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.     

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.     

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.     

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.     

Control of ion selectivity in potassium channels by electrostatic and dynamic properties of carbonyl ligands

Potassium channels are essential for maintaining a normal ionic balance across cell membranes. Central to this function is the ability of such channels to support transmembrane ion conduction at nearly diffusion-limited rates while discriminating for K+ over Na+ by more than a thousand-fold. This selectivity arises because the transfer of the K+ ion into the channel pore is energetically favoured, a feature commonly attributed to a structurally precise fit between the K+ ion and carbonyl groups lining the rigid and narrow pore. But proteins are relatively flexible structures that undergo rapid thermal atomic fluctuations larger than the small difference in ionic radius between K+ and Na+. Here we present molecular dynamics simulations for the potassium channel KcsA, which show that the carbonyl groups coordinating the ion in the narrow pore are indeed very dynamic ('liquid-like') and that their intrinsic electrostatic properties control ion selectivity. This finding highlights the importance of the classical concept of field strength. Selectivity for K+ is seen to emerge as a robust feature of a flexible fluctuating pore lined by carbonyl groups.     

Voltage-sensing residues in the S4 region of a mammalian K+ channel

The ability of ion-channel proteins to respond to a change of the transmembrane voltage is one of the basic mechanisms underlying electrical excitability of nerve and muscle membranes. The voltage sensor has been postulated to be the fourth putative transmembrane segment (S4) of voltage-activated Na+, Ca2+ and K+ channels. Mutations of positively charged residues within S4 alter gating of Na and Shaker-type K+ channels, but quantitative correlations between the charge or a residue in S4 and the gating valence of the channel have not yet been established. Here, with improved resolution of the voltage dependence of steady-state activation, we present estimates of the equivalent gating valence with sufficient precision to allow quantitative examination of the contribution of individual charged residues to the gating valence of a mammalian non-inactivating K+ channel. We conclude that at least part of the gating charge associated with channel activation is indeed contributed by charged residues within the S4 segment.     

Ion permeation through the Na+,K+-ATPase

P-type ATPase pumps generate concentration gradients of cations across membranes in nearly all cells. They provide a polar transmembrane pathway, to which access is strictly controlled by coupled gates that are constrained to open alternately, thereby enabling thermodynamically uphill ion transport (for example, see ref. 1). Here we examine the ion pathway through the Na+,K+-ATPase, a representative P-type pump, after uncoupling its extra- and intracellular gates with the marine toxin palytoxin. We use small hydrophilic thiol-specific reagents as extracellular probes and we monitor their reactions, and the consequences, with cysteine residues introduced along the anticipated cation pathway through the pump. The distinct effects of differently charged reagents indicate that a wide outer vestibule penetrates deep into the Na+,K+-ATPase, where the pathway narrows and leads to a charge-selectivity filter. Acidic residues in this region, which are conserved to coordinate pumped ions, allow the approach of cations but exclude anions. Reversing the charge at just one of those positions converts the pathway from cation selective to anion selective. Close structural homology among the catalytic subunits of Ca2+-, Na+,K+- and H+,K+-ATPases argues that their extracytosolic cation exchange pathways all share these physical characteristics.     

Calcium channel characteristics conferred on the sodium channel by single mutations.

The sodium channel, one of the family of structurally homologous voltage-gated ion channels, differs from other members, such as the calcium and the potassium channels, in its high selectivity for Na+. This selectivity presumably reflects a distinct structure of its ion-conducting pore. We have recently identified two clusters of predominantly negatively charged amino-acid residues, located at equivalent positions in the four internal repeats of the sodium channel as the main determinants of sensitivity to the blockers tetrodotoxin and saxitoxin. All site-directed mutations reducing net negative charge at these positions also caused a marked decrease in single-channel conductance. Thus these two amino-acid clusters probably form part of the extracellular mouth and/or the pore wall of the sodium channel. We report here the effects on ion selectivity of replacing lysine at position 1,422 in repeat III and/or alanine at position 1,714 in repeat IV of rat sodium channel II (ref. 3), each located in one of the two clusters, by glutamic acid, which occurs at the equivalent positions in calcium channels. These amino-acid substitutions, unlike other substitutions in the adjacent regions, alter ion-selection properties of the sodium channel to resemble those of calcium channels. This result indicates that lysine 1,422 and alanine 1,714 are critical in determining the ion selectivity of the sodium channel, suggesting that these residues constitute part of the selectivity filter of the channel.     

Functional analysis of an archaebacterial voltage-dependent K+ channel

All living organisms use ion channels to regulate the transport of ions across cellular membranes. Certain ion channels are classed as voltage-dependent because they have a voltage-sensing structure that induces their pores to open in response to changes in the cell membrane voltage. Until recently, the voltage-dependent K+, Ca2+ and Na+ channels were regarded as a unique development of eukaryotic cells, adapted to accomplish specialized electrical signalling, as exemplified in neurons. Here we present the functional characterization of a voltage-dependent K+ (K(V)) channel from a hyperthermophilic archaebacterium from an oceanic thermal vent. This channel possesses all the functional attributes of classical neuronal K(V) channels. The conservation of function reflects structural conservation in the voltage sensor as revealed by specific, high-affinity interactions with tarantula venom toxins, which evolved to inhibit eukaryotic K(V) channels.     

Energetic optimization of ion conduction rate by the K+ selectivity filter.

The K+ selectivity filter catalyses the dehydration, transfer and rehydration of a K+ ion in about ten nanoseconds. This physical process is central to the production of electrical signals in biology. Here we show how nearly diffusion-limited rates are achieved, by analysing ion conduction and the corresponding crystallographic ion distribution in the selectivity filter of the KcsA K+ channel. Measurements with K+ and its slightly larger analogue, Rb+, lead us to conclude that the selectivity filter usually contains two K+ ions separated by one water molecule. The two ions move in a concerted fashion between two configurations, K+-water-K+-water (1,3 configuration) and water-K+-water-K+ (2,4 configuration), until a third ion enters, displacing the ion on the opposite side of the queue. For K+, the energy difference between the 1,3 and 2,4 configurations is close to zero, the condition of maximum conduction rate. The energetic balance between these configurations is a clear example of evolutionary optimization of protein function.     

Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution.

Ion transport proteins must remove an ion's hydration shell to coordinate the ion selectively on the basis of its size and charge. To discover how the K+ channel solves this fundamental aspect of ion conduction, we solved the structure of the KcsA K+ channel in complex with a monoclonal Fab antibody fragment at 2.0 A resolution. Here we show how the K+ channel displaces water molecules around an ion at its extracellular entryway, and how it holds a K+ ion in a square antiprism of water molecules in a cavity near its intracellular entryway. Carbonyl oxygen atoms within the selectivity filter form a very similar square antiprism around each K+ binding site, as if to mimic the waters of hydration. The selectivity filter changes its ion coordination structure in low K+ solutions. This structural change is crucial to the operation of the selectivity filter in the cellular context, where the K+ ion concentration near the selectivity filter varies in response to channel gating.     

Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel

Voltage-gated sodium (Na(v)) channels are essential for the rapid depolarization of nerve and muscle, and are important drug targets. Determination of the structures of Na(v) channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Na(v) channels, exemplified by the Na(+)-selective channel of bacteria (NaChBac), provides a useful model system for structure-function analysis. Here we report the crystal structure of Na(v)Rh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 (Rickettsiales sp. HIMB114; denoted Rh), at 3.05?? resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr?178 and Leu?179 constitute an inner site within the selectivity filter where a hydrated Ca(2+) resides in the crystal structure. The outer mouth of the Na(+) selectivity filter, defined by Ser?181 and Glu?183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that Na(v)Rh is in an 'inactivated' conformation. Comparison of Na(v)Rh with Na(v)Ab reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.     

Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin

Small-conductance Ca2+-activated K+ channels (SK channels) are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are heteromeric complexes that comprise pore-forming alpha-subunits and the Ca2+-binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM-binding domain (CaMBD), which is located in an intracellular region of the alpha-subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. Here we report the 1.60 A crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three alpha-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.     

Energetics of ion conduction through the K+ channel.

K+ channels are transmembrane proteins that are essential for the transmission of nerve impulses. The ability of these proteins to conduct K+ ions at levels near the limit of diffusion is traditionally described in terms of concerted mechanisms in which ion-channel attraction and ion-ion repulsion have compensating effects, as several ions are moving simultaneously in single file through the narrow pore. The efficiency of such a mechanism, however, relies on a delicate energy balance-the strong ion-channel attraction must be perfectly counterbalanced by the electrostatic ion-ion repulsion. To elucidate the mechanism of ion conduction at the atomic level, we performed molecular dynamics free energy simulations on the basis of the X-ray structure of the KcsA K+ channel. Here we find that ion conduction involves transitions between two main states, with two and three K+ ions occupying the selectivity filter, respectively; this process is reminiscent of the 'knock-on' mechanism proposed by Hodgkin and Keynes in 1955. The largest free energy barrier is on the order of 2-3 kcal mol-1, implying that the process of ion conduction is limited by diffusion. Ion-ion repulsion, although essential for rapid conduction, is shown to act only at very short distances. The calculations show also that the rapidly conducting pore is selective.     

Structural basis for modulation and agonist specificity of HCN pacemaker channels

The family of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are crucial for a range of electrical signalling, including cardiac and neuronal pacemaker activity, setting resting membrane electrical properties and dendritic integration. These nonselective cation channels, underlying the I(f), I(h) and I(q) currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by beta-adrenergic agonists. Here we have investigated the mechanism underlying this modulation by studying a carboxy-terminal fragment of HCN2 containing the cyclic nucleotide-binding domain (CNBD) and the C-linker region that connects the CNBD to the pore. X-ray crystallographic structures of this C-terminal fragment bound to cAMP or cGMP, together with equilibrium sedimentation analysis, identify a tetramerization domain and the mechanism for cyclic nucleotide specificity, and suggest a model for ligand-dependent channel modulation. On the basis of amino acid sequence similarity to HCN channels, the cyclic nucleotide-gated, and eag- and KAT1-related families of channels are probably related to HCN channels in structure and mechanism.     

Rotational movement during cyclic nucleotide-gated channel opening

Cyclic nucleotide-gated (CNG) channels are crucial components of visual, olfactory and gustatory signalling pathways. They open in response to direct binding of intracellular cyclic nucleotides and thus contribute to cellular control of both the membrane potential and intracellular Ca2+ levels. Cytosolic Ni2+ potentiates the rod channel (CNG1) response to cyclic nucleotides and inhibits the olfactory channel (CNG2) response. Modulation is due to coordination of Ni2+ by channel-specific histidines in the C-linker, between the S6 transmembrane segment and the cyclic nucleotide-binding domain. Here we report, using a histidine scan of the initial C-linker of the CNG1 channel, stripes of sites producing Ni2+ potentiation or Ni2+ inhibition, separated by 50 degrees on an alpha-helix. These results suggest a model for channel gating where rotation of the post-S6 region around the channel's central axis realigns the Ni2+-coordinating residues of multiple subunits. This rotation probably initiates movement of the S6 and pore opening.     

Molecular mechanism of cAMP modulation of HCN pacemaker channels.

Hyperpolarization-activated cation channels of the HCN gene family contribute to spontaneous rhythmic activity in both heart and brain. All four family members contain both a core transmembrane segment domain, homologous to the S1-S6 regions of voltage-gated K+ channels, and a carboxy-terminal 120 amino-acid cyclic nucleotide-binding domain (CNBD) motif. Homologous CNBDs are responsible for the direct activation of cyclic nucleotide-gated channels and for modulation of the HERG voltage-gated K+ channel--important for visual and olfactory signalling and for cardiac repolarization, respectively. The direct binding of cyclic AMP to the cytoplasmic site on HCN channels permits the channels to open more rapidly and completely after repolarization of the action potential, thereby accelerating rhythmogenesis. However, the mechanism by which cAMP binding modulates HCN channel gating and the basis for functional differences between HCN isoforms remain unknown. Here we demonstrate by constructing truncation mutants that the CNBD inhibits activation of the core transmembrane domain. cAMP binding relieves this inhibition. Differences in activation gating and extent of cAMP modulation between the HCN1 and HCN2 isoforms result largely from differences in the efficacy of CNBD inhibition.     

Structural basis of water-specific transport through the AQP1 water channel.

Water channels facilitate the rapid transport of water across cell membranes in response to osmotic gradients. These channels are believed to be involved in many physiological processes that include renal water conservation, neuro-homeostasis, digestion, regulation of body temperature and reproduction. Members of the water channel superfamily have been found in a range of cell types from bacteria to human. In mammals, there are currently 10 families of water channels, referred to as aquaporins (AQP): AQP0-AQP9. Here we report the structure of the aquaporin 1 (AQP1) water channel to 2.2 A resolution. The channel consists of three topological elements, an extracellular and a cytoplasmic vestibule connected by an extended narrow pore or selectivity filter. Within the selectivity filter, four bound waters are localized along three hydrophilic nodes, which punctuate an otherwise extremely hydrophobic pore segment. This unusual combination of a long hydrophobic pore and a minimal number of solute binding sites facilitates rapid water transport. Residues of the constriction region, in particular histidine 182, which is conserved among all known water-specific channels, are critical in establishing water specificity. Our analysis of the AQP1 pore also indicates that the transport of protons through this channel is highly energetically unfavourable.     

Gating pore current in an inherited ion channelopathy

Ion channelopathies are inherited diseases in which alterations in control of ion conductance through the central pore of ion channels impair cell function, leading to periodic paralysis, cardiac arrhythmia, renal failure, epilepsy, migraine and ataxia. Here we show that, in contrast with this well-established paradigm, three mutations in gating-charge-carrying arginine residues in an S4 segment that cause hypokalaemic periodic paralysis induce a hyperpolarization-activated cationic leak through the voltage sensor of the skeletal muscle Na(V)1.4 channel. This 'gating pore current' is active at the resting membrane potential and closed by depolarizations that activate the voltage sensor. It has similar permeability to Na+, K+ and Cs+, but the organic monovalent cations tetraethylammonium and N-methyl-D-glucamine are much less permeant. The inorganic divalent cations Ba2+, Ca2+ and Zn2+ are not detectably permeant and block the gating pore at millimolar concentrations. Our results reveal gating pore current in naturally occurring disease mutations of an ion channel and show a clear correlation between mutations that cause gating pore current and hypokalaemic periodic paralysis. This gain-of-function gating pore current would contribute in an important way to the dominantly inherited membrane depolarization, action potential failure, flaccid paralysis and cytopathology that are characteristic of hypokalaemic periodic paralysis. A survey of other ion channelopathies reveals numerous examples of mutations that would be expected to cause gating pore current, raising the possibility of a broader impact of gating pore current in ion channelopathies.     

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.