Does the Na channel conduct ions through a water-filled pore or a condensed-state pathway?

Many investigators assert that the ion-conducting pathway of the Na channel is a water-filled pore. This assertion must be reevaluated to clear the way for more productive approaches to channel gating. The hypothesis of an aqueous pore leaves the questions of voltage-dependent gating and ion selectivity unexplained because a column of water can neither serve as a switch nor provide the necessary selectivity. The price of believing in an aqueous pore therefore is a futile search for separate ad hoc mechanisms for gating and selectivity. The fallacy is to assume that only water is available to carry ions rapidly, ignoring the role of the glycoprotein, which can form an elastomeric phase with water. The elastomer is a state of matter, neither liquid nor solid, in which the molecules of a liquid are threaded together with cross-linked polymer chains; it supports fast ion motion (Owen, 1989). An alternative hypothesis for channel gating, based on condensed-state materials science, already exists (Leuchtag, 1988, 1991a). The ferroelectric-superionic transition hypothesis (FESITH) postulates that the Na channel exists in a metastable ordered (closed) state at resting potential and, on threshold depolarization, undergoes a reversible order-disorder phase transition to a less-ordered, ion-conducting (open) state. The ordered state is ferroelectric; the disordered state is a fast ion conductor selective for Li+ and Na+. The basis of the voltage dependence is elevation of transition temperature with electric field, well established in ferroelectrics. FESITH is consistent with single-channel transitions, gating currents, heat and cold block, and other phenomena observed at channel or membrane level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase transitions and ion currents in a model ferroelectric channel unit

The hypothesis of ferroelectric electrodiffusion is examined mathematically. A thermodynamic potential, the elastic Gibbs function, written in polynomial form, provides the dielectric equation of state for the model. The other equations of electrodiffusion theory complete the model. This system reduces to a second-order partial differential equation, which is formally solved by the phase-plane method. This solution, applied to the Na channel, leads to a propagating phase-transition wave accompanied by movement of ionic charge. This may be readily interpreted as a transmembrane wave traveling along a ferroelectric unit within, and transporting ions through, the channel. Comparison of the temperature dependence of axonal conduction velocity with that of the spontaneous polarization of Rochelle salt suggests that the Na channel of squid axon contains a ferroelectric unit having a lower Curie point, but decomposing before reaching its upper Curie point. Comparison with data from reconstitution experiments suggests that the ferroelectric unit is a carbohydrate enclosed in an intrinsic protein structure to form a glycoprotein channel. The value experimentally estimated for the surface charge of the Na channel is within the range of spontaneous polarizations of typical ferroelectric crystals. It is argued that the ferroelectric probably is a single crystal of the order-disorder type, which undergoes a first-order transition between a ferroelectric and a paraelectric state during excitational activity. The hypothesis of ferroelectric channel units is consistent with the existence and directionality of the observed "gating" currents.     

Voltage-dependent membrane capacitance in rat pituitary nerve terminals due to gating currents

We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.     

Evidence for a population of sleepy sodium channels in squid axon at low temperature

We have studied the effects of temperature changes on Na currents in squid giant axons. Decreases in temperature in the 15-1 degrees C range decrease peak Na current with a Q10 of 2.2. Steady state currents, which are tetrodotoxin sensitive and have the same reversal potential as peak currents, are almost unaffected by temperature changes. After removal of inactivation by pronase treatment, steady state current amplitude has a Q10 of 2.3. Na currents generated at large positive voltages sometimes exhibit a biphasic activation pattern. The first phase activates rapidly and partially inactivates and is followed by a secondary slow current increase that lasts several milliseconds. Peak Na current amplitude can be increased by delivering large positive prepulses, an effect that is more pronounced at low temperatures. The slow activation phase is eliminated after a positive prepulse. The results are consistent with the hypothesis that there are two forms of the Na channel: (a) rapidly activating channels that completely inactivate, and (b) slowly activating "sleepy" channels that inactivate slowly if at all. Some fast channels are assumed to be converted to sleepy channels by cooling, possibly because of a phase transition in the membrane. The existence of sleepy channels complicates the determination of the Q10 of gating parameters and single-channel conductance.     

Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin

The gating charge and voltage dependence of the open state to the inactivated state (O-->I) transition was measured for the voltage- dependent mammalian cardiac Na channel. Using the site 3 toxin, Anthopleurin-A (Ap-A), which selectively modifies the O-->I transition (see Hanck, D. A., and M. F. Sheets. 1995. Journal of General Physiology. 106:601-616), we studied Na channel gating currents (Ig) in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Comparison of Ig recorded in response to step depolarizations before and after modification by Ap-A toxin showed that toxin-modified gating currents decayed faster and had decreased initial amplitudes. The predominate change in the charge-voltage (Q-V) relationship was a reduction in gating charge at positive potentials such that Qmax was reduced by 33%, and the difference between charge measured in Ap-A toxin and in control represented the gating charge associated with Na channels undergoing inactivation by O-->I. By comparing the time course of channel activation (represented by the gating charge measured in Ap-A toxin) and gating charge associated with the O-->I transition (difference between control and Ap-A charge), the influence of activation on the time course of inactivation could be accounted for and the inherent voltage dependence of the O-->I transition determined. The O-->I transition for cardiac Na channels had a valence of 0.75 e-. The total charge of the cardiac voltage-gated Na channel was estimated to be 5 e-. Because charge is concentrated near the opening transition for this isoform of the channel, the time constant of the O-->I transition at 0 mV could also be estimated (0.53 ms, approximately 12 degrees C). Prediction of the mean channel open time-voltage relationship based upon the magnitude and valence of the O- ->C and O-->I rate constants from INa and Ig data matched data previously reported from single Na channel studies in heart at the same temperature.     

Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects.

The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.     

Nonlinear charge movement in mammalian cardiac ventricular cells. Components from Na and Ca channel gating [published erratum appears in J Gen Physiol 1989 Aug;94(2):401]

Intramembrane charge movement was recorded in rat and rabbit ventricular cells using the whole-cell voltage clamp technique. Na and K currents were eliminated by using tetraethylammonium as the main cation internally and externally, and Ca channel current was blocked by Cd and La. With steps in the range of -110 to -150 used to define linear capacitance, extra charge moves during steps positive to approximately -70 mV. With holding potentials near -100 mV, the extra charge moving outward on depolarization (ON charge) is roughly equal to the extra charge moving inward on repolarization (OFF charge) after 50-100 ms. Both ON and OFF charge saturate above approximately +20 mV; saturating charge movement is approximately 1,100 fC (approximately 11 nC/muF of linear capacitance). When the holding potential is depolarized to -50 mV, ON charge is reduced by approximately 40%, with little change in OFF charge. The reduction of ON charge by holding potential in this range matches inactivation of Na current measured in the same cells, suggesting that this component might arise from Na channel gating. The ON charge remaining at a holding potential of -50 mV has properties expected of Ca channel gating current: it is greatly reduced by application of 10 muM D600 when accompanied by long depolarizations and it is reduced at more positive holding potentials with a voltage dependence similar to that of Ca channel inactivation. However, the D600-sensitive charge movement is much larger than the Ca channel gating current that would be expected if the movement of channel gating charge were always accompanied by complete opening of the channel.     

Pharmacological and kinetic analysis of K channel gating currents

We have measured gating currents from the squid giant axon using solutions that preserve functional K channels and with experimental conditions that minimize Na channel contributions to these currents. Two pharmacological agents were used to identify a component of gating current that is associated with K channels. Low concentrations of internal Zn2+ that considerably slow K channel ionic currents with no effect on Na channel currents altered the component of gating current associated with K channels. At low concentrations (10-50 microM) the small, organic, dipolar molecule phloretin has several reported specific effects on K channels: it reduces K channel conductance, shifts the relationship between channel conductance and membrane voltage (Vm) to more positive potentials, and reduces the voltage dependence of the conductance-Vm relation. The K channel gating charge movements were altered in an analogous manner by 10 microM phloretin. We also measured the dominant time constants of the K channel ionic and gating currents. These time constants were similar over part of the accessible voltage range, but at potentials between -40 and 0 mV the gating current time constants were two to three times faster than the corresponding ionic current values. These features of K channel function can be reproduced by a simple kinetic model in which the channel is considered to consist of two, two-state, nonidentical subunits.     

Free-energy landscapes of ion-channel gating are malleable: changes in the number of bound ligands are accompanied by changes in the location of the transition state in acetylcholine-receptor channels

Acetylcholine-receptor channels (AChRs) are allosteric membrane proteins that mediate synaptic transmission by alternatively opening and closing ("gating") a cation-selective transmembrane pore. Although ligand binding is not required for the channel to open, the binding of agonists (for example, acetylcholine) increases the closed right harpoon over left harpoon open equilibrium constant because the ion-impermeable --> ion-permeable transition of the ion pathway is accompanied by a low-affinity --> high-affinity change at the agonist-binding sites. The fact that the gating conformational change of muscle AChRs can be kinetically modeled as a two-state reaction has paved the way to the experimental characterization of the corresponding transition state, which represents a snapshot of the continuous sequence of molecular events separating the closed and open states. Previous studies of fully (di) liganded AChRs, combining single-channel kinetic measurements, site-directed mutagenesis, and data analysis in the framework of the linear free-energy relationships of physical organic chemistry, have suggested a transition-state structure that is consistent with channel opening being an asynchronous conformational change that starts at the extracellular agonist-binding sites and propagates toward the intracellular end of the pore. In this paper, I characterize the gating transition state of unliganded AChRs, and report a remarkable difference: unlike that of diliganded gating, the unliganded transition state is not a hybrid of the closed- and open-state structures but, rather, is almost indistinguishable from the open state itself. This displacement of the transition state along the reaction coordinate obscures the mechanism underlying the unliganded closed right harpoon over left harpoon open reaction but brings to light the malleable nature of free-energy landscapes of ion-channel gating.     

Simple shifts in the voltage dependence of sodium channel gating caused by divalent cations

The effect of elevated divalent cation concentration on the kinetics of sodium ionic and gating currents was studied in voltage-clamped frog skeletal muscle fibers. Raising the Ca concentration from 2 to 40 mM resulted in nearly identical 30-mV shifts in the time courses of activation, inactivation, tail current decay, and ON and OFF gating currents, and in the steady state levels of inactivation, charge immobilization, and charge vs. voltage. Adding 38 mM Mg to the 2 mM Ca bathing a fiber produced a smaller shift of approximately 20 mV in gating current kinetics and the charge vs. voltage relationship. The results with both Ca and Mg are consistent with the hypothesis that elevated concentrations of these alkali earth cations alter Na channel gating by changing the membrane surface potential. The different shifts produced by Ca and Mg are consistent with the hypothesis that the two ions bind to fixed membrane surface charges with different affinities, in addition to possible screening.     

Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers [published erratum appears in J Gen Physiol 1989 Apr;93(4):following 760]

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.     

Gating induces a conformational change in the outer vestibule of ENaC

The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (alpha, beta, and gamma). The channel forms the pathway for Na(+) absorption in the kidney, and mutations cause disorders of Na(+) homeostasis. However, little is known about the mechanisms that control the gating of ENaC. We investigated the gating mechanism by introducing bulky side chains at a position adjacent to the extracellular end of the second membrane spanning segment (549, 520, and 529 in alpha, beta, and gammaENaC, respectively). Equivalent "DEG" mutations in related DEG/ENaC channels in Caenorhabditis elegans cause swelling neurodegeneration, presumably by increasing channel activity. We found that the Na(+) current was increased by mutagenesis or chemical modification of this residue and adjacent residues in alpha, beta, and gammaENaC. This resulted from a change in the gating of ENaC; modification of a cysteine at position 520 in betaENaC increased the open state probability from 0. 12 to 0.96. Accessibility to this side chain from the extracellular side was state-dependent; modification occurred only when the channel was in the open conformation. Single-channel conductance decreased when the side chain contained a positive, but not a negative charge. However, alterations in the side chain did not alter the selectivity of ENaC. This is consistent with a location for the DEG residue in the outer vestibule. The results suggest that channel gating involves a conformational change in the outer vestibule of ENaC. Disruption of this mechanism could be important clinically since one of the mutations that increased Na(+) current (gamma(N530K)) was identified in a patient with renal disease.     

The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4.

Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Q(max)) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of I(Na) was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels. After Na channel modification by Ap-A toxin, decays of I(Na) were slowed to similar values for all four channel mutants. Toxin modification produced a graded effect on gating charge (Q) of mutant channels, reducing Q(max) by 12% for the R1C and R1Q mutants, by 22% for the R2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% reduction seen for wild-type currents. Consistent with these findings, the relationship of Q(max) to G(max) was significantly shallower for R1 mutants than for R2C and R3C mutant Na channels. These data suggest that site-3 toxins primarily inhibit gating charge associated with movement of the S4 in domain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.     

Dynamical properties of the MscL of Escherichia coli: a normal mode analysis

The mechanosensitive channel (MscL) is an integral membrane protein which gates in response to membrane tension. Physiological data have shown that the gating transition involves a very large change in the conformation, and that the open state of the channel forms a large non-specific pore with a high conductance. The Escherichia coli channel structure was first modeled by homology modeling, starting with the X-ray structure of the homologous from Mycobacterium tuberculosis. Then, the dynamical and conformational properties of the channel were explored, using normal mode analysis. Such an analysis was also performed with the different structures proposed recently by Sukharev and co-workers. Similar dynamical behaviors are observed, which are characteristic of the channel architecture, subtle differences being due to the different relative positioning of the structural elements. The ability of particular regions of the channel to deform is discussed with respect to the functional and structural properties, implied in the gating process. Our results show that the first step of the gating mechanism can be described with three low-frequency modes only. The movement associated to these modes is clearly an iris-like movement involving both tilt and twist rotation.     

Mutations in the S6 Gate Isolate a Late Step in the Activation Pathway and Reduce 4-AP Sensitivity in Shaker Kv Channel

K_v channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10–20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker’s sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a α-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.     

Mutations in the S6 Gate Isolate a Late Step in the Activation Pathway and Reduce 4-AP Sensitivity in Shaker Kv Channel

K_v channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10–20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker’s sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a α-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.     

Modifications of human cardiac sodium channel gating by UVA light

Voltage-gated Na(+) channels are membrane proteins responsible for the generation of action potentials. In this report we demonstrate that UVA light elicits gating changes of human cardiac Na+ channels. First, UVA irradiation hampers the fast inactivation of cardiac Nav1.5 Na(+) channels expressed in HEK293t cells. A maintained current becomes conspicuous during depolarization and reaches its maximal quasi steady-state level within 5-7 min. Second, the activation time course is slowed by UVA light; modification of the activation gating by UVA irradiation continues for 20 min without reaching steady state. Third, along with the slowed activation time course, the peak current is reduced progressively. Most Na(+) currents are eliminated during 20 min of UVA irradiation. Fourth, UVA light increases the holding current nonlinearly; this phenomenon is slow at first but abruptly fast after 20 min. Other skeletal muscle Nav1.4 isoforms and native neuronal Na(+) channels in rat GH(3) cells are likewise sensitive to UVA irradiation. Interestingly, a reactive oxygen metabolite (hydrogen peroxide at 1.5%) and an oxidant (chloramine-T at 0.5 mM) affect Na(+) channel gating similarly, but not identically, to UVA. These results together suggest that UVA modification of Na(+) channel gating is likely mediated via multiple reactive oxygen metabolites. The potential link between oxidative stress and the impaired Na(+) channel gating may provide valuable clues for ischemia/reperfusion injury in heart and in CNS.     

Long-range interactions, voltage sensitivity, and ion conduction in S4 segments of excitable channels.

Forces acting on the S4 segments of the channel, the voltage-sensing structures, are analyzed. The conformational change in the Na channel is modeled as a helix-coil transition in the four S4 segments, coupled to the membrane voltage by electrical forces. In the model, repulsions between like charges make the S4 segment unstable, but field-dependent forces hold it in an alpha-helix configuration at resting potential. At threshold depolarization, the S4 helices cooperatively expand into random coils, breaking the hydrogen bonds connecting adjacent loops of the alpha helices. Exposed electron pairs left on the carbonyl oxygens constitute sites at which cations can bind selectively. The first hydrogen bond to break is at the channel exterior, then the second breaks, and so on in a zipper-like motion along the entire segment. The Na+ ions hop from one site to the next until all H bonds are broken and all sites are filled with ions. This completes the pathway over which the permeant ions move through the channel, driven by the electrochemical potential difference across the membrane. This microscopic mechanism is consistent with the thermodynamic explanation of ion-channel gating previously formulated as the ferroelectric-superionic transition hypothesis.     

Capacitance fluctuations causing channel noise reduction in stochastic Hodgkin-Huxley systems

Voltage-dependent ion channels determine the electric properties of axonal cell membranes. They not only allow the passage of ions through the cell membrane, but also contribute to an additional charging of the cell membrane resulting in the so-called capacitance loading. The switching of the channel gates between an open and a closed configuration is intrinsically related to the movement of gating charge within the cell membrane. At the beginning of an action potential, the transient gating current is opposite to the direction of the current of sodium ions through the membrane. Therefore, the excitability is expected to become reduced due to the influence of a gating current. Our stochastic Hodgkin-Huxley-like modeling takes into account both the channel noise-i.e. the fluctuations of the number of open ion channels-and the capacitance fluctuations that result from the dynamics of the gating charge. We investigate the spiking dynamics of membrane patches of a variable size and analyze the statistics of the spontaneous spiking. As a main result, we find that the gating currents yield a drastic reduction of the spontaneous spiking rate for sufficiently large ion channel clusters. Consequently, this demonstrates a prominent mechanism for channel noise reduction.     

K(+)-dependent composite gating of the yeast K(+) channel, Tok1

TOK1 encodes an outwardly rectifying K(+) channel in the plasma membrane of the budding yeast Saccharomyces cerevisiae. It is capable of dwelling in two kinetically distinct impermeable states, a near-instantaneously activating R state and a set of related delayed activating C states (formerly called C(2) and C(1), respectively). Dwell in the R state is dependent on membrane potential and both internal and external K(+) in a manner consistent with the K(+) electrochemical potential being its determinant, where dwell in the C states is dependent on voltage and only external K(+). Whereas activation from the C states showed high temperature dependencies, typical of gating transitions in other Shaker-like channels, activation from the R state had a temperature dependence nearly as low as that of simple ionic diffusion. These findings lead us to conclude that although the C states reflect the activity of an internally oriented channel gate, the R state results from an intrinsic gating property of the channel filter region.