Position and motions of the S4 helix during opening of the Shaker potassium channel

The four voltage sensors in voltage-gated potassium (Kv) channels activate upon membrane depolarization and open the pore. The location and motion of the voltage-sensing S4 helix during the early activation steps and the final opening transition are unresolved. We studied Zn(2+) bridges between two introduced His residues in Shaker Kv channels: one in the R1 position at the outer end of the S4 helix (R362H), and another in the S5 helix of the pore domain (A419H or F416H). Zn(2+) bridges readily form between R362H and A419H in open channels after the S4 helix has undergone its final motion. In contrast, a distinct bridge forms between R362H and F416H after early S4 activation, but before the final S4 motion. Both bridges form rapidly, providing constraints on the average position of S4 relative to the pore. These results demonstrate that the outer ends of S4 and S5 remain in close proximity during the final opening transition, with the S4 helix translating a significant distance normal to the membrane plane.     

S4 charges move close to residues in the pore domain during activation in a K channel.

Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET(+)). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 A) to a position close (8 A) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.     

Molecular movement of the voltage sensor in a K channel

The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 A) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 A) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model.     

Double gaps along Shaker S4 demonstrate omega currents at three different closed states

The aim of the present study was to investigate in detail how the voltage sensor in the Shaker potassium channel moves during the gating process. After the publication of the open channel structure from the crystallized KvAP channel in 2003, an alternative so-called "paddle" model was put forward in contrast to the existing helical screw model. The voltage sensor S4 contains 4 arginine residues relevant for gating, R1(362), R2(365), R3(368) and R4(371), each separated by 2 neutral residues. These charged residues coil as one of three threads on the S4-α-helix. Based on a previous finding that the mutation R1S leads to the so-called omega leak current through a "gating-pore" in the closed state, we introduced gaps systematically along the arginine thread substituting long arginines by short serines. Mutations R2S or R3S did neither create transient nor steady leaks. The fact that the native residue A359, which is located three amino acids in front of R1, is a short one, motivated us to check its role. Mutation of A359 to arginine blocked the omega current in R1S mutant indicating that the omega pore is occupied by A359 and R1. Introducing further double gaps (RR to SS) at sequential positions (0+1, 1+2, 2+3), produced clear leak currents which were remarkably stable over a wide voltage range. These leaks contradict that S4 would swing together with S3 in lipid according to the paddle hypothesis. Rather, our results show that during gating the S4 segment moves in 3 helical steps through a fixed pore formed by the channel protein.     

Atomic constraints between the voltage sensor and the pore domain in a voltage-gated K+ channel of known structure

In voltage-gated K(+) channels (Kv), membrane depolarization promotes a structural reorganization of each of the four voltage sensor domains surrounding the conducting pore, inducing its opening. Although the crystal structure of Kv1.2 provided the first atomic resolution view of a eukaryotic Kv channel, several components of the voltage sensors remain poorly resolved. In particular, the position and orientation of the charged arginine side chains in the S4 transmembrane segments remain controversial. Here we investigate the proximity of S4 and the pore domain in functional Kv1.2 channels in a native membrane environment using electrophysiological analysis of intersubunit histidine metallic bridges formed between the first arginine of S4 (R294) and residues A351 or D352 of the pore domain. We show that histidine pairs are able to bind Zn(2+) or Cd(2+) with high affinity, demonstrating their close physical proximity. The results of molecular dynamics simulations, consistent with electrophysiological data, indicate that the position of the S4 helix in the functional open-activated state could be shifted by approximately 7-8 A and rotated counterclockwise by 37 degrees along its main axis relative to its position observed in the Kv1.2 x-ray structure. A structural model is provided for this conformation. The results further highlight the dynamic and flexible nature of the voltage sensor.     

Relative Movements of Transmembrane Regions at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Pore during Channel Gating

Multiple transmembrane (TM) segments line the pore of the cystic fibrosis transmembrane conductance regulator Cl(-) channel; however, the relative alignment of these TMs and their relative movements during channel gating are unknown. To gain three-dimensional structural information on the outer pore, we have used patch clamp recording to study the proximity of pairs of cysteine side chains introduced into TMs 6 and 11, using both disulfide cross-linking and Cd(2+) coordination. Following channel activation, disulfide bonds could apparently be formed between three cysteine pairs (of 15 studied): R334C/T1122C, R334C/G1127C, and T338C/S1118C. To examine the state dependence of cross-linking, we combined these cysteine mutations with a nucleotide-binding domain mutation (E1371Q) that stabilizes the channel open state. Investigation of the effects of the E1371Q mutation on disulfide bond formation and Cd(2+) coordination suggests that although R334C/T1122C and T338C/S1118C are closer together in the channel open state, R334C/G1127C are close together and can form disulfide bonds only when the channel is closed. These results provide important new information on the three-dimensional structure of the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore: TMs 6 and 11 are close enough together to form disulfide bonds in both open and closed channels. Moreover, the altered relative locations of residues in open and in closed channels that we infer allow us to propose that channel opening and closing may be associated with a relative translational movement of TMs 6 and 11, with TM6 moving "down" (toward the cytoplasm) during channel opening.     

Voltage-insensitive Gating after Charge-neutralizing Mutations in the S4 Segment of Shaker Channels

Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from −160 to +80 mV with a constant opening probability (P _o). Although P _o is low (∼0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca²⁺ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb⁺ is substituted for K⁺ as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive P _o seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain.     

The orientation and molecular movement of a k(+) channel voltage-sensing domain

Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.     

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.     

Amino acid substitution within the S2 and S4 transmembrane segments in Shaker potassium channel modulates channel gating

To investigate of the gating properties in the voltage-activated potassium channel, we have mutated a variety of S2 and S4 residues in the Shaker potassium protein. Results showed that the R365C and R368C, but not the E283C, R362C, R365S, R368S or the ShB-IR, were sensitive to micromolar concentrations of Cd(2+) ions. This indicates that R365 and R368 play a crucial role in the channel gating due to a conformational modulation of the channel structure. Doubly mutated channels of the E283C/R365E and E283C/R368E caused a transient increase in current amplitude, which reached a peak within a few seconds and then decreased toward initial levels, despite the continual presence of Cd(2+). Taken together, our results suggest that E283, R365, and R368 form a network of strong, local, and electrostatic interactions that relate closely to the mechanism of the channel gating.     

Localization of the K+ lock-In and the Ba2+ binding sites in a voltage-gated calcium-modulated channel. Implications for survival of K+ permeability.

Using Ba2+ as a probe, we performed a detailed characterization of an external K+ binding site located in the pore of a large conductance Ca2+-activated K+ (BKCa) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba2+ blocks BKCa channels and decreasing external K+ using a K+ chelator, (+)-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba2+-blocked events. Average Ba2+ dwell time changes from 10 s at 10 mM external K+ to 100 ms in the limit of very low [K+]. Using a model where external K+ binds to a site hindering the exit of Ba2+ toward the external side (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-568), we calculated a dissociation constant of 2.7 mircoM for K) at this lock-in site. We also found that BK(Ca) channels enter into a long-lasting nonconductive state when the external [K+] is reduced below 4 microM using the crown ether. Channel activity can be recovered by adding K+, Rb+, Cs+, or NH4+ to the external solution. These results suggest that the BK(Ca) channel stability in solutions of very low [K+] is due to K+ binding to a site having a very high affinity. Occupancy of this site by K+ avoids the channel conductance collapse and the exit of Ba2+ toward the external side. External tetraethylammonium also reduced the Ba2+ off rate and impeded the channel from entering into the long-lasting nonconductive state. This effect requires the presence of external K+. It is explained in terms of a model in which the conduction pore contains Ba2+, K+, and tetraethylammonium simultaneously, with the K+ binding site located internal to the tetraethylammonium site. Altogether, these results and the known potassium channel structure (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) imply that the lock-in site and the Ba2+ sites are the external and internal ion sites of the selectivity filter, respectively.     

Gating pore currents in DIIS4 mutations of NaV1.4 associated with periodic paralysis: saturation of ion flux and implications for disease pathogenesis

S4 voltage–sensor mutations in CaV1.1 and NaV1.4 channels cause the human muscle disorder hypokalemic periodic paralysis (HypoPP). The mechanism whereby these mutations predispose affected sarcolemma to attacks of sustained depolarization and loss of excitability is poorly understood. Recently, three HypoPP mutations in the domain II S4 segment of NaV1.4 were shown to create accessory ionic permeation pathways, presumably extending through the aqueous gating pore in which the S4 segment resides. However, there are several disparities between reported gating pore currents from different investigators, including differences in ionic selectivity and estimates of current amplitude, which in turn have important implications for the pathological relevance of these aberrant currents. To clarify the features of gating pore currents arising from different DIIS4 mutants, we recorded gating pore currents created by HypoPP missense mutations at position R666 in the rat isoform of Nav1.4 (the second arginine from the outside, at R672 in human NaV1.4). Extensive measurements were made for the index mutation, R666G, which created a gating pore that was permeable to K⁺ and Na⁺. This current had a markedly shallow slope conductance at hyperpolarized voltages and robust inward rectification, even when the ionic gradient strongly favored outward ionic flow. These characteristics were accounted for by a barrier model incorporating a voltage-gated permeation pathway with a single cation binding site oriented near the external surface of the electrical field. The amplitude of the R666G gating pore current was similar to the amplitude of a previously described proton-selective current flowing through the gating pore in rNaV1.4-R663H mutant channels. Currents with similar amplitude and cation selectivity were also observed in R666S and R666C mutant channels, while a proton-selective current was observed in R666H mutant channels. These results add support to the notion that HypoPP mutations share a common biophysical profile comprised of a low-amplitude inward current at the resting potential that may contribute to the pathological depolarization during attacks of weakness.     

Intersegment hydrogen bonds as possible structural determinants of the N/Q/R site in glutamate receptors.

Specific electrophysiological and pharmacological properties of ionic channels in NMDA, AMPA, and kainate subtypes of ionotropic glutamate receptors (GluRs) are determined by the Asn (N), Gln (Q), and Arg (R) residues located at homologous positions of the pore-lining M2 segments (the N/Q/R site). Presumably, the N/Q/R site is located at the apex of the reentrant membrane loop and forms the narrowest constriction of the pore. Although the shorter Asn residues are expected to protrude in the pore to a lesser extent than the longer Gln residues, the effective dimension of the NMDA channel (corresponding to the size of the largest permeant organic cation) is, surprisingly, smaller than that of the AMPA channel. To explain this paradox, we propose that the N/Q/R residues form macrocyclic structures (rings) stabilized by H-bonds between a NH(2) group in the side chain of a given M2 segment and a C==O group of the main chain in the adjacent M2 segment. Using Monte Carlo minimization, we have explored conformational properties of the rings. In the Asn, but not in the Gln ring, the side-chain oxygens protruding into the pore may facilitate ion permeation and accept H-bonds from the blocking drugs. In this way, the model explains different electrophysiological and pharmacological properties of NMDA and non-NMDA GluR channels. The ring of H-bonded polar residues at the pore narrowing resembles the ring of four Thr(75) residues observed in the crystallographic structure of the KcsA K(+) channel.     

Gating of voltage-dependent potassium channels

Activation of voltage-dependent ion channels is primarily controlled by the applied potential difference across the membrane. For potassium channels the Drosophila Shaker channel has served as an archetype of all other potassium channels in studies of activation mechanisms. In the Shaker potassium channel much of the voltage sensitivity is conferred by the S4 transmembrane helix, which contains seven positively charged residues. During gating, the movement of these charges produces gating currents. Mutagenic and fluorescence studies indicate that four of these residues are particularly important and contribute to the majority of gating charge, R362, R365, R368 and R371. The channel is thought to dwell in several closed states prior to opening. Ionic-charge pairing with negatively charged residues in the S2 and S3 helices is thought to be important in regulating these closed states and detailed kinetic models have attempted to define the kinetics and charge of the transitions between these states. Neutral residues throughout the S4 and S5 helices are thought to control late steps in channel opening and may have important roles in modulating the stability of the open state and late closed states. In response to depolarization, the S4 helix is thought to undergo a rotational translation and this movement is also important in studies of the movement of the pore helices, S5 and S6, during opening. This review will examine residues that are important during activation as well as kinetic models that have attempted to quantitatively define the activation pathway of voltage-dependent potassium channels.     

Dynamic interaction of S5 and S6 during voltage-controlled gating in a potassium channel

A gain-of-function mutation in the Caenorhabditis elegans exp-2 K(+)-channel gene is caused by a cysteine-to-tyrosine change (C480Y) in the sixth transmembrane segment of the channel (Davis, M.W., R. Fleischhauer, J.A. Dent, R.H. Joho, and L. Avery. 1999. Science. 286:2501-2504). In contrast to wild-type EXP-2 channels, homotetrameric C480Y mutant channels are open even at -160 mV, explaining the lethality of the homozygous mutant. We modeled the structure of EXP-2 on the 3-D scaffold of the K(+) channel KcsA. In the C480Y mutant, tyrosine 480 protrudes from S6 to near S5, suggesting that the bulky side chain may provide steric hindrance to the rotation of S6 that has been proposed to accompany the open-closed state transitions (Perozo, E., D.M. Cortes, and L.G. Cuello. 1999. Science. 285:73-78). We tested the hypothesis that only small side chains at position 480 allow the channel to close, but that bulky side chains trap the channel in the open state. Mutants with small side chain substitutions (Gly and Ser) behave like wild type; in contrast, bulky side chain substitutions (Trp, Phe, Leu, Ile, Val, and His) generate channels that conduct K(+) ions at potentials as negative as -120 mV. The side chain at position 480 in S6 in the pore model is close to and may interact with a conserved glycine (G421) in S5. Replacement of G421 with bulky side chains also leads to channels that are trapped in an active state, suggesting that S5 and S6 interact with each other during voltage-dependent open-closed state transitions, and that bulky side chains prevent the dynamic changes necessary for permanent channel closing. Single-channel recordings show that mutant channels open frequently at negative membrane potentials indicating that they fail to reach long-lasting, i.e., stable, closed states. Our data support a "two-gate model" with a pore gate responsible for the brief, voltage-independent openings and a separately located, voltage-activated gate (Liu, Y., and R.H. Joho. 1998. Pflügers Arch. 435:654-661).     

Coupling between Residues on S4 and S1 Defines the Voltage-Sensor Resting Conformation in NaChBac

The voltage sensor is a four-transmembrane helix bundle (S1-S4) that couples changes in membrane potential to conformational alterations in voltage-gated ion channels leading to pore opening and ion conductance. Although the structure of the voltage sensor in activated potassium channels is available, the conformation of the voltage sensor at rest is still obscure, limiting our understanding of the voltage-sensing mechanism. By employing a heterologously expressed Bacillus halodurans sodium channel (NaChBac), we defined constraints that affect the positioning and depolarization-induced outward motion of the S4 segment. We compared macroscopic currents mediated by NaChBac and mutants in which E43 on the S1 segment and the two outermost arginines (R1 and R2) on S4 were substituted. Neutralization of the negatively charged E43 (E43C) had a significant effect on channel gating. A double-mutant cycle analysis of E43 and R1 or R2 suggested changes in pairing during channel activation, implying that the interaction of E43 with R1 stabilizes the voltage sensor in its closed/available state, whereas interaction of E43 with R2 stabilizes the channel open/unavailable state. These constraints on S4 dynamics that define its stepwise movement upon channel activation and positioning at rest are novel, to the best of our knowledge, and compatible with the helical-screw and electrostatic models of S4 motion.     

Of cilia,titin, and neurosteroids

Missense mutations at arginine residues in the S4 voltage-sensor domains of Na_V1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K⁺. Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K⁺ during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K⁺, yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na⁺ current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type Na_V1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na⁺ was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼10 µS/cm² or 1% of the total resting conductance.Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na⁺ current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na⁺ current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of Na_V, Ca_V, and K_V channels associated with human disease.     

Structural determinants of Kvbeta1.3-induced channel inactivation: a hairpin modulated by PIP2

Inactivation of voltage-gated Kv1 channels can be altered by Kvbeta subunits, which block the ion-conducting pore to induce a rapid ('N-type') inactivation. Here, we investigate the mechanisms and structural basis of Kvbeta1.3 interaction with the pore domain of Kv1.5 channels. Inactivation induced by Kvbeta1.3 was antagonized by intracellular PIP(2). Mutations of R5 or T6 in Kvbeta1.3 enhanced Kv1.5 inactivation and markedly reduced the effects of PIP(2). R5C or T6C Kvbeta1.3 also exhibited diminished binding of PIP(2) compared with wild-type channels in an in vitro lipid-binding assay. Further, scanning mutagenesis of the N terminus of Kvbeta1.3 revealed that mutations of L2 and A3 eliminated N-type inactivation. Double-mutant cycle analysis indicates that R5 interacts with A501 and T480 of Kv1.5, residues located deep within the pore of the channel. These interactions indicate that Kvbeta1.3, in contrast to Kvbeta1.1, assumes a hairpin structure to inactivate Kv1 channels. Taken together, our findings indicate that inactivation of Kv1.5 is mediated by an equilibrium binding of the N terminus of Kvbeta1.3 between phosphoinositides (PIPs) and the inner pore region of the channel.     

A direct demonstration of closed-state inactivation of K+ channels at low pH

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K⁺ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.     

Benzo[c]phenanthrene adducts and nogalamycin inhibit DNA transesterification by vaccinia topoisomerase

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N2-deoxyguanosine and N6-deoxyadenosine positions within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the +6 and +5 base pairs, slowed 4-fold by intercalation between the +5 and +4 base pairs, and virtually abolished by BcPh intercalation between the +4 and +3 base pairs and the +3 and +2 base pairs. Intercalation between the +2 and +1 base pairs by the +2R BcPh dA adduct abolished transesterification, whereas the overlapping +1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the +1 and -1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the -1 and -2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the -2 and -3 base pairs had little effect. The anthracycline drug nogalamycin, a non-covalent intercalator with preference for 5'-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC50 of 0.7 microM. Nogalamycin was most effective when the drug was pre-incubated with DNA and when the cleavage target site was 5'-CCCTT/G instead of 5'-CCCTT/A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.