Whole-cell clamp of dissociated photoreceptors from the eye of Lima scabra

Voltage-dependent membrane currents were investigated in enzymatically dissociated photoreceptors of Lima scabra using the whole-cell clamp technique. Depolarizing steps to voltages more positive than -10 mV elicit a transient inward current followed by a delayed, sustained outward current. The outward current is insensitive to replacement of a large fraction of extracellular Cl- with the impermeant anion glucuronate. Superfusion with tetraethylammonium and 4-aminopyridine reversibly abolishes the outward current, and internal perfusion with cesium also suppresses it, indicating that it is mediated by potassium channels. Isolation of the inward current reveals a fast activation kinetics, the peak amplitude occurring as early as 4-5 ms after stimulus onset, and a relatively rapid, though incomplete inactivation. Within the range of voltages examined, spanning up to +90 mV, reversal was not observed. The inward current is not sensitive to tetrodotoxin at concentrations up to 10 microM, and survives replacement of extracellular Na with tetramethylammonium. On the other hand, it is completely eliminated by calcium removal from the perfusing solution, and it is partially blocked by submillimolar concentrations of cadmium, suggesting that it is entirely due to voltage-dependent calcium channels. Analysis of the kinetics and voltage dependence of the isolated calcium current indicates the presence of two components, possibly reflecting the existence of separate populations of channels. Barium and strontium can pass through these channels, though less easily than calcium. Both the activation and the inactivation become significantly more sluggish when these ions serve as the charge carrier. A large fraction of the outward current is activated by preceding calcium influx. Suppression of this calcium-dependent potassium current shows a small residual component resembling the delayed rectifier. In addition, a transient outward current sensitive to 4-aminopyridine (Ia) could also be identified. The relevance of such conductance mechanisms in the generation of the light response in Lima photoreceptors is discussed.     

Effect of sotalol on transmembrane ionic currents responsible for repolarization in cardiac ventricular myocytes from rabbit and guinea pig

The effects of sotalol, a β-adrenoceptor blocker and class III antiarrhythmic agent, on transmembrane ionic currents were examined in single rabbit and guinea pig ventricular myocytes using whole-cell voltage-clamp techniques. In neither of these species did 60 μM sotalol appreciably effect the inward rectifier, the transient outward or the inward calcium currents. In addition, sotalol did not elicit a slowly inactivating component of the sodium current as did 1 μg/ml veratrine. In guinea pig ventricular myocytes, sotalol also significantly depressed the outward delayed rectifier current. An outward delayed rectifier current was not observed in rabbit ventricular myocytes examined at room temperature; and, under these conditions sotalol did not lengthen action potential duration. Sotalol induced lengthening of cardiac action potential duration can, therefore, be explained by depression the outward delayed rectifier current.     

Slow components of potassium tail currents in rat skeletal muscle

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.     

Sulfur dioxide derivative modulation of potassium channels in rat ventricular myocytes

The effects of sulfur dioxide (SO2) derivatives (bisulfite and sulfite, 1:3 M/M) on voltage-dependent potassium current in isolated adult rat ventricular myocyte were investigated using the whole cell patch-clamp technique. SO2 derivatives (10 microM) increased transient outward potassium current (I(to)) and inward rectifier potassium current (I(K1)), but did not affect the steady-state outward potassium current (I(ss)). SO2 derivatives significantly shifted the steady-state activation curve of I(to) toward the more negative potential at the V(h) point, but shifted the inactivation curve to more positive potential. SO2 derivatives markedly shifted the curve of time-dependent recovery of I(to) from the steady-state inactivation to the left, and accelerated the recovery of I(to) from inactivation. In addition, SO2 derivatives also significantly change the inactivation time constants of I(to) with increasing fast time constant and decreasing slow time constant. These results indicated a possible correlation between the change of properties of potassium channel and SO2 inhalation toxicity, which might cause cardiac myocyte injury through increasing extracellular potassium via voltage-gated potassium channels.     

Characterization and functional consequences of delayed rectifier current transient in ventricular repolarization

Although inactivation of the rapidly activating delayed rectifier current (I(Kr)) limits outward current on depolarization, the role of I(Kr) (and recovery from inactivation) during repolarization is uncertain. To characterize I(Kr) during ventricular repolarization (and compare with the inward rectifier current, I(K1)), voltage-clamp waveforms simulating the action potential were applied to canine ventricular, atrial, and Purkinje myocytes. In ventricular myocytes, I(Kr) was minimal at plateau potentials but transiently increased during repolarizing ramps. The I(Kr) transient was unaffected by repolarization rate and maximal after 150-ms depolarizations (+25 mV). Action potential clamps revealed the I(Kr) transient terminating the plateau. Although peak I(Kr) transient density was relatively uniform among myocytes, potentials characterizing the peak transients were widely dispersed. In contrast, peak inward rectifier current (I(K1)) density during repolarization was dispersed, whereas potentials characterizing I(K1) defined a narrower (more negative) voltage range. In summary, rapidly activating I(Kr) provides a delayed voltage-dependent (and functionally time-independent) outward transient during ventricular repolarization, consistent with rapid recovery from inactivation. The heterogeneous voltage dependence of I(Kr) provides a novel means for modulating the contribution of this current during repolarization.     

Calcium currents in a fast-twitch skeletal muscle of the rat

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.     

Potassium currents in cultured cells of the rat retinal pigment epithelium

Whole-cell currents were investigated in cultured rat retinal pigment epithelial (RPE) cells. Two voltage-dependent conductances were discriminated. First, at potentials more positive than −30 mV, a time-dependent outward current was activated. Inhibition by Ba²⁺ (10 mM) and 4-aminopyridine (10 mM) indicated that this current was carried by potassium ions. This current showed no inactivation during 5 sec depolarizations. Second, an inward current, sensitive to Ba²⁺ (10 mM) and 4-aminopyridine (10 mM), was activated at potentials more negative than — 70 mV. Under extra- and intracellular potassium-free conditions, both currents disappeared. In summary, cultured rat RPE cells expressed one potassium conductance similar to the delayed rectifier and one similar to the inward rectifier. The delayed rectifier expressed characteristics comparable with those known in mammalian species and different from those in non-mammalian species.     

A model for human ventricular tissue

The experimental and clinical possibilities for studying cardiac arrhythmias in human ventricular myocardium are very limited. Therefore, the use of alternative methods such as computer simulations is of great importance. In this article we introduce a mathematical model of the action potential of human ventricular cells that, while including a high level of electrophysiological detail, is computationally cost-effective enough to be applied in large-scale spatial simulations for the study of reentrant arrhythmias. The model is based on recent experimental data on most of the major ionic currents: the fast sodium, L-type calcium, transient outward, rapid and slow delayed rectifier, and inward rectifier currents. The model includes a basic calcium dynamics, allowing for the realistic modeling of calcium transients, calcium current inactivation, and the contraction staircase. We are able to reproduce human epicardial, endocardial, and M cell action potentials and show that differences can be explained by differences in the transient outward and slow delayed rectifier currents. Our model reproduces the experimentally observed data on action potential duration restitution, which is an important characteristic for reentrant arrhythmias. The conduction velocity restitution of our model is broader than in other models and agrees better with available data. Finally, we model the dynamics of spiral wave rotation in a two-dimensional sheet of human ventricular tissue and show that the spiral wave follows a complex meandering pattern and has a period of 265 ms. We conclude that the proposed model reproduces a variety of electrophysiological behaviors and provides a basis for studies of reentrant arrhythmias in human ventricular tissue.     

Time-dependent outward current in guinea pig ventricular myocytes. Gating kinetics of the delayed rectifier

Several conflicting models have been used to characterize the gating behavior of the cardiac delayed rectifier. In this study, whole-cell delayed rectifier currents were measured in voltage-clamped guinea pig ventricular myocytes, and a minimal model which reproduced the observed kinetic behavior was identified. First, whole-cell potassium currents between -10 and +70 mV were recorded using external solutions designed to eliminate Na and Ca currents and two components of time-dependent outward current were found. One component was a La3(+)-sensitive current which inactivated and resembled the transient outward current described in other cell types; single-channel observations confirmed the presence of a transient outward current in these guinea pig ventricular cells (gamma = 9.9 pS, [K]o = 4.5 mM). Analysis of envelopes of tail amplitudes demonstrated that this component was absent in solutions containing 30-100 microM La3+. The remaining time-dependent current, IK, activated with a sigmoidal time course that was well-characterized by three time constants. Nonlinear least-squares fits of a four-state Markovian chain model (closed - closed - closed - open) to IK activation were therefore compared to other models previously used to characterize IK gating: n2 and n4 Hodgkin-Huxley models and a Markovian chain model with only two closed states. In each case the four-state model was significantly better (P less than 0.05). The failure of the Hodgkin-Huxley models to adequately describe the macroscopic current indicates that identical and independent gating particles should not be assumed for this K channel. The voltage-dependent terms describing the rate constants for the four-state model were then derived using a global fitting approach for IK data obtained over a wide range of potentials (-80 to +70 mV). The fit was significantly improved by including a term representing the membrane dipole forces (P less than 0.01). The resulting rate constants predicted long single-channel openings (greater than 1 s) at voltages greater than 0 mV. In cell-attached patches, single delayed rectifier channels which had a mean chord conductance of 5.4 pS at +60 mV ([K]o = 4.5 mM) were recorded for brief periods. These channels exhibited behavior predicted by the four-state model: long openings and latency distributions with delayed peaks. These results suggest that the cardiac delayed rectifier undergoes at least two major transitions between closed states before opening upon depolarization.     

Voltage-dependent conductances in Limulus ventral photoreceptors

The voltage-dependent conductances of Limulus ventral photoreceptors have been investigated using a voltage-clamp technique. Depolarization in the dark induces inward and outward currents. The inward current is reduced by removing Na+ or Ca2+ and is abolished by removing both ions. These results suggest that both Na+ and Ca2+ carry voltage-dependent inward current. Inward current is insensitive to tetrodotoxin but is blocked by external Ni2+. The outward current has a large transient component that is followed by a smaller maintained component. Intracellular tetraethylammonium preferentially reduces the maintained component, and extracellular 4-amino pyridine preferentially reduces the transient component. Neither component is strongly affected by removal of extracellular Ca2+ or by intracellular injection of EGTA. It is concluded that the photoreceptors contain at least three separate voltage-dependent conductances: 1) a conductance giving rise to inward currents; 2) a delayed rectifier giving rise to maintained outward K+ current; and 3) a rapidly inactivating K+ conductance similar to the A current of molluscan neurons.     

Voltage Clamp Studies on the Slow Inward Current during the Excitation of Nitellopsis obtusa

The ionic currents across the plasmalemma of Nitellopsis obtusawere measured in voltage clamp experiments. Depolarization ofthe cell by 30–100 mV from the level of the resting potentialresulted in (1) a rapid inward current, (2) a subsequent slowinward current, and (3) a stationary outward current. The firstcurrent component changed sign at –20 to –30 mV.The second component decreased to a minimum at this clampedlevel. With increasing depolarizing steps some slow transientcurrent component reappeared without changing sign. This transientinward current occurred also when the potential was clampedeither at large depolarizing (+80 mV) or at large hyperpolarizing(–300 mV) potentials. In cases when the slow inward currentcomponent was evident cessation of protoplasmic streaming wasobserved. The ATPase inhibitor dicyclohexylcarbodiimide (DCCD)at a concentration of 2 x 10^–5 M in the external mediuminhibited the slow transient inward current without affectingthe first rapid current component. It is suggested that theirreversible slow transient current component reflects the onsetof some active ion-transport system in the plasmalemma duringcell excitation.     

氧自由基致豚鼠心室肌细胞跨膜电位变化的离子电流基础

目的：旨在提示氧自由基参与缺血／再灌注性心委失常发生的离子电流基础。方法：采用膜片钳全细胞式记录技术,观察Ｈ２Ｏ２（１ｍｍｏｌ／Ｌ）对豚鼠心室肌细胞跨膜电位和相关离子电流的影响。结果：Ｈ２Ｏ２使豚鼠心肌单细胞的静息电位（ＲＰ）降低,动作电位时程（ＡＳＤ）显著缩短,对动作电位幅度（ＡＰＡ）和超射（ＯＳ）及钠电流的峰值（ＩＮａ）均无明显影响;明显抑制内向整流钾电流（ＩＫ１）,尤其在超极化时;增强延迟外     

Muscarinic activation of transient inward current and contraction in canine colon circular smooth muscle cells

Muscarinic receptor mediated membrane currents and contractions were studied in isolated canine colon circular smooth muscle cells. Carbachol (10(-5) M) evoked a slow transient inward current that was superimposed by a transient outward current at holding potentials greater than -50 mV. Carbachol contracted the cells by 70 +/- 2%. The effects of carbachol were blocked by atropine (10(-6) M), tetraethyl ammonium (20 mM), and BAPTA-AM (25 mM applied for 20 min). The inward current and contraction were not sensitive to diltiazem (10(-5) M), nitrendipine (3 x 10(-7) M), niflumic acid (10(-5) M), or N-phenylanthranilic acid (10(-4) M), but were gradually inhibited after repetitive stimulations in Ca2+ free solution. Ni2+ (2 mM) inhibited the inward current by 67 +/- 4%. The inward current reversed at +15 mV. The outward component could be selectively inhibited by iberiotoxin (20 nM) or by intracellular Cs+. Repeated stimulation in the presence of cyclopiazonic acid (CPA, 3 microM) inhibited the carbachol-induced outward current and partially inhibited contraction. CPA did not inhibit the inward current. In conclusion, muscarinic receptor stimulation evoked a CPA-sensitive calcium release that caused contraction and a CPA-insensitive transient inward current was activated that is primarily carried by Ca2+ ions and is sensitive to Ni2+.     

Electrical remodeling of cardiac myocytes from mice with heart failure due to the overexpression of tumor necrosis factor-alpha

Mice that overexpress the inflammatory cytokine tumor necrosis factor-alpha in the heart (TNF mice) develop heart failure characterized by atrial and ventricular dilatation, decreased ejection fraction, atrial and ventricular arrhythmias, and increased mortality (males > females). Abnormalities in Ca2+ handling, prolonged action potential duration (APD), calcium alternans, and reentrant atrial and ventricular arrhythmias were previously observed with the use of optical mapping of perfused hearts from TNF mice. We therefore tested whether altered voltage-gated outward K+ and/or inward Ca2+ currents contribute to the altered action potential characteristics and the increased vulnerability to arrhythmias. Whole cell voltage-clamp recordings of K+ currents from left ventricular myocytes of TNF mice revealed an approximately 50% decrease in the rapidly activating, rapidly inactivating transient outward K+ current Ito and in the rapidly activating, slowly inactivating delayed rectifier current IK,slow1, an approximately 25% decrease in the rapidly activating, slowly inactivating delayed rectifier current IK,slow2, and no significant change in the steady-state current Iss compared with controls. Peak amplitudes and inactivation kinetics of the L-type Ca2+ current ICa,L were not altered. Western blot analyses revealed a reduction in the proteins underlying Kv4.2, Kv4.3, and Kv1.5. Thus decreased K+ channel expression is largely responsible for the prolonged APD in the TNF mice and may, along with abnormalities in Ca2+ handling, contribute to arrhythmias.     

Modelling action potentials and membrane currents of mammalian skeletal muscle fibres in coherence with potassium concentration changes in the T-tubular system

During prolonged activity the action potentials of skeletal muscle fibres change their shape. A model study was made as to whether potassium accumulation and removal in the tubular space is important with respect to those variations. Classical Hodgkin-Huxley type sodium and (potassium) delayed rectifier currents were used to determine the sarcolemmal and tubular action potentials. The resting membrane potential was described with a chloride conductance, a potassium conductance (inward rather than outward rectifier) and a sodium conductance (minor influence) in both sarcolemmal and tubular membranes. The two potassium conductances, the Na-K pump and the potassium diffusion between tubular compartments and to the external medium contributed to the settlement of the potassium concentration in the tubular space. This space was divided into 20 coupled concentric compartments. In the longitudinal direction the fibre was a cable series of 56 short segments. All the results are concerned with one of the middle segments. During action potentials, potassium accumulates in the tubular space by outward current through both the delayed and inward rectifier potassium conductances. In between the action potentials the potassium concentration decreases in all compartments owing to potassium removal processes. In the outer tubular compartment the diffusion-driven potassium export to the bathing solution is the main process. In the inner tubular compartment, potassium removal is mainly effected by re-uptake into the sarcoplasm by means of the inward rectifier and the Na-K pump. This inward transport of potassium strongly reduces the positive shift of the tubular resting membrane potential and the consequent decrease of the action potential amplitude caused by inactivation of the sodium channels. Therefore, both potassium removal processes maintain excitability of the tubular membrane in the centre of the fibre, promote excitation-contraction coupling and contribute to the prevention of fatigue. Received: 5 May 1998 / Revised version: 27 October 1998 / Accepted: 19 January 1999     

Plasmalemmal,voltage-dependent ionic currents from excitable pulvinar motor cells ofMimosa pudica

Summary Plasmalemmal ionic currents from excitable motor cells of the primary pulvinus ofMimosa pudica were investigated by patch-clamp techniques. In almost all of the enzymatically isolated protoplasts, a delayed rectifier potassium current was activated by depolarization, while no currents were detected upon hyperpolarization. This sustained outward current was reversibly blocked by Ba and TEA and serves to repolarize the membrane potential. Outward single channel currents that very likely underly the macroscopic outward potassium current had an elementary conductance of 20 pS. In addition, in a few protoplasts held at hyperpolarized potentials, depolarization-activated transient inward currents were observed, and under current clamp, action potential-like responses were triggered by depolarizing current injections or by mechanical perturbations. The activation characteristics of both inward currents and spikes showed striking similarities compared to those of action potentialsin situ.     

三氯化铝对大鼠海马CA1区锥体细胞瞬间外向钾电流和延迟整流钾电流的影响

采用全细胞膜片钳技术,研究了三氯化铝(AlCl3)对急性分离的大鼠海马CA1区神经元钾通道的影响。结果表明,AlCl3对钾电流有明显的抑制作用,具有一定的浓度依赖性,1000μmol／AlCl3可改变IA和IX激活曲线和失活曲线的Vb和k值,使钾电流激活曲线右移,使失活曲线左移。这些结果表明AlCl3对大鼠海马CA1区神经元K^ 通道有抑制作用,它可能是铝引起中枢神经系统损伤的机制之一。     

Passive properties and membrane currents of canine ventricular myocytes

The membrane potential and membrane currents of single canine ventricular myocytes were studied using either single microelectrodes or suction pipettes. The myocytes displayed passive membrane properties and an action potential configuration similar to those described for multicellular dog ventricular tissue. As for other cardiac cells, in canine ventricular myocytes: (a) an inward rectifier current plays an important role in determining the resting membrane potential and repolarization rate; (b) a tetrodotoxin-sensitive Na current helps maintain the action potential plateau; and (c) the Ca current has fast kinetics and a large amplitude. Unexpected findings were the following: (a) in approximately half of the myocytes, there is a transient outward current composed of two components, one blocked by 4-aminopyridine and the other by Mn or caffeine; (b) there is clearly a time-dependent outward current (delayed rectifier current) that contributes to repolarization; and (c) the relationship of maximum upstroke velocity of phase 0 to membrane potential is more positive and steeper than that observed in cardiac tissues from Purkinje fibers.     

Ionic currents in cardiac myocytes of squid, Alloteuthis subulata

This paper provides the first study of voltage-sensitive membrane currents present in heart myocytes from cephalopods. Whole cell patch clamp recordings have revealed six different ionic currents in myocytes freshly dissociated from squid cardiac tissues (branchial and systemic hearts). Three types of outward potassium currents were identified: first, a transient outward voltage-activated A-current (I_A), blocked by 4-aminopyridine, and inactivated by holding the cells at a potential of −40 mV; second, an outward, voltage-activated, delayed rectifier current with a sustained time course (I_K); and third, an outward, calcium-dependent, potassium current (I_K(Ca)) sensitive to Co²⁺ and apamin, and with the characteristic N-shaped current voltage relationship. Three inward voltage-activated currents were also identified. First, a rapidly activating and inactivating, sodium current (I_Na), blocked by tetrodotoxin, inactivated at holding potentials more positive than −40 mV, and abolished when external sodium was replaced by choline. Second, an L-type calcium current (I_Ca,L) with a sustained time course, suppressed by nifedipine or Co²⁺, and enhanced by substituting Ca²⁺ for Ba²⁺ in the external medium. The third inward current was also carried by calcium ions, but could be distinguished from the L-type current by differences in its voltage dependence. It also had a more transient time course, was activated at more negative potentials, and resembled the previously described low-voltage-activated, T-type calcium current. Accepted: 24 September 1999     

Inward rectification of the minK potassium channel

The minK protein induces a slowly activating voltage-dependent potassium current when expressed in Xenopus oocytes. We have used macroscopic minK currents to determine the open channel current-voltage relationship for the channel, and have found that the minK current is inwardly rectifying. The channel passes inward current at least 20fold more readily than outward current. Both rat and human minK exhibit this property. The rectification of minK is similar to that reported for a slow component of the cardiac delayed rectifier, strengthening the hypothesis that minK is responsible for that current.We would like to thank Drs. Steve Goldstein and Chris Miller for the artificial rat minK gene, and Dr. Rick Swanson for the human minK construct. This work was supported by NIH grant GM-48851 to L.K.K.