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.     

Reconstructing voltage sensor-pore interaction from a fluorescence scan of a voltage-gated K+ channel

X-ray crystallography has made considerable recent progress in providing static structures of ion channels. Here we describe a complementary method-systematic fluorescence scanning-that reveals the structural dynamics of a channel. Local protein motion was measured from changes in the fluorescent intensity of a fluorophore attached at one of 37 positions in the pore domain and in the S4 voltage sensor of the Shaker K+ channel. The local rearrangements that accompany activation and slow inactivation were mapped onto the homologous structure of the KcsA channel and onto models of S4. The results place clear constraints on S4 location, voltage-dependent movement, and the mechanism of coupling of S4 motion to the operation of the slow inactivation gate in the pore domain.     

The N-terminus of the K channel KAT1 controls its voltage-dependent gating by altering the membrane electric field.

Functional roles of different domains (pore region, S4 segment, N-terminus) of the KAT1 potassium channel in its voltage-dependent gating were electrophysiologically studied in Xenopus oocytes. The KAT1 properties did not depend on the extracellular K+ concentration or on residue H267, equivalent to one of the residues known to be important in C-type inactivation in Shaker channels, indicating that the hyperpolarization-induced KAT1 inward currents are related to the channel activation rather than to recovery from inactivation. Neutralization of a positively charged amino acid in the S4 domain (R176S) reduced the gating charge movement, suggesting that it acts as a voltage-sensing residue in KAT1. N-terminal deletions alone (e.g., delta20-34) did not affect the gating charge movement. However, the deletions paradoxically increased the voltage sensitivity of the R176S mutant channel, but not that of the wild-type channel. We propose a simple model in which the N-terminus determines the KAT1 voltage sensitivity by contributing to the electric field sensed by the voltage sensor.     

Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3-S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.     

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.     

Three-dimensional structure of a voltage-gated potassium channel at 2.5 nm resolution

BACKGROUND: The voltage-gated potassium channel Shaker from Drosophila consists of a tetramer of identical subunits, each containing six transmembrane segments. The atomic structure of a bacterial homolog, the potassium channel KcsA, is much smaller than Shaker. It does not have a voltage sensor and other important domains like the N-terminal tetramerization (T1) domain. The structure of these additional elements has to be studied in the more complex voltage-gated channels. RESULTS: We determined the three-dimensional structure of the entire Shaker channel at 2.5 nm resolution using electron microscopy. The four-fold symmetric structure shows a large and a small domain linked by thin 2 nm long connectors. To interpret the structure, we used the crystal structures of the isolated T1 domain and the KcsA channel. A unique density assignment was made based on the symmetry and dimensions of the crystal structures and domains, identifying the smaller domain as the cytoplasmic mass of Shaker containing T1 and the larger domain as embedded in the membrane. CONCLUSIONS: The two-domain architecture of the Shaker channel is consistent with the recently proposed "hanging gondola" model for the T1 domain, putting the T1 domain at a distance from the membrane domain but attached to it by thin connectors. The space between the two domains is sufficient to permit cytoplasmic access of ions and the N-terminal inactivation domain to the pore region. A hanging gondola architecture has also been observed in the nicotinic acetylcholine receptor and the KcsA structure, suggesting that it is a common element of ion channels.     

Functional interactions at the interface between voltage-sensing and pore domains in the Shaker K(v) channel

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.     

Tethered blockers as molecular 'tape measures' for a voltage-gated K+ channel

The propagation of electrical signals in excitable cells is orchestrated by a molecular family of voltage-dependent ion channel proteins. These K+, Na+, and Ca++ channels are all composed of four identical or similar units, each containing six transmembrane segments (S1-S6) in a roughly four-fold symmetric structure. The S5-S6 sequences fold into a central pore unit, which is surrounded by a voltage-gating module composed of S1-S4. The recent structure of KcsA, a two-transmembrane bacterial K+ channel, illuminates the physical character of the pore unit, but little is known about the arrangement of the surrounding S1-S4 sequences. To locate regions of this gating module in space, we synthesized a series of compounds of varying length that function as molecular 'tape measures': quaternary ammonium (QA) pore blockers that can be tethered to specific test residues. We show that in a Shaker K+ channel, the extracellular ends of S1 and S3 are approximately 30 ? from the tetraethylammonium (TEA) blocking site at the external opening of the pore. A portion of the S3-S4 loop is, at 17-18 ?, considerably closer.     

Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons.

We have cloned a functional cDNA encoding the cyclic nucleotide-gated channel selectively expressed in catfish olfactory sensory neurons. The cyclic nucleotide-gated channels share sequence and structural features with the family of voltage-gated ion channels. This homology is most evident in transmembrane region S4, the putative voltage sensor domain, and the H5 domain, thought to form the channel pore. We have characterized the single-channel properties of the cloned catfish channel and compared these properties with the channel in native catfish olfactory sensory neurons. The channel is activated equally well by cAMP and cGMP, shows only a slight voltage dependence of gating, and exhibits a pH- and voltage-dependent subconductance state similar to that observed for the voltage-gated L-type calcium channel.     

Conserved gating hinge in ligand- and voltage-dependent K+ channels

Ion channels open and close their pore in a process called gating. On the basis of crystal structures of two voltage-independent K(+) channels, KcsA and MthK, a conformational change for gating has been proposed whereby the inner helix bends at a glycine hinge point (gating hinge) to open the pore and straightens to close it. Here we ask if a similar gating hinge conformational change underlies the mechanics of pore opening of two eukaryotic voltage-dependent K(+) channels, Shaker and BK channels. In the Shaker channel, substitution of the gating hinge glycine with alanine and several other amino acids prevents pore opening, but the ability to open is recovered if a secondary glycine is introduced at an adjacent position. A proline at the gating hinge favors the open state of the Shaker channel as if by preventing inner helix straightening. In BK channels, which have two adjacent glycine residues, opening is significantly hindered in a graded manner with single and double mutations to alanine. These results suggest that K(+) channels, whether ligand- or voltage-dependent, open when the inner helix bends at a conserved glycine gating hinge.     

Gating charges in the activation and inactivation processes of the HERG channel

The hERG channel has a relatively slow activation process but an extremely fast and voltage-sensitive inactivation process. Direct measurement of hERG's gating current (Piper, D.R., A. Varghese, M.C. Sanguinetti, and M. Tristani-Firouzi. 2003. PNAS. 100:10534-10539) reveals two kinetic components of gating charge transfer that may originate from two channel domains. This study is designed to address three questions: (1) which of the six positive charges in hERG's major voltage sensor, S4, are responsible for gating charge transfer during activation, (2) whether a negative charge in the cytoplasmic half of S2 (D466) also contributes to gating charge transfer, and (3) whether S4 serves as the sole voltage sensor for hERG inactivation. We individually mutate S4's positive charges and D466 to cysteine, and examine (a) effects of mutations on the number of equivalent gating charges transferred during activation (z(a)) and inactivation (z(i)), and (b) sidedness and state dependence of accessibility of introduced cysteine side chains to a membrane-impermeable thiol-modifying reagent (MTSET). Neutralizing the outer three positive charges in S4 and D466 in S2 reduces z(a), and cysteine side chains introduced into these positions experience state-dependent changes in MTSET accessibility. On the other hand, neutralizing the inner three positive charges in S4 does not affect z(a). None of the charge mutations affect z(i). We propose that the scheme of gating charge transfer during hERG's activation process is similar to that described for the Shaker channel, although hERG has less gating charge in its S4 than in Shaker. Furthermore, channel domain other than S4 contributes to gating charge involved in hERG's inactivation process.     

Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics

The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.     

Tracking S4 movement by gating pore currents in the bacterial sodium channel NaChBac

Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1–R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.     

A novel current pathway parallel to the central pore in a mutant voltage-gated potassium channel

Voltage-gated potassium channels are proteins composed of four subunits consisting of six membrane-spanning segments S1-S6, with S4 as the voltage sensor. The region between S5 and S6 forms the potassium-selective ion-conducting central α-pore. Recent studies showed that mutations in the voltage sensor of the Shaker channel could disclose another ion permeation pathway through the voltage-sensing domain (S1-S4) of the channel, the ω-pore. In our studies we used the voltage-gated hKv1.3 channel, and the insertion of a cysteine at position V388C (Shaker position 438) generated a current through the α-pore in high potassium outside and an inward current at hyperpolarizing potentials carried by different cations like Na(+), Li(+), Cs(+), and NH(4)(+). The observed inward current looked similar to the ω-current described for the R1C/S Shaker mutant channel and was not affected by some pore blockers like charybdotoxin and tetraethylammonium but was inhibited by a phenylalkylamine blocker (verapamil) that acts from the intracellular side. Therefore, we hypothesize that the hKv1.3_V388C mutation in the P-region generated a channel with two ion-conducting pathways. One, the α-pore allowing K(+) flux in the presence of K(+), and the second pathway, the σ-pore, functionally similar but physically distinct from the ω-pathway. The entry of this new pathway (σ-pore) is presumably located at the backside of Y395 (Shaker position 445), proceeds parallel to the α-pore in the S6-S6 interface gap, ending between S5 and S6 at the intracellular side of one α-subunit, and is blocked by verapamil.     

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates Shaker K⁺ channels and voltage-gated Ca²⁺ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP₂ regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP₂ regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K⁺ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP₂ regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K⁺ channel family. Open-state stabilization by PIP₂ has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP₂ strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP₂ has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4–S5 linker) and R479 near the S6 activation gate are required for PIP₂ to inhibit voltage activation. The ability of PIP₂ to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP₂ on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP₂-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP₂ can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP₂ regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.     

Transfer of ion binding site from ether-à-go-go to Shaker: Mg2+ binds to resting state to modulate channel opening

In ether-à-go-go (eag) K⁺ channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are ∼4 Å apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular Mg²⁺. In I287D+F324D, Mg²⁺ significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that Mg²⁺ binds to the voltage sensor in the resting conformation. Supporting this conclusion, Mg²⁺ shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for Mg²⁺ binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation.     

A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans.

We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of an up to now unique prokaryotic potassium ion channel (KcsA). It has a rectifying current-voltage relationship and displays subconductance states, the largest of which amounts to A approximately equal to 90 pS. The channel is blocked by Cs- ions and gating requires the presence of Mg2+ ions. The kcsA gene has been identified in the gram-positive soil bacterium Streptomyces lividans. It encodes a predicted 17.6 kDa protein with two potential membrane-spanning helices linked by a central domain which shares a high degree of similarity with the H5 segment conserved among eukaryotic ion channels. Multiple alignments of deduced amino acids suggest that the novel channel has the closest kinship to the S5, H5 and S6 regions of voltage-gated K+ channel families, mainly to the subfamily represented by the Shaker protein from Drosophila melanogaster. Moreover, KcsA is most distantly related to eukaryotic inwardly rectifying channels with two putative predicted transmembrane segments.     

Removal of the outermost arginine in IVS4 segment of the Ca(V)3.1 channel affects amplitude but not voltage dependence of gating current

Positively charged amino acids in S4 segments of voltage-dependent Ca(V)3.1 channel form putative voltage sensor. Previously we have shown that exchange of uppermost positively charged arginine in IVS4 segment for cysteine (mutation R1717C) affected deactivation and inactivation, but not activation of macroscopic current. Now we compared gating currents from both channels. Maximal amplitude of charge movement in R1717C channel decreased but voltage-dependent characteristics of charge movement were not significantly altered. We concluded that mutation of R1717C affects the coupling between S4 activation and pore opening, but not the S4 activation itself.     

Deletion of the S3-S4 linker in the Shaker potassium channel reveals two quenching groups near the outside of S4

When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (DeltaF) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391-408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3-S4 linker. In the deletion mutant, the maximal DeltaF/F seen was diminished 10-fold, and the DeltaF at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3-S4 linker. The residual DeltaF showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of -90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a DeltaF at extreme hyperpolarizations (more negative than -90 mV) only. A signal from the interaction with this group in the wt S3-S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic DeltaF as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment.     

Depolarization induces intersubunit cross-linking in a S4 cysteine mutant of the Shaker potassium channel

Voltage-gated potassium (K(v)) channels are integral membrane proteins, composed of four subunits, each comprising six (S1-S6) transmembrane segments. S1-S4 comprise the voltage-sensing domain, and S5-S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K channel and expressed the mutant channel in Xenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the channel. The latter two effects suggest stabilization of the open state of the channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K channel is reduced.