphenylhydantoin (DPH) inhibits the expressed channel in a voltage- and use-dependent manner, much like the effect seen in primary mammalian neuronal preparations. The inhibition of the expressed human sodium channel by DPH can be described by models previously developed to explain block of Na channels by local anesthetics. The preferential block of Na channels during depolarization helps explain the selectivity of DPH for neurons involved in seizure activity.

The bacterial K+ channel structure and its implications for neuronal channels.

Voltage-gated K+ (Kv) channels play a central role in generating action potentials and rhythmic patterns, as well as in dendritic signal processing in neurons. Recently, the first structure of a member of the K+ channel family was solved. Although this channel is from bacteria and has a streamlined body plan with no voltage gating, it establishes the architecture of the functional core of the voltage-gated (K+) channels and their relatives. This architecture explains the crucial features of ion permeation and blockade, and gives some strong hints about gating. The bacterial K+ channel structure is the central piece in a puzzle; it remains to be seen how it will fit together with other domains of the Kv channels, with auxiliary subunits, and with other signal transduction molecules.

Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells.

Single channel currents through Ca2+-activated K+ channels of bovine chromaffin cells were measured to determine the effects of small ions on permeation through the channel. The channel selects strongly for K+ over Na+ and Cs+, and Rb+ carries a smaller current through the channel than K+. Tetraethylammonium ion (TEA+) blocks channel currents when applied to either side of the membrane; it is effective at lower concentrations when applied externally. Millimolar concentrations of internal Na+ reduce the average current through the channel and produce large fluctuations (flicker) in the open channel currents. This flickery block is analyzed by a new method, amplitude distribution analysis, which can measure block and unblock rates in the microsecond time range even though individual blocking events are not time-resolved by the recording system. The analysis shows that the rate of block by Na+ is very voltage dependent, but the unblock rate is voltage independent. These results can be explained easily by supposing that current flow through the channel is diffusion limited, a hypothesis consistent with the large magnitude of the single channel current.

Relief of Na+ block of Ca2+-activated K+ channels by external cations.

The flickery block of single Ca2+-activated K+ channels that is produced by internally applied Na+ can be relieved by millimolar concentrations of external K+. This effect of K+ on the kinetics of Na+ block was studied by the method of amplitude distribution analysis described in the companion paper (Yellen, G., 1984b, J. Gen. Physiol., 84:157-186). It appears that K+ relieves block by increasing the exit rate of the blocking ion from the channel, not by competitively slowing its entrance rate. This suggests that a K ion that enters the channel from the outside can expel the blocking Na ion, which entered the channel from the inside. Cs+, which cannot carry current through the channel, and Rb+, which carries a reduced current through the channel, are just as effective as K+ in relieving the block by internal Na+. The kinetics of block by internal nonyltriethylammonium (C9) are unaffected by the presence of these ions in the external bathing solution.

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.

Blocker state dependence and trapping in hyperpolarization-activated cation channels: evidence for an intracellular activation gate.

Hyperpolarization-activated cation currents (I(h)) are key determinants of repetitive electrical activity in heart and nerve cells. The bradycardic agent ZD7288 is a selective blocker of these currents. We studied the mechanism for ZD7288 blockade of cloned I(h) channels in excised inside-out patches. ZD7288 blockade of the mammalian mHCN1 channel appeared to require opening of the channel, but strong hyperpolarization disfavored blockade. The steepness of this voltage-dependent effect (an apparent valence of approximately 4) makes it unlikely to arise solely from a direct effect of voltage on blocker binding. Instead, it probably indicates a differential affinity of the blocker for different channel conformations. Similar properties were seen for ZD7288 blockade of the sea urchin homologue of I(h) channels (SPIH), but some of the blockade was irreversible. To explore the molecular basis for the difference in reversibility, we constructed chimeric channels from mHCN1 and SPIH and localized the structural determinant for the reversibility to three residues in the S6 region likely to line the pore. Using a triple point mutant in S6, we also revealed the trapping of ZD7288 by the closing of the channel. Overall, the observations led us to hypothesize that the residues responsible for ZD7288 block of I(h) channels are located in the pore lining, and are guarded by an intracellular activation gate of the channel.

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.

Expression of Torpedo nicotinic acetylcholine receptor subunits in yeast is enhanced by use of yeast signal sequences.

We have produced the four subunits of the nicotinic acetylcholine receptor of Torpedo californica, an integral membrane protein, in the yeast Saccharomyces cerevisiae. Two of the subunits (alpha and delta) were readily produced from their cDNAs after simply subcloning them into a yeast shuttle vector adjacent to a yeast promoter. The other two protein subunits (beta and gamma) were not produced by this strategy, although the amounts of mRNA produced from these expression constructs are similar to those for alpha and delta. Replacing the DNA coding for the normal N-terminal signal sequences for the beta and gamma subunits with DNA coding for the signal sequence of yeast invertase results in successful protein synthesis. The yeast signal sequence allows these subunits to be translocated across the membrane of the endoplasmic reticulum and to be glycosylated. The appropriate final size of the subunit proteins suggests that the yeast signal sequence has been properly cleaved after translocation.