Office hysteroscopy: A report of 2402 cases.

INTRODUCTION: Hysteroscopy is the gold standard for evaluation of uterine cavity. It can be performed either as office setting or as day care procedure under general anaesthesia. Objective of this study is to assess feasibility and acceptability of office hysteroscopy without anaesthesia. MATERIALS AND METHODS: This retrospective observational study took place in the gynaecologic unit of a teaching hospital. Women who had had an office hysteroscopy from 2010 to 2013 were included. RESULTS: Two thousand four hundred and two office hysteroscopies were carried out. Indications were menorrhagia (32.2%), postoperative evaluation (20.8%), infertility (15.8%), postmenopausal bleeding (10.9%) and other indications (20.3%). Women's mean age was 39.4 [39.0-39.9] and significantly higher among women with a failure of the office hysteroscopy (47.3 vs. 38.6, P<0.01). The failure rate was 9.5%, significantly higher in women with postmenopausal bleeding and lower in women for a postoperative evaluation. Assessment of an abnormal uterine cavity was done in 56.0% of cases with 28.7% of myomas, 27.2% of polyps, 17.7% of synechiaes, 14.7% of endometrial hypertrophies, 9.0% of trophoblastic retentions and 7.7% of uterine malformation. The complication rate of office hysteroscopy was 0.05%. Mean pain score during the examination was 3.57 out of 10 [3.48-3.66] and 0.89 [0.83-0.95] five minutes later. CONCLUSION: Office hysteroscopy is safe and feasible with little pain. A failure rate of 9.5% is reported, mainly for older women with postmenopausal bleeding.

Use of voltage clamp fluorimetry in understanding potassium channel gating: a review of Shaker fluorescence data.

Voltage clamp fluorimetry (VCF) utilizes fluorescent probes that covalently bind to cysteine residues introduced into proteins and emit light as a function of their environment. Measurement of this emitted light during membrane depolarization reveals changes in the emission level as the environment of the labelled residue changes. This allows for the correlation of channel gating events with movement of specific protein moieties, at nanosecond time resolution. Since the pioneering use of this technique to investigate Shaker potassium channel activation movements, VCF has become an invaluable technique used to understand ion channel gating. This review summarizes the theory and some of the data on the application of the VCF technique. Although its usage has expanded beyond voltage-gated potassium channels and VCF is now used in a number of other voltage- and ligand-gated channels, we will focus on studies conducted in Shaker potassium channels, and what they have told us about channel activation and inactivation gating.

Localization and trafficking of cardiac voltage-gated potassium channels.

The proper trafficking and localization of cardiac potassium channels is profoundly important to the regulation of the regionally distinct action potentials across the myocardium. These processes are only beginning to be unravelled and involve modulators of channel synthesis and assembly, post-translational processing, various molecular motors and an increasing number of modifying enzymes and molecular anchors. The roles of anchoring proteins, molecular motors and kinases are explored and recent findings on channel internalization and trafficking are presented.

Voltage clamp fluorimetry studies of mammalian voltage-gated K(+) channel gating.

VCF (voltage clamp fluorimetry) provides a powerful technique to observe real-time conformational changes that are associated with ion channel gating. The present review highlights the insights such experiments have provided in understanding Kv (voltage-gated potassium) channel gating, with particular emphasis on the study of mammalian Kv1 channels. Further applications of VCF that would contribute to our understanding of the modulation of Kv channels in health and disease are also discussed.

Gating charge immobilization caused by the transition between inactivated states in the Kv1.5 channel.

Sustained Na(+) or Li(+) conductance is a feature of the inactivated state in wild-type (WT) and nonconducting Shaker and Kv1.5 channels, and has been used here to investigate the cause of off-gating charge immobilization in WT and Kv1.5-W472F nonconducting mutant channels. Off-gating immobilization in response to brief pulses in cells perfused with NMG/NMG is the result of a more negative voltage dependence of charge recovery (V(1/2) is -96 mV) compared with on-gating charge movement (V(1/2) is -6.3 mV). This shift is known to be associated with slow inactivation in Shaker channels and the disparity is reduced by 40 mV, or approximately 50% in the presence of 135 mM Cs. Off-gating charge immobilization is voltage-dependent with a V(1/2) of -12 mV, and correlates well with the development of Na(+) conductance on repolarization through C-type inactivated channels (V(1/2) is -11 mV). As well, the time-dependent development of the inward Na(+) tail current and gating charge immobilization after depolarizing pulses of different durations has the same time constant (tau = 2.7 ms). These results indicate that in Kv1.5 channels the transition to a stable C-type inactivated state takes only 2-3 ms and results in strong charge immobilization in the absence of Group IA metal cations, or even in the presence of Na. Inclusion of low concentrations of Cs delays the appearance of Na(+) tail currents in WT channels, prevents transition to inactivated states in Kv1.5-W472F nonconducting mutant channels, and removes charge immobilization. Higher concentrations of Cs are able to modulate the deactivating transition in Kv1.5 channels and prevent the residual slowing of charge return.

Altered state dependence of c-type inactivation in the long and short forms of human Kv1.5.

Evidence from both human and murine cardiomyocytes suggests that truncated isoforms of Kv1.5 can be expressed in vivo. Using whole-cell patch-clamp recordings, we have characterized the activation and inactivation properties of Kv1.5DeltaN209, a naturally occurring short form of human Kv1.5 that lacks roughly 75% of the T1 domain. When expressed in HEK 293 cells, this truncated channel exhibited a V(1/2) of -19.5 +/- 0.9 mV for activation and -35.7 +/- 0.7 mV for inactivation, compared with a V(1/2) of -11.2 +/- 0.3 mV for activation and -0.9 +/- 1.6 mV for inactivation in full-length Kv.15. Kv1.5DeltaN209 channels exhibited several features rarely observed in voltage-gated K(+) channels and absent in full-length Kv1.5, including a U-shaped voltage dependence of inactivation and "excessive cumulative inactivation," in which a train of repetitive depolarizations resulted in greater inactivation than a continuous pulse. Kv1.5DeltaN209 also exhibited a stronger voltage dependence to recovery from inactivation, with the time to half-recovery changing e-fold over 30 mV compared with 66 mV in full-length Kv1.5. During trains of human action potential voltage clamps, Kv1.5DeltaN209 showed 30-35% greater accumulated inactivation than full-length Kv1.5. These results can be explained with a model based on an allosteric model of inactivation in Kv2.1 (Klemic, K.G., C.-C. Shieh, G.E. Kirsch, and S.W. Jones. 1998. Biophys. J. 74:1779-1789) in which an absence of the NH(2) terminus results in accelerated inactivation from closed states relative to full-length Kv1.5. We suggest that differential expression of isoforms of Kv1.5 may contribute to K(+) current diversity in human heart and many other tissues.

A discrete amino terminal domain of Kv1.5 and Kv1.4 potassium channels interacts with the spectrin repeats of alpha-actinin-2.

The interaction between the amino terminus of Kv1-type potassium channels and alpha-actinin-2 has been investigated. Using a combination of yeast two-hybrid analysis and in vitro binding assays, alpha-actinin-2 was found to bind to the N-termini of both Kv1.4 and Kv1.5 but not to the equivalent segments of Kv1.1, Kv1.2 or Kv1.3. Deletion analysis in the in vitro binding assays delineated the actinin binding region of Kv1.5 to between amino acids 73 and 148 of the channel. The Kv1.5 binding sites in alpha-actinin-2 were found to lie within actinin's internal spectrin repeats. Unlike the reported interaction between actinin and the NMDA receptor, calmodulin was found to have no effect on actinin binding to Kv1.5.

Modulation of Kv1.5 potassium channel gating by extracellular zinc.

Zinc ions are known to induce a variable depolarizing shift of the ionic current half-activation potential and substantially slow the activation kinetics of most K(+) channels. In Kv1.5, Zn(2+) also reduces ionic current, and this is relieved by increasing the external K(+) or Cs(+) concentration. Here we have investigated the actions of Zn(2+) on the gating currents of Kv1.5 channels expressed in HEK cells. Zn(2+) shifted the midpoint of the charge-voltage (Q-V) curve substantially more (approximately 2 times) than it shifted the V(1/2) of the g-V curve, and this amounted to +60 mV at 1 mM Zn(2+). Both Q1 and Q2 activation charge components were similarly affected by Zn(2+), which indicated free access of Zn(2+) to channel closed states. The maximal charge movement was also reduced by 1 mM Zn(2+) by approximately 15%, from 1.6 +/- 0.5 to 1.4 +/- 0.47 pC (n = 4). Addition of external K(+) or Cs(+), which relieved the Zn(2+)-induced ionic current reduction, decreased the extent of the Zn(2+)-induced Q-V shift. In 135 mM extracellular Cs(+), 200 microM Zn(2+) reduced ionic current by only 8 +/- 1%, compared with 71% reduction in 0 mM extracellular Cs(+), and caused a comparable shift in both the g-V and Q-V relations (17.9 +/- 0.6 mV vs. 20.8 +/- 2.1 mV, n = 6). Our results confirm the presence of two independent binding sites involved in the Zn(2+) actions. Whereas binding to one site accounts for reduction of current and binding to the other site accounts for the gating shift in ionic current recordings, both sites contribute to the Zn(2+)-induced Q-V shift.

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.

External K(+) relieves the block but not the gating shift caused by Zn(2+) in human Kv1.5 potassium channels.

1. We used the whole-cell recording technique to examine the effect of extracellular Zn(2+) on macroscopic currents due to Kv1.5 channels expressed in the human embryonic kidney cell line HEK293. 2. Fits of a Boltzmann function to tail current amplitudes showed that 1 mM Zn2+ shifted the half-activation voltage from -10.2 +/- 0.4 to 21.1 +/- 0.7 mV and the slope factor increased from 6.8 +/- 0.4 to 9.4 +/- 0.7 mV. The maximum conductance in 1 mM Zn2+ and with 3.5 mM K(+)o was 33 +/- 7 % of the control value. 3. In physiological saline the apparent KD for the Zn(2+) block was 650 +/- 24 M and was voltage independent. A Hill coefficient of 1.0 +/- 0.03 implied that block is mediated by the occupation of a single binding site. 4. Increasing the external concentration of K(+) ([K(+)]o) inhibited the block by Zn(2+). Estimates of the apparent K(D) of the Zn(2+) block in 0, 5 and 135 mM K(+) were 69, 650 and 2100 M, respectively. External Cs(+) relieved the Zn(2+) block but was less effective than K(+). Changing [K(+)]o did not affect the Zn(2+)-induced gating shift. 5. A model of allosteric inhibition fitted to the relationship between the block by Zn(2+) and the block relief by external K(+) gave KD estimates of approximately 70 M for Zn(2+) and approximately 500 M for K(+). 6. We propose that the gating shift and the block caused by Zn(2+) are mediated by two distinct sites and that the blocking site is located in the external mouth of the pore.

Influence of permeating ions on Kv1.5 channel block by nifedipine.

Nifedipine can block K(+) currents through Kv1.5 channels in an open-channel manner (32). Replacement of internal and external K(+) with equimolar Rb(+) or Cs(+) reduced the potency of nifedipine block of Kv1.5 from an IC(50) of 7.3 microM (K(+)) to 16.0 microM (Rb(+)) and 26.9 microM (Cs(+)). The voltage dependence of block was unaffected, and a single binding site block model was used to describe block for all three ions. By varying ion species at the intra- and extracellular mouth of the channel and by using a nonconducting W472F-Kv1.5 mutant, we demonstrated that block was conditioned by the ion permeating the pore and, to a lesser extent, by the extracellular ion species alone. In Kv1.5, the outer pore mutations R487V and R487Y reduced nifedipine potency close to that of Kv4.2 and other Kv channels with an equivalent valine. Although changing this residue can affect C-type inactivation of Kv channels, the normalized reduction and time course of currents blocked by nifedipine in 5, 135, and 300 mM extracellular K(+) concentration was the same. Similarly, a mean recovery time constant from nifedipine block of 316 ms was unchanged (332 ms) after 5-s prepulses to allow C-type inactivation. This is consistent with the conclusion that nifedipine block and C-type inactivation in the Kv1.5 channel can coexist but are mediated by distinct mechanisms coordinated by outer pore conformation.

A high-Na(+) conduction state during recovery from inactivation in the K(+) channel Kv1.5.

Na(+) conductance through cloned K(+) channels has previously allowed characterization of inactivation and K(+) binding within the pore, and here we have used Na(+) permeation to study recovery from C-type inactivation in human Kv1.5 channels. Replacing K(+) in the solutions with Na(+) allows complete Kv1.5 inactivation and alters the recovery. The inactivated state is nonconducting for K(+) but has a Na(+) conductance of 13% of the open state. During recovery, inactivated channels progress to a higher Na(+) conductance state (R) in a voltage-dependent manner before deactivating to closed-inactivated states. Channels finally recover from inactivation in the closed configuration. In the R state channels can be reactivated and exhibit supernormal Na(+) currents with a slow biexponential inactivation. Results suggest two pathways for entry to the inactivated state and a pore conformation, perhaps with a higher Na(+) affinity than the open state. The rate of recovery from inactivation is modulated by Na(+)(o) such that 135 mM Na(+)(o) promotes the recovery to normal closed, rather than closed-inactivated states. A kinetic model of recovery that assumes a highly Na(+)-permeable state and deactivation to closed-inactivated and normal closed states at negative voltages can account for the results. Thus these data offer insight into how Kv1. 5 channels recover their resting conformation after inactivation and how ionic conditions can modify recovery rates and pathways.

alpha-actinin-2 couples to cardiac Kv1.5 channels, regulating current density and channel localization in HEK cells.

Voltage-gated K(+) (Kv) channels are particularly important in the physiology of excitable cells in the heart and the brain. PSD-95 is known to cluster Shaker channels and NMDA receptors and the latter is known to couple through alpha-actinin-2 to the post-synaptic cytoskeleton [Wyszynski et al. (1997) Nature 385, 439-442], but the mechanisms by which Kv channels are linked to the actin cytoskeleton and clustered at specific sites in the heart are unknown. Here we provide evidence that Kv1.5 channels, widely expressed in the cardiovascular system, bind with alpha-actinin-2. Human Kv1.5 interacts via its N-terminus/core region and can be immunoprecipitated with alpha-actinin-2 both after in vitro translation and from HEK cells expressing both proteins. The ion channels and alpha-actinin-2 co-localize at the membrane in HEK cells, where disruption of the actin cytoskeleton and antisense constructs to alpha-actinin-2 modulate the ion and gating current density.

Regulation of transient Na+ conductance by intra- and extracellular K+ in the human delayed rectifier K+ channel Kv1.5.

1. Significant Na+ conductance has been described in only a few native and cloned K+ channels, but has been used to characterize inactivation and K+ binding within the permeation pathway, and to refine models of K+ flux through multi-ion pores. Here we use Na+ permeation of the delayed rectifier K+ channel Kv1.5 to study extra- and intracellular K+ (K+o and K+i, respectively) regulation of conductance and inactivation, using whole-cell recording from human embryonic kidney (HEK)-293 cells. 2. Kv1.5 Na+ currents in the absence of K+o and K+i were confirmed by: (i) resistance of outward Na+ currents to dialysis by K+-free solutions; (ii) tail current reversal potential changes with Na+o with a slope of 55.8 mV per decade; (iii) block by 4-aminopyridine (50 % at 50 microM), and resistance to Cl- channel inhibition. 3. Na+ currents were transient followed by a small sustained current. An envelope test confirmed that activated Kv1.5 channels conducted Na+, and that rapid current decay reflected C-type inactivation. Sustained currents ( approximately 13 % of peak) represented Na+ flux through inactivated Kv1.5 channels. 4. K+o could modulate the maximum available Na+ conductance in the stable cell line while channels were closed. Before the first pulse of a train, increasing K+o concentration increased the subsequent Na+ conductance from approximately 15 (0 mM K+o) to 30 nS (5 mM K+o), with a Kd of 23 microM. Repeated low rate depolarizations in Na+i/Na+o solutions induced a use-dependent loss of Kv1.5 channel Na+ conductance, distinct from that caused by C-type inactivation. K+o binding that sensed little of the electric field could prevent this secondary loss of available Kv1.5 channels with a Kd of 230 microM. These two effects on conductance were both voltage independent, and had no effect on channel inactivation rate. 5. K+o concentrations >= 0.3 mM slowed the inactivation rate in a strongly voltage-dependent manner. This suggested it could compete for binding at a K+ site or sites deeper in the pore, as well as restoring the Na+ conductance. K+i was able to modulate the inactivation rate but was unable to affect conductance. 6. Mutation of arginine 487 in the outer pore region of the channel to valine (R487V) greatly reduced C-type inactivation in Na+ solutions, caused loss of channel use dependence, and prevented any conductance increase upon the addition of 0.1 mM K+o. Our results confirm the existence of a high affinity binding site at the selectivity filter that regulates inactivation, and also reveals the presence of at least one additional high affinity outer mouth site that predominantly regulates conductance of resting channels, and protects channels activated by depolarization when they conduct Na+.

Sequential gating in the human heart K(+) channel Kv1.5 incorporates Q(1) and Q(2) charge components.

On-gating current from the Kv1.5 cardiac delayed rectifier K(+) channel expressed in HEK-293 cells was separated into two distinct charge systems, Q(1) and Q(2), obtained from double Boltzmann fits to the charge-voltage relationship. Q(1) and Q(2) had characteristic voltage dependence and sensitivity with half-activation potentials of -29.6 +/- 1.6 and -2.19 +/- 2.09 mV and effective valences of 1. 87 +/- 0.15 and 5.53 +/- 0.27 e(-), respectively. The contribution to total gating charge was 0.20 +/- 0.04 for Q(1) and 0.80 +/- 0.04 (n = 5) for Q(2). At intermediate depolarizations, heteromorphic gating current waveforms resulted from relatively equal contributions from Q(1) and Q(2), but with widely different kinetics. Prepulses to -20 mV moved only Q(1), simplified on-gating currents, and allowed rapid Q(2) movement. Voltage-dependent on-gating current recovery in the presence of 4-aminopyridine (1 mM) suggested a sequentially coupled movement of the two charge systems during channel activation. This allowed the construction of a linear five-state model of Q(1) and Q(2) gating charge movement, which predicted experimental on-gating currents over a wide potential range. Such models are useful in determining state-dependent mechanisms of open and closed channel block of cardiac K(+) channels.

Non-specific action of methoxamine on Ito, and the cloned channels hKv 1.5 and Kv 4.2.

The alpha1-adrenoceptor agonist methoxamine acted independently of receptor activation to reduce Ito and the sustained outward current in rat ventricular myocytes, and hKv 1.5 and Kv 4.2 cloned K+ channel currents. Two hundred microM methoxamine reduced Ito by 36% in the presence of 2 microM prazosin, and by 37 and 38% after preincubation of myocytes with either N-ethylmaleimide or phenoxybenzamine (n=6). The EC50 values at +60 mV for direct reduction of Ito, hKv 1.5, and Kv 4.2 by methoxamine were 239, 276, and 363 microM, respectively, with Hill coefficients of 0.87-1.5. Methoxamine accelerated Ito and Kv 4.2 current inactivation in a concentration- and voltage-dependent manner. Apparent rate constants for methoxamine binding and unbinding gave Kd values in agreement with EC50 values measured from dose-response relations. The voltage-dependence of block supported charged methoxamine binding to a putative intracellular site that sensed approximately 20% of the transmembrane electrical field. In the presence of methoxamine, deactivating Kv 4.2 tail currents displayed a distinct rising phase, and were slowed relative to control, such that tail current crossover was observed. These observations support a dominant mechanism of open channel block, although closed channel block could not be ruled out. Single-channel data from hKv 1.5 patches revealed increased closed times with blank sweeps and decreased burst duration in the presence of drug, and a reduction of mean channel open time from 1.8 ms in control to 0.4 ms in 500 microM methoxamine. For this channel, therefore, both open and closed channel block appeared to be important mechanisms for the action of methoxamine.

Modulation of slow inactivation in human cardiac Kv1.5 channels by extra- and intracellular permeant cations.

1. The properties and regulation of slow inactivation by intracellular and extracellular cations in the human heart K+ channel hKv1.5 have been investigated. Extensive NH2- and COOH-terminal deletions outside the central core of transmembrane domains did not affect the degree of inactivation. 2. The voltage dependence of steady-state inactivation curves of hKv1.5 channels was unchanged in Rb+ and Cs+, compared with K+, but biexponential inactivation over 10 s was reduced from approximately 100 % of peak current in Na+ to approximately 65 % in K+, approximately 50 % in Rb+ and approximately 30 % in Cs+. This occurred as a result of a decrease in both fast and slow components of inactivation, with little change in inactivation time constants. 3. Changes in extracellular cation species and concentration (5-300 mM) had only small effects on the rates of inactivation and recovery from inactivation (tau recovery approximately 1 s). Mutation of residues at a putative regulatory site at R487 in the outer pore mouth did not affect slow inactivation or recovery from inactivation of hKv1.5, although sensitivity to extracellular TEA was conferred. 4. Symmetrical reduction of both intra- and extracellular cation concentrations accelerated and augmented both components of inactivation of K+ (Kd = 34.7 mM) and Cs+ (Kd = 20.5 mM) currents. These effects could be quantitatively accounted for by unilateral reduction of intracellular K+ (K+i) (Kd = 43.4 mM) or Cs+i with constant 135 mM external ion concentrations. 5. We conclude that inactivation and recovery from inactivation in hKv1.5 were not typically C-type in nature. However, the ion species dependence of inactivation was still closely coupled to ion permeation through the pore. Intracellular ion modulatory actions were more potent than extracellular actions, although still of relatively low affinity. These results suggest the presence of ion binding sites capable of regulating inactivation located on both intracellular and extracellular sides of the pore selectivity filter.

Gating current studies reveal both intra- and extracellular cation modulation of K+ channel deactivation.

1. The presence of permeant ions can modulate the rate of gating charge return in wild-type human heart K+ (hKv1.5) channels. Here we employ gating current measurements in a non-conducting mutant, W472F, of the hKv1.5 channel to investigate how different cations can modulate charge return and whether the actions can be specifically localized at the internal as well as the external mouth of the channel pore. 2. Intracellular cations were effective at accelerating charge return in the sequence Cs+ > Rb+ > K+ > Na+ > NMG+. Extracellular cations accelerated charge return with the selectivity sequence Cs+ > Rb+ > Na+ = NMG+. 3. Intracellular and extracellular cation actions were of relatively low affinity. The Kd for preventing slowing of the time constant of the off-gating current decay (tau off) was 20.2 mM for intracellular Cs+ (Cs+i) and 358 mM for extracellular Cs+ (Cs+o). 4. Both intracellular and extracellular cations can regulate the rate of charge return during deactivation of hKv1.5, but intracellular cations are more effective. We suggest that ion crystal radius is an important determinant of this action, with larger ions preventing slowing more effectively. Important parallels exist with cation-dependent modulation of slow inactivation of ionic currents in this channel. However, further experiments are required to understand the exact relationship between acceleration of charge return and the slowing of inactivation of ionic currents by cations.

The 1997 Stevenson Award Lecture. Cardiac K+ channel gating: cloned delayed rectifier mechanisms and drug modulation.

K+ channels are ubiquitous membrane proteins, which have a central role in the control of cell excitability. In the heart, voltage-gated delayed rectifier K+ channels, like Kv1.5, determine repolarization and the cardiac action potential plateau duration. Here we review the broader properties of cloned voltage-gated K+ channels with specific reference to the hKv1.5 channel in heart. We discuss the basic structural components of K+ channels such as the pore, voltage sensor, and fast inactivation, all of which have been extensively studied. Slow, or C-type, inactivation and the structural features that control pore opening are less well understood, although recent studies have given new insight into these problems. Information about channel transitions that occur prior to opening is provided by gating currents, which reflect charge-carrying transitions between kinetic closed states. By studying modulation of the gating properties of K+ channels by cations and with drugs, we can make a more complete interpretation of the state dependence of drug and ion interactions with the channel. In this way we can uncover the detailed mechanisms of action of K+ channel blockers such as tetraethylammonium ions and 4-aminopyridine, and antiarrhythmic agents such as nifedipine and quinidine.