Subunit composition determines Kv1 potassium channel surface expression

Shaker-related or Kv1 voltage-gated K(+) channels play critical roles in regulating the excitability of mammalian neurons. Native Kv1 channel complexes are octamers of four integral membrane alpha subunits and four cytoplasmic beta subunits, such that a tremendous diversity of channel complexes can be assembled from the array of alpha and beta subunits expressed in the brain. However, biochemical and immunohistochemical studies have demonstrated that only certain complexes predominate in the mammalian brain, suggesting that regulatory mechanisms exist that ensure plasma membrane targeting of only physiologically appropriate channel complexes. Here we show that Kv1 channels assembled as homo- or heterotetrameric complexes had distinct surface expression characteristics in both transfected mammalian cells and hippocampal neurons. Homotetrameric Kv1.1 channels were localized to endoplasmic reticulum, Kv1.4 channels to the cell surface, and Kv1.2 channels to both endoplasmic reticulum and the cell surface. Heteromeric assembly with Kv1.4 resulted in dose-dependent increases in cell surface expression of coassembled Kv1.1 and Kv1.2, while coassembly with Kv1.1 had a dominant-negative effect on Kv1.2 and Kv1.4 surface expression. Coassembly with Kv beta subunits promoted cell surface expression of each Kv1 heteromeric complex. These data suggest that subunit composition and stoichiometry determine surface expression characteristics of Kv1 channels in excitable cells.     

Modulation of Kv4-encoded K(+) currents in the mammalian myocardium by neuronal calcium sensor-1

Voltage-gated K(+) channels are multimeric proteins, consisting of four pore-forming alpha-subunits alone or in association with accessory subunits. Recently, for example, it was shown that the accessory Kv channel interacting proteins form complexes with Kv4 alpha-subunits and modulate Kv4 channel activity. The experiments reported here demonstrate that the neuronal calcium sensor protein-1 (NCS-1), another member of the recoverin-neuronal calcium sensor superfamily, is expressed in adult mouse ventricles and that NCS-1 co-immunoprecipitates with Kv4.3 from (adult mouse) ventricular extracts. In addition, co-expression studies in HEK-293 cells reveal that NCS-1 increases membrane expression of Kv4 alpha-subunits and functional Kv4-encoded K(+) current densities. Co-expression of NCS-1 also decreases the rate of inactivation of Kv4 alpha-subunit-encoded K(+) currents. In contrast to the pronounced effects of Kv channel interacting proteins on Kv4 channel gating, however, NCS-1 co-expression does not measurably affect the voltage dependence of steady-state inactivation or the rate of recovery from inactivation of Kv4-encoded K(+) currents. Taken together, these results suggest that NCS-1 is an accessory subunit of Kv4-encoded I(to,f) channels that functions to regulate I(to,f) density in the mammalian myocardium.     

Structural compatibility between the putative voltage sensor of voltage-gated K+ channels and the prokaryotic KcsA channel

Sequence similarity among and electrophysiological studies of known potassium channels, along with the three-dimensional structure of the Streptomyces lividans K(+) channel (KcsA), support the tenet that voltage-gated K(+) channels (Kv channels) consist of two distinct modules: the "voltage sensor" module comprising the N-terminal portion of the channel up to and including the S4 transmembrane segment and the "pore" module encompassing the C-terminal portion from the S5 transmembrane segment onward. To substantiate this modular design, we investigated whether the pore module of Kv channels may be replaced with the pore module of the prokaryotic KcsA channel. Biochemical and immunocytochemical studies showed that chimeric channels were expressed on the cell surface of Xenopus oocytes, demonstrating that they were properly synthesized, glycosylated, folded, assembled, and delivered to the plasma membrane. Unexpectedly, surface-expressed homomeric chimeras did not exhibit detectable voltage-dependent channel activity upon both hyperpolarization and depolarization regardless of the expression system used. Chimeras were, however, strongly dominant-negative when coexpressed with wild-type Kv channels, as evidenced by the complete suppression of wild-type channel activity. Notably, the dominant-negative phenotype correlated well with the formation of stable, glycosylated, nonfunctional, heteromeric channels. Collectively, these findings imply a structural compatibility between the prokaryotic pore module and the eukaryotic voltage sensor domain that leads to the biogenesis of non-responsive channels. Our results lend support to the notion that voltage-dependent channel gating depends on the precise coupling between both protein domains, probably through a localized interaction surface.     

A family of K+ channel ancillary subunits regulate taste sensitivity in Caenorhabditis elegans

We have identified a family of ancillary subunits of K(+) channels in Caenorhabditis elegans. MPS-1 and its related members MPS-2, MPS-3, and MPS-4 are detected in the nervous system of the nematode. Electrophysiological analysis in ASE neurons and mammalian cells and epigenetic inactivation by double-stranded RNA interference (RNAi) in vivo show that each MPS can associate with and functionally endow the voltage-gated K(+) channel KVS-1. In the chemosensory neuron ADF, three different MPS subunits combine with KVS-1 to form both binary (MPS-1.KVS-1) and ternary (MPS-2.MPS-3.KVS-1) complexes. RNAi of mps-2, mps-3, or both, enhance the taste of the animal for sodium without altering the susceptibility to other attractants. When sodium is introduced in the test plate as background or as antagonist attractant, the nematode loses the ability to recognize a second attractant. Thus, it appears that the chemosensory apparatus of C. elegans uses sensory thresholds and that a voltage-gated K(+) channel is specifically required for this mechanism.     

Multiple Kv channel-interacting proteins contain an N-terminal transmembrane domain that regulates Kv4 channel trafficking and gating

Kv channel-interacting proteins (KChIPs) are auxiliary subunits of the heteromultimeric channel complexes that underlie neuronal I(SA), the subthreshold transient K(+) current that dynamically regulates membrane excitability, action potential firing properties, and long term potentiation. KChIPs form cytoplasmic associations with the principal pore-forming Kv4 subunits and typically mediate enhanced surface expression and accelerated recovery from depolarization-induced inactivation. An exception is KChIP4a, which dramatically suppresses Kv4 inactivation while promoting neither surface expression nor recovery. These unusual properties are attributed to the effects of a K channel inactivation suppressor domain (KISD) encoded within the variable N terminus of KChIP4a. Here, we have functionally and biochemically characterized two brain KChIP isoforms, KChIP2x and KChIP3x (also known as KChIP3b) and show that they also contain a functional KISD. Like KChIP4a and in contrast with non-KISD-containing KChIPs, both KChIP2x and KChIP3x strongly suppress inactivation and slow activation and inhibit the typical increases in surface expression of Kv4.2 channels. We then examined the properties of the KISD to determine potential mechanisms for its action. Subcellular fractionation shows that KChIP4a, KChIP2x, and KChIP3x are highly associated with the membrane fraction. Fluorescent confocal imaging of enhanced green fluorescent proteins (eGFP) N-terminally fused with KISD in HEK293T cells indicates that KISDs of KChIP4a, KChIP2x, and KChIP3x all autonomously target eGFP to intracellular membranes. Cell surface biotinylation experiments on KChIP4a indicate that the N terminus is exposed extracellularly, consistent with a transmembrane KISD. In summary, KChIP4a, KChIP2x, and KChIP3x comprise a novel class of KChIP isoforms characterized by an unusual transmembrane domain at their N termini that modulates Kv4 channel gating and trafficking.     

An arrhythmia susceptibility gene in Caenorhabditis elegans

kcne are evolutionarily conserved genes that encode accessory subunits of voltage-gated K(+) (Kv) channels. Missense mutations in kcne1, kcne2, and kcne3 are linked to congenital and acquired channelopathies in Homo sapiens. Here we show an unique example of conservation of kcne activities at genetic, physiological, functional, and pathophysiological level in Caenorhabditis elegans. Thus, mps-4 is the homologue of kcne1 that operates in human heart and inner ear. Like its KCNE relatives, MPS-4 assembles with a Kv channel, EXP-2, to form a complex that controls pharyngeal muscle contractility. MPS-4 modulates EXP-2 function in a similar fashion as KCNE proteins endow human channels. When defective, MPS-4, can induce abnormal repolarization by mechanisms that resemble the way KCNE proteins are thought to provoke arrhythmia in human heart. Mutation of a conserved aspartate residue associated with human disease (MPS-4-D74N) alters the functional attributes of the C. elegans current. Taken together these data underscore a significant conservation of KCNE activities in different pumps. This implies that C. elegans can develop into a system to study the molecular and genetic basis of KCNE-mediated muscle contractility and disease states.     

MinK, MiRP1, and MiRP2 diversify Kv3.1 and Kv3.2 potassium channel gating

High frequency firing in mammalian neurons requires ultra-rapid delayed rectifier potassium currents generated by homomeric or heteromeric assemblies of Kv3.1 and Kv3.2 potassium channel alpha subunits. Kv3.1 alpha subunits can also form slower activating channels by coassembling with MinK-related peptide 2 (MiRP2), a single transmembrane domain potassium channel ancillary subunit. Here, using channel subunits cloned from rat and expressed in Chinese hamster ovary cells, we show that modulation by MinK, MiRP1, and MiRP2 is a general mechanism for slowing of Kv3.1 and Kv3.2 channel activation and deactivation and acceleration of inactivation, creating a functionally diverse range of channel complexes. MiRP1 also negatively shifts the voltage dependence of Kv3.1 and Kv3.2 channel activation. Furthermore, MinK, MiRP1, and MiRP2 each form channels with Kv3.1-Kv3.2 heteromers that are kinetically distinct from one another and from MiRP/homomeric Kv3 channels. The findings illustrate a mechanism for dynamic expansion of the functional repertoire of Kv3.1 and Kv3.2 potassium currents and suggest roles for these alpha subunits outside the scope of sustained rapid neuronal firing.     

Amino-terminal determinants of U-type inactivation of voltage-gated K+ channels

The T1 domain is a cytosolic NH2-terminal domain present in all Kv (voltage-dependent potassium) channels, and is highly conserved between Kv channel subfamilies. Our characterization of a truncated form of Kv1.5 (Kv1.5deltaN209) expressed in myocardium demonstrated that deletion of the NH2 terminus of Kv1.5 imparts a U-shaped inactivation-voltage relationship to the channel, and prompted us to investigate the NH2 terminus as a regulatory site for slow inactivation of Kv channels. We examined the macroscopic inactivation properties of several NH2-terminal deletion mutants of Kv1.5 expressed in HEK 293 cells, demonstrating that deletion of residues up to the T1 boundary (Kv1.5deltaN19, Kv1.5deltaN91, and Kv1.5deltaN119) did not alter Kv1.5 inactivation, however, deletion mutants that disrupted the T1 structure consistently exhibited inactivation phenotypes resembling Kv1.5deltaN209. Chimeric constructs between Kv1.5 and the NH2 termini of Kv1.1 and Kv1.3 preserved the inactivation kinetics observed in full-length Kv1.5, again suggesting that the Kv1 T1 domain influences slow inactivation. Furthermore, disruption of intersubunit T1 contacts by mutation of residues Glu(131) and Thr(132) to alanines resulted in channels exhibiting a U-shaped inactivation-voltage relationship. Fusion of the NH2 terminus of Kv2.1 to the transmembrane segments of Kv1.5 imparted a U-shaped inactivation-voltage relationship to Kv1.5, whereas fusion of the NH2 terminus of Kv1.5 to the transmembrane core of Kv2.1 decelerated Kv2.1 inactivation and abolished the U-shaped voltage dependence of inactivation normally observed in Kv2.1. These data suggest that intersubunit T1 domain interactions influence U-type inactivation in Kv1 channels, and suggest a generalized influence of the T1 domain on U-type inactivation between Kv channel subfamilies.     

Isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations. Targeting of Kv1.5 to caveolae

The precise subcellular localization of ion channels is often necessary to ensure rapid and efficient integration of both intracellular and extracellular signaling events. Recently, we have identified lipid raft association as a novel mechanism for the subcellular sorting of specific voltage-gated K(+) channels to regions of the membrane rich in signaling complexes. Here, we demonstrate isoform-specific targeting of voltage-gated K(+) (Kv) channels to distinct lipid raft populations with the finding that Kv1.5 specifically targets to caveolae. Multiple lines of evidence indicate that Kv1.5 and Kv2.1 exist in distinct raft domains: 1) channel/raft association shows differential sensitivity to increasing concentrations of Triton X-100; 2) unlike Kv2.1, Kv1.5 colocalizes with caveolin on the cell surface and redistributes with caveolin following microtubule disruption; and 3) immunoisolation of caveolae copurifies Kv1.5 channel. Both depletion of cellular cholesterol and inhibition of sphingolipid synthesis alter Kv1.5 channel function by inducing a hyperpolarizing shift in the voltage dependence of activation and inactivation. The differential targeting of Kv channel subtypes to caveolar and noncaveolar rafts within a single membrane represents a unique mechanism of compartmentalization, which may permit isoform-specific modulation of K(+) channel function.     

Kvbeta subunits increase expression of Kv4.3 channels by interacting with their C termini

Auxiliary Kvbeta subunits form complexes with Kv1 family voltage-gated K(+) channels by binding to a part of the N terminus of channel polypeptide. This association influences expression and gating of these channels. Here we show that Kv4.3 proteins are associated with Kvbeta2 subunits in the brain. Expression of Kvbeta1 or Kvbeta2 subunits does not affect Kv4.3 channel gating but increases current density and protein expression. The increase in Kv4.3 protein is larger at longer times after transfection, suggesting that Kvbeta-associated channel proteins are more stable than those without the auxiliary subunits. This association between Kv4.3 and Kvbeta subunits requires the C terminus but not the N terminus of the channel polypeptide. Thus, Kvbeta subunits utilize diverse molecular interactions to stimulate the expression of Kv channels from different families.     

KCNE1 subunits require co-assembly with K+ channels for efficient trafficking and cell surface expression

KCNE peptides are a class of type I transmembrane beta subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K(+) channels. Accordingly, mutations that disrupt the assembly and trafficking of KCNE-K(+) channel complexes give rise to disease. The cellular mechanisms responsible for ensuring that KCNE peptides assemble with voltage-gated K(+) channels have yet to be elucidated. Using enzymatic deglycosylation, immunofluorescence, and quantitative cell surface labeling experiments, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with specific K(+) channel subunits; co-assembly mediates KCNE1 progression through the secretory pathway and results in cell surface expression. We also address an apparent discrepancy between our results and a previous study in human embryonic kidney cells, which showed wild type KCNE1 peptides can reach the plasma membrane without exogenously expressed K(+) channel subunits. By comparing KCNE1 trafficking in three cell lines, our data suggest that the errant KCNE1 trafficking observed in human embryonic kidney cells may be due, in part, to the presence of endogenous voltage-gated K(+) channels in these cells.     

Association of Kv1.5 and Kv1.3 contributes to the major voltage-dependent K+ channel in macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.     

Differential targeting of Shaker-like potassium channels to lipid rafts

Ion channel targeting within neuronal and muscle membranes is an important determinant of electrical excitability. Recent evidence suggests that there exists within the membrane specialized microdomains commonly referred to as lipid rafts. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction proteins such as nitric-oxide synthase, ligand-gated receptors, and multiple protein kinases. Here, we demonstrate that the voltage-gated K(+) channel Kv2.1, but not Kv4.2, targets to lipid rafts in both heterologous expression systems and rat brain. The Kv2.1 association with lipid rafts does not appear to involve caveolin. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1 associated rafts and shifts the midpoint of Kv2.1 inactivation by nearly 40 mV without affecting peak current density or channel activation. The differential targeting of Kv channels to lipid rafts represents a novel mechanism both for the subcellular sorting of K(+) channels to regions of the membrane rich in signaling complexes and for modulating channel properties via alterations in lipid content.     

Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage

Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 microM-100 microM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.     

Molecular proximity of Kv1.3 voltage-gated potassium channels and beta(1)-integrins on the plasma membrane of melanoma cells: effects of cell adherence and channel blockers

Tumor cell membranes have multiple components that participate in the process of metastasis. The present study investigates the physical association of beta1-integrins and Kv1.3 voltage-gated potassium channels in melanoma cell membranes using resonance energy transfer (RET) techniques. RET between donor-labeled anti-beta1-integrin and acceptor-labeled anti-Kv1.3 channels was detected on LOX cells adherent to glass and fibronectin-coated coverslips. However, RET was not observed on LOX cells in suspension, indicating that molecular proximity of these membrane molecules is adherence-related. Several K(+) channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between beta1-integrins and Kv1.3 channels. However, the irrelevant K(+) channel blocker apamin had no effect on RET between beta1-integrins and Kv1.3 channels. Based on these findings, we speculate that the lateral association of Kv1.3 channels with beta1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.     

Molecular coupling in the human ether-a-go-go-related gene-1 (hERG1) K+ channel inactivation pathway

Emerging evidence suggests that K(+) channel inactivation involves coupling between residues in adjacent regions of the channel. Human ether-a-go-go-related gene-1 (hERG1) K(+) channels undergo a fast inactivation gating process that is crucial for maintaining electrical stability in the heart. The molecular mechanisms that drive inactivation in hERG1 channels are unknown. Using alanine scanning mutagenesis, we show that a pore helix residue (Thr-618) that points toward the S5 segment is critical for normal inactivation gating. Amino acid substitutions at position 618 modulate the free energy of inactivation gating, causing enhanced or reduced inactivation. Mutation of an S5 residue that is predicted to be adjacent to Thr-618 (W568L) abolishes inactivation and alters ion selectivity. The introduction of the Thr-618-equivalent residue in Kv1.5 enhances inactivation. Molecular dynamic simulations of the Kv1.2 tetramer reveal van der Waals coupling between hERG1 618- and 568-equivalent residues and a significant increase in interaction energies when threonine is introduced at the 618-equivalent position. We propose that coupling between the S5 segment and pore helix may participate in the inactivation process in hERG1 channels.     

PSD-95 and SAP97 exhibit distinct mechanisms for regulating K(+) channel surface expression and clustering

Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K(+) channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95-induced clusters of Kv1 K(+) channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K(+) channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvbeta subunits. Consistent with a mechanism of plasma membrane channel-PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3-5 microm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.     

The electrically silent Kv6.4 subunit confers hyperpolarized gating charge movement in Kv2.1/Kv6.4 heterotetrameric channels

The voltage-gated K(+) (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ~40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K(+) equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits.     

Hanging gondola structure of the T1 domain in a voltage-gated K(+) channel

The T1 domain is a approximately 100-residue sequence in the cytoplasmic N-terminal region of K(v)-type K(+) channels. The structure of the isolated domain is known, but it is uncertain whether the structure of this domain is maintained in the fully assembled, membrane-associated, homotetrameric channel protein. We use the structure of the isolated domain as a guide for designing disulfide bonds to cross-link Shaker K(+) channels through the T1 domain. Six pairs of residues with side chains closely apposed across the T1 subunit interface were selected for replacement by cysteine. Of these, three pairs formed cross-links upon air oxidation of cysteine-substituted Shaker channels expressed in Xenopus oocyte membranes. Two of these cross-linked channels were examined electrophysiologically and were found to have gating properties only slightly altered from wild-type. The results show that the structure of the isolated T1 domain exists in the mature ion channel. They also demand that this domain is attached to the membrane-embedded part of the protein as a cytoplasmic "hanging gondola", and that ions gain access to the pore through four "windows" formed by the linker connecting T1 to the channel's first transmembrane helix.     

Sp-tetraKCNG: A novel cyclic nucleotide gated K(+) channel

The sequence of a novel cGMP-regulated, tetrameric, K(+) selective channel (Sp-tetraKCNG) was discovered in the sea urchin Strongylocentrotus purpuratus. The Sp-tetraKCNG is a single polypeptide made of four KCNG domains similar to voltage-dependent Na(+) and Ca(2+) channels. Each KCNG domain has six transmembrane segments (S1-S6), the ion pore having the K(+) selectivity signature GYGD and a cyclic nucleotide-binding domain (CNBD). This novel channel is evolutionary located between K(+)-selective and voltage-dependent EAG channels and voltage-independent cationic CNG channels. Bilayer reconstitutions demonstrate such a cGMP-regulated K(+) selective channel in sea urchin spermatozoa.