Heteromultimeric channels formed by rat brain potassium-channel proteins

An important step towards understanding the molecular basis of the functional diversity of voltage-gated K+ channels in the mammalian brain has been the discovery of a family of genes encoding rat brain K+ channel-forming (RCK) proteins. All species of these RCK proteins form homomultimeric voltage-gated K+ channels with distinct functional characteristics in Xenopus laevis oocytes following injection of the respective cRNAs. RCK-specific mRNAs are coexpressed in several regions of the brain, suggesting that RCK proteins also assemble into heteromultimeric K+ channels. In addition expression experiments with fractionated poly(A)+ mRNA have suggested that heteromultimeric K+ channels may occur in mammalian brain. We report here that heteromultimeric K+ channels composed of two different RCK proteins (RCK1 and RCK4) assemble after cotransfection of HeLa cells with the corresponding cDNAs and after coinjection of the corresponding cRNAs into Xenopus oocytes. The heteromultimeric RCK1, 4 channel mediates a transient potassium outward current, similar to the RCK4 channel but inactivates more slowly, has a larger conductance and is more sensitive to block by dendrotoxin and tetraethylammonium chloride.     

Determination of the subunit stoichiometry of a voltage-activated potassium channel.

The voltage-activated K+, Na+ and Ca2+ channels are responsible for the generation and propagation of electrical signals in cell membranes. The K+ channels are multimeric membrane proteins formed by the aggregation of an unknown number of independent subunits. By studying the interaction of a scorpion toxin with coexpressed wild-type and toxin-insensitive mutant Shaker K+ channels, the subunit stoichiometry can be determined. The Shaker K+ channel is found to have a tetrameric structure. This is consistent with the sequence relationship between a K+ channel and each of the four internally homologous repeats of Na+ and Ca2+ channels.     

Existence of distinct sodium channel messenger RNAs in rat brain

The sodium channel is a voltage-gated ionic channel essential for the generation of action potentials. It has been reported that the sodium channels purified from the electric organ of Electrophorus electricus (electric eel) and from chick cardiac muscle consist of a single polypeptide of relative molecular mass (Mr) approximately 260,000 (260K), whereas those purified from rat brain and skeletal muscle contain, in addition to the large polypeptide, two or three smaller polypeptides of Mr 37-45K. Recently, we have elucidated the primary structure of the Electrophorus sodium channel by cloning and sequencing the DNA complementary to its messenger RNA. Despite the apparent homogeneity of the purified sodium channel preparations, several types of tetrodotoxin (or saxitoxin) binding sites or sodium currents have been observed in many excitable membranes. The occurrence of distinguishable populations of sodium channels may be attributable to different states of the same channel protein or to distinct channel proteins. We have now isolated complementary DNA clones derived from two distinct rat brain mRNAs encoding sodium channel large polypeptides and present here the complete amino-acid sequences of the two polypeptides (designated sodium channels I and II), as deduced from the cDNA sequences. A partial DNA sequence complementary to a third homologous mRNA from rat brain has also been cloned.     

Intracellular gate opening in Shaker K+ channels defined by high-affinity metal bridges

Voltage-gated potassium channels such as Shaker help to control electrical signalling in neurons by regulating the passage of K+ across cell membranes. Ion flow is controlled by a voltage-dependent gate at the intracellular side of the pore, formed by the crossing of four alpha-helices--the inner-pore helices. The prevailing model of gating is based on a comparison of the crystal structures of two bacterial channels--KcsA in a closed state and MthK in an open state--and proposes a hinge motion at a conserved glycine that splays the inner-pore helices wide open. We show here that two types of intersubunit metal bridge, involving cysteines placed near the bundle crossing, can occur simultaneously in the open state. These bridges provide constraints on the open Shaker channel structure, and on the degree of movement upon opening. We conclude that, unlike predictions from the structure of MthK, the inner-pore helices of Shaker probably maintain the KcsA-like bundle-crossing motif in the open state, with a bend in this region at the conserved proline motif (Pro-X-Pro) not found in the bacterial channels. A narrower opening of the bundle crossing in Shaker K+ channels may help to explain why Shaker has an approximately tenfold lower conductance than its bacterial relatives.     

Putative receptor for the cytoplasmic inactivation gate in the Shaker K+ channel

Inactivation of ion channels is important in the control of membrane excitability. For example, delayed-rectifier K+ channels, which regulate action potential repolarization, are inactivated only slowly, whereas A-type K+ channels, which affect action potential duration and firing frequency, have both fast and slow inactivation. Fast inactivation of Na+ and K+ channels may result from the blocking of the permeation pathway by a positively charged cytoplasmic gate such as the one encoded by the first 20 amino acids of the Shaker B (ShB) K+ channel. We report here that mutation of five highly conserved residues between the proposed membrane-spanning segments S4 and S5 (also termed H4) of ShB affects the stability of the inactivated state and alters channel conductance. One such mutation stabilizes the inactivated state of ShB as well as the inactivated state induced in the delayed-rectifier type K+ channel drk1 by the cytoplasmic application of the ShB N-terminal peptide. The S4-S5 loop, therefore, probably forms part of a receptor for the inactivation gate and lies near the channel's permeation pathway.     

The contribution of Shaker K+ channels to the information capacity of Drosophila photoreceptors

An array of rapidly inactivating voltage-gated K+ channels is distributed throughout the nervous systems of vertebrates and invertebrates. Although these channels are thought to regulate the excitability of neurons by attenuating voltage signals, their specific functions are often poorly understood. We studied the role of the prototypical inactivating K+ conductance, Shaker, in Drosophila photoreceptors by recording intracellularly from wild-type and Shaker mutant photoreceptors. Here we show that loss of the Shaker K+ conductance produces a marked reduction in the signal-to-noise ratio of photoreceptors, generating a 50% decrease in the information capacity of these cells in fully light-adapted conditions. By combining experiments with modelling, we show that the inactivation of Shaker K+ channels amplifies voltage signals and enables photoreceptors to use their voltage range more effectively. Loss of the Shaker conductance attenuated the voltage signal and induced a compensatory decrease in impedance. Our results demonstrate the importance of the Shaker K+ conductance for neural coding precision and as a mechanism for selectively amplifying graded signals in neurons, and highlight the effect of compensatory mechanisms on neuronal information processing.     

Alteration of ionic selectivity of a K+ channel by mutation of the H5 region

The high ionic selectivity of K+ channels is a unifying feature of this diverse class of membrane proteins. Though K+ channels differ widely in regulation and kinetics, physiological studies have suggested a common structure: a single file pore containing multiple ion-binding sites and having broader vestibules at both ends. We have used site-directed mutagenesis and single-channel recordings to identify a molecular region that influences ionic selectivity in a cloned A-type K+ channel from Drosophila. Single amino-acid substitutions in H5, the fifth hydrophobic region, enhanced the passage of NH4+ and Rb+, ions with diameters larger than K+, without compromising the ability of the channel to exclude the smaller cation, Na+. The mutations that substantially altered selectivity had little effect on the gating properties of the channel. We conclude that the H5 region is likely to line the pore of the K+ channel.     

Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement

Voltage-gated ion channels are responsible for generating electrical impulses in nerves and other excitable cells. The fourth transmembrane helix (S4) in voltage-gated channels is the primary voltage-sensing unit that mediates the response to a changing membrane electric field. The molecular mechanism of voltage sensing, particularly with respect to the magnitude of the transmembrane movement of S4, remains controversial. To determine the extent of this transmembrane movement, we use fluorescent resonance energy transfer between the S4 domain and a reference point in the lipid bilayer. The lipophilic ion dipicrylamine distributes on either side of the lipid bilayer depending on the membrane potential, and is used here as a resonance-energy-transfer acceptor from donor molecules attached to several positions in the Shaker K+ channel. A voltage-driven transmembrane movement of the donor should produce a transient fluorescence change because the acceptor also translocates as a function of voltage. In Shaker K+ channels no such transient fluorescence is observed, indicating that the S4 segment does not translocate across the lipid bilayer. Based on these observations, we propose a molecular model of voltage gating that can account for the observed 13e gating charge with limited transmembrane S4 movement.     

Stereospecific binding of a disordered peptide segment mediates BK channel inactivation

A number of functionally important actions of proteins are mediated by short, intrinsically disordered peptide segments, but the molecular interactions that allow disordered domains to mediate their effects remain a topic of active investigation. Many K+ channel proteins, after initial channel opening, show a time-dependent reduction in current flux, termed 'inactivation', which involves movement of mobile cytosolic peptide segments (approximately 20-30 residues) into a position that physically occludes ion permeation. Peptide segments that produce inactivation show little amino-acid identity and tolerate appreciable mutational substitutions without disrupting the inactivation process. Solution nuclear magnetic resonance of several isolated inactivation domains reveals substantial conformational heterogeneity with only minimal tendency to ordered structures. Channel inactivation mechanisms may therefore help us to decipher how intrinsically disordered regions mediate functional effects. Whereas many aspects of inactivation of voltage-dependent K+ channels (Kv) can be described by a simple one-step occlusion mechanism, inactivation of the voltage-dependent large-conductance Ca2+-gated K+ (BK) channel mediated by peptide segments of auxiliary β-subunits involves two distinguishable kinetic steps. Here we show that two-step inactivation mediated by an intrinsically disordered BK β-subunit peptide involves a stereospecific binding interaction that precedes blockade. In contrast, blocking mediated by a Shaker Kv inactivation peptide is consistent with direct, simple occlusion by a hydrophobic segment without substantial steric requirement. The results indicate that two distinct types of molecular interaction between disordered peptide segments and their binding sites produce qualitatively similar functions.     

Cloning of a probable potassium channel gene from mouse brain

Potassium channels comprise a diverse class of ion channels important for neuronal excitability and plasticity. The recent cloning of the Shaker locus from Drosophila melanogaster has provided a starting point for molecular studies of potassium channels. Predicted Shaker proteins appear to be integral membrane proteins and have a sequence similar to the sequence of the S4 segment of the vertebrate sodium channel, where the S4 segment has been proposed to be the voltage sensor. Expression studies in frog oocytes confirm that Shaker encodes a component of a potassium channel (the A channel) that conducts a fast transient potassium current. Here we report the isolation of complementary DNA clones from the mouse brain, the nucleotide sequences of which predict a protein remarkably similar to the Shaker protein. The strong conservation of the predicted protein sequence in flies and mammals suggests that these mouse clones encode a potassium channel component and that the conserved amino acids may be essential to some aspect of potassium channel function.     

Enzymatic activation of voltage-gated potassium channels

Voltage-gated ion channels in excitable nerve, muscle, and endocrine cells generate electric signals in the form of action potentials. However, they are also present in non-excitable eukaryotic cells and prokaryotes, which raises the question of whether voltage-gated channels might be activated by means other than changing the voltage difference between the solutions separated by the plasma membrane. The search for so-called voltage-gated channel activators is motivated in part by the growing importance of such agents in clinical pharmacology. Here we report the apparent activation of voltage-gated K+ (Kv) channels by a sphingomyelinase.     

Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors

Many voltage-dependent K+ channels open when the membrane is depolarized and then rapidly close by a process called inactivation. Neurons use inactivating K+ channels to modulate their firing frequency. In Shaker-type K+ channels, the inactivation gate, which is responsible for the closing of the channel, is formed by the channel's cytoplasmic amino terminus. Here we show that the central cavity and inner pore of the K+ channel form the receptor site for both the inactivation gate and small-molecule inhibitors. We propose that inactivation occurs by a sequential reaction in which the gate binds initially to the cytoplasmic channel surface and then enters the pore as an extended peptide. This mechanism accounts for the functional properties of K+ channel inactivation and indicates that the cavity may be the site of action for certain drugs that alter cation channel function.     

External pH regulates the inward K+ channels in Brassica pollen protoplasts

Patch-clamp whole-cell recording techniques were used to investigate the regulation of the inward K* channels by external pH in Brassica pollen protoplasts. The inward K+ currents, conductance and voltage-independent maximum conductance were not significantly affected when the external pH was changed from 5.8 to 4.5. However, compared with the results obtained at pH 5.8, the inward K+ currents and conductance were significantly inhibited at pH 8 .5 . The activation kinetics of the inward K * channels was also significantly regulated by external pH. The channel activation time was shorter at the lower external pH than that at higher pH. Since external pH is a critical factor to regulate pollen germination, the results suggest that external pH regulation of the inward K+ channels may be one of potential physiological mechanisms for the regulation of pollen germination.     

Detection of K+ and Cl-channels from calf cardiac sarcolemma in planar lipid bilayer membranes

The ionic currents underlying the cardiac action potential are believed to be much more complex than those in nerve. During the cardiac action potential, various membrane channels control the flow of K+, Na+, Ca2+ and Cl- across the sarcolemma of cardiac muscle cells. Thus, it has become increasingly clear that a detailed knowledge of the mechanisms that activate (or inactivate) heart channels is required to understand cardiac excitability. We report here the use of planar lipid bilayer techniques to detect and characterize K+ and Cl- channels in purified heart sarcolemma membrane vesicles. We have identified four different types of channel on the basis of their selectivity, conductance and gating kinetics. We present in some detail the properties of a K+ channel and a Cl- channel. We have tentatively identified the K+ channel with the ix type of current found in Purkinje, myocardial ventricular and atrial fibres. The chloride channel might be related to the transient chloride current found in Purkinje fibres.     

Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy

Voltage-gated ion channels are transmembrane proteins that are essential for nerve impulses and regulate ion flow across cell membranes in response to changes in membrane potential. They are made up of four homologous domains or subunits, each of which contains six transmembrane segments. Studies of potassium channels have shown that the second (S2) and fourth (S4) segments contain several charged residues, which sense changes in voltage and form part of the voltage sensor. Although these regions clearly undergo conformational changes in response to voltage, little is known about the nature of these changes because voltage-dependent distance changes have not been measured. Here we use lanthanide-based resonance energy transfer to measure distances between Shaker potassium channel subunits at specific residues. Voltage-dependent distance changes of up to 3.2 A were measured at several sites near the S4 segment. These movements directly correlated with electrical measurements of the voltage sensor, establishing the link between physical changes and electrical charge movement. Measured distance changes suggest that the region associated with the S4 segment undergoes a rotation and possible tilt, rather than a large transmembrane movement, in response to voltage. These results demonstrate the first in situ measurement of atomic scale movement in a trans-membrane protein.     

Neuropeptide modulation of single calcium and potassium channels detected with a new patch clamp configuration

Calcium channel activity is crucial for secretion and synaptic transmission, but it has been difficult to study Ca2+ channel modulation because survival and regulation of some of these channels require cytoplasmic constituents that are lost with the formation of cell-free patches. Here we report a new patch clamp configuration in which activity and regulation of channels are maintained after removal from cells. A pipette containing the pore-forming agent nystatin is sealed onto a cell and withdrawn to form an enclosed vesicle. The resulting perforated vesicle, formed from pituitary tumour cells, contains Ca2+ and K+ channels. Ca2(+)-activated K+ channels in the vesicle are activated by cyclic AMP analogues, and by a neuropeptide (thyrotropin-releasing hormone) that stimulates phosphatidylinositol turnover and inositol trisphosphate-gated Ca2+ release from intracellular organelles. Thus, the perforated vesicle retains signal transduction systems necessary for ion channel modulation. Functional dihydropyridine-sensitive Ca2+ channels (L-type) are maintained in the vesicle, and their gating is inhibited by thyrotropin-releasing hormone. Hence, this new patch clamp configuration has allowed a direct detection of the single-channel basis of transmitter-induced inhibition of L-type Ca2+ channels. The modulation of Ca2(+)-channel gating may be an important mechanism for regulating hormone secretion from pituitary cells.     

Atomic structure of a Na+- and K+-conducting channel

Ion selectivity is one of the basic properties that define an ion channel. Most tetrameric cation channels, which include the K+, Ca2+, Na+ and cyclic nucleotide-gated channels, probably share a similar overall architecture in their ion-conduction pore, but the structural details that determine ion selection are different. Although K+ channel selectivity has been well studied from a structural perspective, little is known about the structure of other cation channels. Here we present crystal structures of the NaK channel from Bacillus cereus, a non-selective tetrameric cation channel, in its Na+- and K+-bound states at 2.4 A and 2.8 A resolution, respectively. The NaK channel shares high sequence homology and a similar overall structure with the bacterial KcsA K+ channel, but its selectivity filter adopts a different architecture. Unlike a K+ channel selectivity filter, which contains four equivalent K+-binding sites, the selectivity filter of the NaK channel preserves the two cation-binding sites equivalent to sites 3 and 4 of a K+ channel, whereas the region corresponding to sites 1 and 2 of a K+ channel becomes a vestibule in which ions can diffuse but not bind specifically. Functional analysis using an 86Rb flux assay shows that the NaK channel can conduct both Na+ and K+ ions. We conclude that the sequence of the NaK selectivity filter resembles that of a cyclic nucleotide-gated channel and its structure may represent that of a cyclic nucleotide-gated channel pore.     

X-ray structure of a voltage-dependent K+ channel

Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.