Activation and Inactivation of Sodium Currents.

The total membrane current in a node of Ranvier consists of a capacity current and an ionic current. The ionic current has three components: In addition to current flow through ionic channels selective for Na⁺ and K⁺, a leakage current of unknown origin regularly is present in nodal voltage-clamp recordings. By definition, the leakage current has a linear potential dependence and does not show activation and inactivation kinetics. Moreover, the leakage current as yet cannot be blocked by specific substances. Sodium currents can be isolated from the other membrane currents by measuring the tetrodotoxin (TTX)-sensitive current, as demonstrated in Figure 13-4 . TTX specifically blocks sodium currents in nodal membranes of frog (Hille, 1968 ) and rat (Neumcke et al., 1987 ) nerve fibers. After sodium channel blockage with TTX, a small-capacity current becomes

fig. 13-1. Action potentials recorded in a rat node of Ranvier. A, Action potentials recorded at 20°C. The action potentials were elicited with a 0.1 millisecond depolarizing current stimulus before and after exchange of the normal solutions with external TEA and internal CsCl. B, A local response and an action potential elicited at 37°C in Ringer’s solution with 30 μs sub- and suprathreshold current stimuli. Note the different time scales in A and B. (From Schwarz and Eikhof, 1987 ).

fig. 13-2. Membrane currents in a rat and a frog nerve fiber induced by depolarizing pulses in Ringer’s solution. Capacity and leakage currents have been subtracted. Each positive pulse was preceded by a 50 mV negative pulse of 50 milliseconds (E_pre). E_H, holding potential of −75 mV in the rat and −70 mV in the frog. (From Röper and Schwarz, 1989 .)

Sodium conductance of the nodal membrane increases upon depolarization and decreases upon repolarization. However, Figure 13-4 shows that the sodium current also decreases during a maintained depolarization. This process is called sodium conductance inactivation and leaves the membrane refractory for a few milliseconds. In the Hodgkin-Huxley model (Frankenhaeuser and Huxley, 1964 ; Hodgkin and Huxley, 1952 ), this behavior of sodium currents is described by assuming the existence of two independent gating processes, activation and inactivation, that involve two kinds of gating particles, called m and h. In the rat node of Ranvier three m particles control activation and one h particle controls inactivation (Neumcke and Stämpfli, 1982 ; Neumcke et al., 1987 ). The probability that a sodium channel is in its open state is therefore m ³ h, and the macroscopic sodium current (I _Na) can be described by $$I_{\text{Na}}=m^{3}h\cdot g_{\text{Na}}\left(E-E_{\text{Na}}\right)$$ where E = the membrane potential, E _Na = the sodium equilibrium potential, and g _Na = the sodium-limiting conductance. As will be shown below, single-channel recordings demonstrate that there is indeed a homogeneous population of sodium channels. With a single-channel conductance γ_Na and a given number of sodium channels N, the equation can be written as $$I_{\text{Na}}=m^{3}h\cdot N\cdot {\gamma }_{\text{Na}}\left(E-E_{\text{Na}}\right)$$

To obtain standard data for the calculation of action potentials, a voltage-clamp analysis of sodium and potassium conductances within a potential range from E = −120 to +80 mV must be performed (Frankenhaeuser and Huxley, 1964 ). The design of the pulse protocols is very similar to that first described by Hodgkin and Huxley ( 1952 ). Frankenhaeuser ( 1960 ) described sodium currents in the node of Ranvier of the toad Xenopus laevis by equations similar to those used for the description of membrane currents in the squid giant axon by Hodgkin and Huxley ( 1952 ). These equations are still very useful for describing the macroscopic membrane currents of amphibian as well as mammalian myelinated axons.

Chiu et al. ( 1979 ) showed that sodium inactivation is faster in rabbit compared with frog nerve fibers. Furthermore, these authors found that the action potential duration in frog and rabbit is similar. They explained

fig. 13-3. Action potential and membrane currents recorded from the same fiber isolated from a human ulnar nerve. Left, Action potential elicited with a 0.5 millisecond depolarizing current stimulus. Right, Membrane currents recorded upon depolarizing potential steps from a holding potential of −80 mV to membrane potentials between −20 and +100 mV in steps of 20 mV. Each depolarization was preceded by a 50 millisecond hyperpolarization to −120 mV. Leakage and capacity currents were not subtracted. (From Schwarz et al., 1993 .)

fig. 13-4. Membrane currents induced by a 50 mV depolarizing potential step in a single myelinated rat nerve fiber at 20°C following a 50 mV hyperpolarizing potential of 50 milliseconds. Top, Pulse protocol. A, Membrane current in normal Ringer’s solution. B, Same protocol as in A, except that 300 nM TTX was added to the Ringer’s solution. C, TTX-sensitive current obtained by subtracting the membrane current shown in B from that in A. One data point is plotted every 50 μs during the first 1.5 milliseconds, and thereafter every 0.5 millisecond. The continuous line was calculated assuming two time constants for sodium inactivation.

To describe sodium and potassium currents in the node of Ranvier, a better approximation often is obtained when permeabilities instead of conductances are calculated with the Goldman equation (Frankenhaeuser, 1960, 1963 ). A complete analysis of sodium permeability activation and inactivation in rat nerve fibers has been made by Schwarz and Eikhof ( 1987 ) and Neumcke et al. ( 1987 ). An analysis of potassium conductance in rat nerve fibers based on that done in the frog (Dubois, 1981 ) can be found in the paper by Röper and Schwarz ( 1989 ).

Temperature Dependence.

In vivo, action potentials in mammalian myelinated nerve fibers are generated and propagated at 37°C. Figure 13-1B shows that the duration of an action potential recorded at 37°C is about 0.3 milliseconds, in contrast to 1.5 milliseconds at 20°C (Schwarz and Eikhof, 1987 ). The temperature coefficient, Q ₁₀, for action potential duration is dependent on temperature, decreasing from 3.7 between 0° and 10°C to 2.2 between 20° and 40°C (Schwarz, 1986 ). In addition to the change in duration, the action potential recorded at 37°C has a smaller amplitude than that recorded at 20°C. This difference is due to the larger leakage conductance and the lower sodium equilibrium potential measured at 37°C. Both changes could be due to experimental artifacts, which occur more easily at 37° than at 20°C. The smaller action potential amplitude also could be explained by differences in the Q₁₀ of the rate constants for sodium activation and inactivation. Indeed, in amphibian nerve fibers, Frankenhaeuser and Moore ( 1963 ) found that the Q ₁₀ for sodium inactivation rate constants α _h and β _h are 2.8 and 2.9, respectively, for a temperature range between 5° and 20°C. The rate constants for sodium activation, α _m and β _m , were less temperature dependent, the Q ₁₀ being 1.8 and 1.7, respectively. This difference in Q ₁₀ also was found in rat (Schwarz and Eikhof, 1987 ) and squid (Kimura and Meves, 1979 ) axons.

Figure 13-2 shows that large outward-rectifying potassium currents can be elicited in frog nerve fibers. These potassium currents first were described quantitatively by Frankenhaeuser ( 1963 ) using the Hodgkin-Huxley equations. The probability that a potassium channel will be in an open state is expressed by the product r ^a k, where n denotes the potassium current activation and k the potassium current inactivation parameter. Analogous to the sodium current and assuming a homogeneous population of potassium channels, the macroscopic potassium current (I _K) can be described by the equations $$I_{\text{K}}=n^{a}k\cdot g_{\text{K}}\left(E-E_{\text{K}}\right)$$ and $$I_{\text{K}}=n^{a}k\cdot N\cdot {\gamma }_{\text{K}}\left(E-E_{\text{K}}\right)$$ where N is the number of potassium channels, which vary with membrane potential and time, and with a single-channel conductance γ_K. A value greater than unity is necessary for the exponent a of the potassium current activation parameter n to take account of the sigmoidal activation of the potassium current during a depolarizing potential step. The value of a varies between 2 for nodes of Ranvier of Xenopus laevis (Frankenhaeuser, 1963 ) and 4 for Rana fibers (Dodge, 1963 ; Hille, 1968 ; Koppenhöfer, 1967 ). At the end of depolarizations lasting several seconds, potassium currents are inactivated. The potassium inactivation parameter k decreases exponentially with time for macroscopic potassium currents (Frankenhaeuser, 1963 ) and is the sum of two exponential components, k ₁ and k ₂, the earliest indication of more than one component of potassium conductance (Schwarz and Vogel, 1971 ).

Fast Potassium Currents.

A depolarization to +60 mV in the frog node of Ranvier in normal Ringer’s solution elicits a large outward-rectifying potassium current, which in turn induces a large potassium tail current upon repolarization to the holding potential (Figure 13-7 ). To avoid potassium accumulation outside the nodal membrane, the same experiment was repeated in isotonic KC1. After a depolarization to 0 mV, the potassium equilibrium potential in isotonic KCl, a large inward potassium tail current occurs on repolarization consisting of a fast and a slow tail current component, the former being selectively blocked by 4-aminopyridine (4-AP) in frog (Dubois, 1981 ) and rat (Röper and Schwarz, 1989 ) nerve fibers. Plotting the amplitudes of the instantaneous fast potassium tail currents recorded after a conditioning depolarization of various amplitudes (p. 263 )

fig. 13-7. Delayed-rectifier potassium currents of toad node of Ranvier. Top, Pulse protocols. A, Outward potassium current in Ringer’s solution. B, Potassium tail currents in isotonic KCl solution following a 100 millisecond conditioning pulse to E = 0 mV. The dashed lines indicate zero current; capacity and leakage currents have been subtracted. (Modified from Bräu et al., 1990 .)

The existence of three different voltage-dependent potassium channel types recently has been confirmed with the patch-clamp technique (I, F, and S channels; see section on single-channel potassium currents later in this chapter). The two fast potassium tail current components, f₁ and f₂, can be separated pharmacologically in frog nerve fibers. The f₂ component can be blocked selectively with capsaicin (Dubois, 1982 ) and the f₁ component with dendrotoxin (DTX) or mast cell degranulating peptide (MCDP; Bräu et al., 1990 ). Capsaicin and DTX were not found to be as selective in rat as in frog nerve fibers (Corrette et al., 1991 ; Röper and Schwarz, 1989 ). Figure 13-8 also shows that the relative contribution of f₁ channels is larger in motor than in sensory fibers in frog peripheral nerve (Dubois, 1981 ).

fig. 13-8. Total fast potassium conductance (circles) in toad peripheral nerve as calculated from the amplitude of fast potassium tail currents recorded at the holding potential of E = −90 mV after 100 millisecond conditioning depolarizations of various amplitudes. The values of the f₁ conductance (squares) were calculated from the values obtained using a holding potential of 0 mV. The difference between total potassium conductance and f₁ conductance yields the f₂ conductance. Note that the relative amplitudes of the f₁ and f₂ conductances are different in motor and sensory fibers. (From Dubois, 1981 .)

Slow Potassium Current.

At large depolarizations of an intact rat node of Ranvier, a slowly increasing potassium current of small amplitude is apparent (Figure 13-2 ). The amplitude of this slow potassium current can be increased by increasing the KCl concentration in the external solution. On depolarizing potential steps, potassium currents exhibiting slow activation kinetics can be recorded. Hyperpolarizing potential steps induce slowly deactivating potassium currents. Further analysis shows that about 50% of this slow potassium conductance is activated at −60 mV (Figure 13-9C ). The activation curve is flat; therefore, a considerable amount of the slow potassium conductance already is activated at the resting potential of about −80 mV (Brismar and Schwarz, 1985 ). The slow potassium conductance can be blocked with TEA (Figure 13-9Ab ). Because of the absence of fast potassium channels in rat and human nodal membranes, that part of the slow potassium conductance already activated at the resting potential assists, together with the leakage conductance, in repolarizing the action potential. As shown in our laboratories and by Kocsis et al. ( 1987 ), activation of additional slow potassium conductance is involved in action potential frequency adaptation (see Figure 13-21 ).

A small potassium current is activated on large negative pulses and superimposed on the deactivating slow potassium current (Figure 13-9Aa ). This small current could be due to an inward-rectifying potassium current. This assumption is supported by reports about the presence of an inward-rectifying potassium current in dorsal root fibers (Baker et al., 1987 ; Birch et al., 1991 ), optic nerve (Eng et al., 1990 ), and sciatic nerve (Gordon et al., 1991 ) of the rat. The predominant location of the inward-rectifying potassium channels has been assigned to the internode.

Distribution of Potassium Channels in Mammalian Myelinated Nerve Fibers.

Whereas only small outward potassium currents and potassium tail currents can be elicited in intact mammalian nerve fibers, large outward potassium currents emerge after paranodal demyelination. Early attempts to induce acute demyelination were made by using osmotic shock or mechanical stretch (Chiu et al., 1979 ). A more reliable method is the superfusion of the node with 0.2% pronase or 0.2% lysolecithin (Figure 13-10 ). Kinetic and pharmacological analysis of potassium tail currents recorded in rat nerve fibers after paranodal demyelination suggest the existence of two fast and one slow potassium channel populations, very (p. 264 )

fig. 13-9. Potassium currents in rat peripheral myelinated axon recorded during 80 millisecond depolarizing and hyperpolarizing pulses of increasing amplitude. The positive pulses were from −45 to 0 mV in steps of 15 mV and one pulse to +25 mV; the negative pulses were from −105 to −150 mV in steps of 15 mV. A, measurements were made in the presence of 300 nM TTX. Aa: measurements in isotonic KCl; Ab: measurements after addition of 10 mM TEA to the isotonic KCl solution; Ac: pulse protocol. Leakage and capacity currents were not subtracted. B, Current-voltage relation of the steady-state currents in Aa (open circles) and Ab (solid circles). The straight line denotes the leakage current as measured in the same fiber in Ringer’s solution. The potassium current (I _K) was determined from the difference between the steady-state currents and the leakage current. C, The normalized slow potassium conductance (g _K,s∞) was calculated from the equation g _K = I _K/(E − E _K), and plotted against membrane potential. The curve was calculated from the fitted parameters of a Boltzmann equation (g _K,s∞ = 1/[1 + exp ((E_s − E)/k)]) with the inflection point at E _s = −59.6 mV and the slope factor k = 18.2 mV. The holding potential (E _H) was −75 mV. (From Röper and Schwarz, 1989 .)

fig. 13-10. Potassium currents (upper traces) recorded during depolarizing pulses of increasing amplitude before and after paranodal demyelination of rat peripheral myelinated axon with 0.2% lysolecithin for 15 minutes. Pulse potentials were between −55 and +80 mV in steps of 22.5 mV. Capacity and leakage currents (lower traces) were elicited by a potential step to −150 mV. External solution: Ringer’s solution +300 nM TTX; holding potential: −77 mV. (From Röper and Schwarz, 1989 .)

Single-channel measurements have provided unequivocal evidence for the existence of different potassium channel types. Their main electrophysiological and pharmacological characteristics are listed in Table 13-1 . There are voltage-dependent potassium channels that can be distinguished by their intermediate, fast, and slow time course of deactivation; they are termed I, F, and S, respectively. The activity of another voltage-gated potassium channel additionally depends on the internal calcium concentration, and it is called the K_Ca channel. Furthermore, three less voltage-sensitive background potassium channels have been observed, an ATP-sensitive, a flicker, and a sodium-activated potassium channel. It will be shown that the potassium current components f₁, f₂, and s closely resemble the potassium channel types I, F, and S, respectively, in their electrophysiological and pharmacological properties.

I Channel.

In Figure 13-11 , recordings from potassium channel types I, F, and S are shown. Their different kinetic behavior is evident during the deactivation process. A depolarizing pulse to E = 0 had opened the channels, but no potassium current flows at the reversal potential in high-potassium internal and external solution. At the beginning of repolarization (see arrows at top recordings of Figure 13-11A and B), instantaneous potassium currents flow and closing of channels starts. In the channel type that is presented in the left-hand column, deactivation occurs within some tens of milliseconds. Because of the time constant of deactivation, this channel is called the intermediate (I) channel, as compared to a faster (F, right-hand column) and a slower (S, lower recording in left-hand column) channel. The current-voltage relation of the single I channel amplitudes (circles in Figure 13-11C ) reveals a conductance of 23 pS (−120 mV < E < −40 mV, high-potassium external solution, 13° to 15°C), It is the I channel type that is observed most frequently in frog nerve fibers in nodal as well as in paranodal regions, whereas, in mammalian nerve fibers, I channels are located predominantly in the paranodal region (see section on distribution of potassium channels in mammalian myelinated nerve fibers). The I channel is selectively permeable to K⁺ ions. With sustained depolarization, I channels inactivate slowly, in about 1 minute at −60 mV. Activation proceeds steeply in a potential range between −60 and −40 mV (Jonas et al., 1989 ).

The delayed rectifier is well known for its sensitivity to TEA (Hille, 1967 ; Koppenhöfer and Vogel, 1969 ). TEA reduced the amplitude of unitary I currents to a 50% value at a concentration of 0.6 mM (IC₅₀; D.-S. Koh, B. Safronov, and W. Vogel, unpublished results). A Hill coefficient of 1 revealed binding of one blocker molecule to one channel. The reduction of the single-channel current amplitude by TEA, as seen also with other potassium channel types (see sections on the calcium-activated potassium channel, sodium-activated potassium channel, and flicker potassium channel; see also Figure 13-15 ), suggests a fast blocking mode (Hille, 1992 ). The TEA block of the I channel did not depend on either membrane potential or direction of ionic current in the physiological potential range between −80 and +40 mV. However, the macroscopic I currents were suppressed selectively by much lower concentrations of DTX and MCDP. These blockers reduced the macroscopic current amplitude but did not change the time course of the current. The unitary I current amplitudes also were not affected by these blockers. A half-maximal reduction of macroscopic I current was observed at IC₅₀ = 7 nM for DTX and at 42 nM for MCDP, which also compares well with earlier voltage-clamp data for component f ₁ of delayed rectifier currents in peripheral nerve obtained by Bräu et al. ( 1990 ).

Barium at millimolar concentrations suppressed I currents by reducing the unitary amplitudes. This block depended weakly on membrane potential. In contrast, the blocking effect of cesium was strongly dependent on membrane potential. At negative potentials, the amplitudes of inwardly directed unitary I currents were reduced considerably in the presence of external cesium, whereas the block was much weaker for outward currents at positive potentials. Externally applied 4-AP at a concentration of 1 mM was effective in suppressing I channel activity at negative membrane potentials, producing a flicker-type block of inward currents. However, only a weak effect of 4-AP was observed at positive potentials. Capsaicin has been described as a specific blocker of the f₂ component (corresponding closely to potassium channels of the F type; see section on the F channel later in this chapter) of macroscopic potassium current (Dubois, 1982 ). However, the same concentration (10 μM) of the blocker suppressed I as well as F currents at positive membrane potentials. A specific blocker of some calcium-activated potassium channel types, charybdotoxin (500 nM), suppressed I currents in a manner similar to that of DTX. Glibenclamide, known as a specific blocker of ATP-sensitive potassium channels, failed to block I channels at concentrations up to 1 μM. A more complete characterization of I channels in amphibian axons is in preparation (D.-S. Koh, B. Safronov, and W. Vogel).

I channels in rat and in human fibers have been described (p. 266 )

fig. 13-11. Discrimination of delayed-rectifier potassium channels I, F, and S by deactivation in Xenopus nerve in high-potassium solution at 15°C. Upper line gives pulse programs: From a holding potential of E = −80 mV to E = 0 (to activate the channels), repolarization (at which deactivation proceeds) was made to −70, −105, and −120 mV (from top to bottom recording) in A and to −65, −105, and −115 mV in B. Duration of the first pulse was 100 milliseconds in A and 5 milliseconds in B. Three current recordings show deactivation of single-channel events of I channels in A, including an S channel event at −120 mV, and F channels in B. Dashed lines give zero current level. High-potassium solution (105 mM) in pipette and in bath. C, Amplitude (i) of single-channel currents plotted versus membrane potential for I channels (circles), F channels (diamonds), and S channels (squares). Solid symbols, external solution containing DTX to block I channels. Each point represents the mean current amplitude of 3 to 100 events. (From Jonas et al., 1989 .)

F Channel.

A second potassium channel type can be seen in the right-hand column of Figure 13-11 . It deactivates within a few milliseconds at potentials between −120 and −65 mV. This comparatively fast-deactivating potassium channel is called the F channel (Jonas et al., 1989 ). Its conductance in high-potassium solution is 30 pS. F channels activate less steeply than I channels and at more positive potentials, between −40 and +40 mV. During sustained depolarization, F channels inactivate within seconds. They are less sensitive to blockage by DTX. F channels are not seen as regularly as I channels; normally, they are observed together with other delayed-rectifier potassium channels. They become visible if the bulk of I channels are blocked by DTX. Recordings in Ringer’s solution are shown in Figure 13-12 . F channels can be recognized from their larger single-channel current amplitude and a more stable open state. F channels have been described in Xenopus fibers (Jonas et al., 1989 ) and in rat fibers (Safronov et al., 1993 ).

S Channel.

The third type of voltage-gated potassium channel can be seen in the bottom recording of Figure 13-11A as small events. It was observed in about 20% of the patches and had a conductance of 7 pS, which is clearly lower than that of either the I or F channel (Jonas et al., 1989 ). Its deactivation proceeded very slowly, hence the name S channel. The potential dependence of the S channel is very flat, and the channel already is active at potentials around −70 mV (i.e., at about the resting potential). Its TEA sensitivity is in the millimolar range (see Table 13-1 ).

From the peak S current of nearly 2.5 nA in Figure 13-9B and the corresponding single-channel current of 1 pA (see Figure 13-11C ), a density of 2500 S channels per node may be estimated. Assuming a nodal area of 25 μm² (Berthold and Rydmark, 1983 ), there may be around 100 S channels per square μm² in the nodal membrane (see Safronov et al., 1993 ).

The functional role of the voltage-gated potassium channel types I, F, and S may be repolarization of the membrane potential during the falling phase of the action potential in amphibian fibers. The lower potassium current density in rat and human fibers, as mentioned in the previous section, reduces this function in these preparations. Together with other potassium-selective channels (see sections on the sodium-activated potassium channel, flicker potassium channel, and functional aspects of channel distribution), the S channels contribute to the resting potential.

Calcium-Activated Potassium Channel.

This channel type is gated not only by potential but also by micromolar (p. 268 )

fig. 13-12. Activity of F and I channels in rat nerve in Ringer’s solution at 21° to 23°C. A, Single-channel recordings of the F and I channels activated by long-lasting voltage steps from a holding potential of −90 mV to +20 mV and 0 mV. Capacity and leakage currents were not subtracted. Bath solution contained 8 nM DTX to reduce the number of active I channels. B, i-E data for the F (open symbols) and I (solid symbols) channels fitted by straight lines corresponding to 19 pS (F channels) and 11 pS (I channels). Each point is based on 3 to 33 measurements from three out-side-out patches. (From Safronov et al., 1993 .)

K_Ca channels have been identified in about one third of all membrane patches. The maximum number of channels ever observed in one patch (40 MΩ pipette resistance) was 17, which means that they are clearly less frequent than the most prominent potassium channel of peripheral amphibian nerve fiber, the I channel.

Activation of K_Ca channels is likely to have effects on the time course of the action potential and on the recovery process following impulse conduction, but, in a healthy resting myelinated nerve fiber, the internal concentration of calcium is probably too low to allow significant activity. However, in critical situations such as hypoxia or hypoglycemia, a pronounced increase in internal calcium concentration could occur. A more detailed description of this K_Ca channel is given elsewhere (Jonas et al., 1991 ).

ATP-Sensitive Potassium Channel.

Openings of this channel typically occur in bursts. With 105 mM potassium solution on both sides of the membrane, its single-channel (p. 269 )

fig. 13-13. Calcium-activated potassium channels in Xenopus nerve at 15°C. A, Recording from an inside-out patch during change of bath solution from high-K⁺ _i + 3 mM EGTA to high-K⁺ _i + 10 μM Ca²⁺ _i and back to high-K⁺ _i + EGTA. The patch contained 12 calcium-activated potassium channels; pipette solution, 105 mM K⁺ _o ; E = +40 mV. B, i-E data with 105 mM K⁺ _o (open circles, five outside-out patches) and Ringer-TTX (solid circles, one outside-out patch) in the bath and high-K⁺ _i + 1 μM [Ca²⁺ _i in the pipette. C, Open probability as a function of membrane potential; P_open was calculated from the maximum number of simultaneously open channels observed in a patch. Bath: high-K⁺ _i + EGTA (circles), high-K⁺ _i containing 10⁻⁵ M calcium (triangles), 10⁻⁴ M calcium (diamonds), and 10⁻³ M calcium (inverted triangles); pipette: 105 mM K_o. The curves represent the function P_open (E) = 1/[1 + exp((E _mid − E)/k)], with E _mid = 40.4, 21.7, and 3.2 mV and k = 14.3 mV. (Modified from Jonas et al., 1991 .)

fig. 13-14. ATP-sensitive potassium channel in Xenopus nerve at 15°C. A, Consecutive recordings from inside-out patch with different bath solutions: high-K⁺ _i + EGTA (control; upper), high-K⁺ _i + EGTA containing 96 μM ATP (middle), and control again (lower trace); E = −80 mV. B, Fractional block versus ATP concentration, 11 measurements on four patches. Block estimated as the relative reduction of open probability. The curve represents the fractional block f(c) = 1/[1 + (IC₅₀/c) ^a ], with half-maximal inhibitory concentration (IC₅₀) = 35 μM and Hill coefficient (a) = 1.5 as obtained by least-squares fit. (Modified from Jonas et al., 1991 .)

K_ATP channels occur nearly as frequently as K_Ca channels, and they also may have effects on the time course of the action potential and on the recovery process following (p. 270 ) impulse conduction. In situations with low energy supply or after high activity of signal conduction, the function of both the K_Ca and the K_ATP channels might consist in counteracting failure of the Na K pump, thus protecting the axon from progressive depolarization and paroxysmal discharge. A more detailed description of the K_ATP channel has been given elsewhere (Jonas et al., 1991 ).

Sodium-Activated Potassium Channel.

The single channel conductance of the K_Na channel is 88 pS in symmetrical high-potassium solution and 34 pS with Ringer’s solution on the outer side of the membrane. The reversal potential was shifted from 0 to −75 mV when the external high-potassium solution was exchanged for 2.5-mM potassium Ringer’s solution, suggesting a high selectivity of this channel for K⁺ over Na⁺ ions. The open probability of the channel only weakly depends on membrane potential. It is 0.37 at E = −90 mV and 0.59 at E = +40 mV, with intracellular Na⁺ (Na⁺ _i ) equal to 100 mM.

Channel activity was studied at different internal sodium concentrations with inside-out patches. At 2 mM Na⁺ _i , the channel openings seldom could be observed. At 20 mM Na⁺ _i , channel openings were long enough to measure the apparent single-channel conductance and frequent substates could be observed. The activity of the channel saturated at internal sodium concentrations higher than 50 mM. A least-squares fit of the activation curve based on the open probability of the K_Na channel gave a half-maximal axoplasmic sodium concentration of 3.3 mM Na⁺ _i and a Hill coefficient of 2.9, suggesting that the channel has more than two binding sites for Na⁺ ions. Replacement of sodium by lithium could not activate the axonal K_Na channels. One prominent gating property of the K_Na channel is the frequent occurrence of substates; at least nine and a main open state could be identified.

TEA reduced only the apparent single-channel conductance but not the open probability of the channels. Compared to other potassium channels in this preparation (except the flicker channel; see later in this chapter), the K_Na channel is relatively insensitive to this blocker, the IC₅₀ being 21 mM TEA. The channel is more sensitive to external cesium (10 mM) at negative membrane potentials. Barium at a concentration of 1 mM profoundly reduced the open probability without any significant reduction of the single-channel current amplitude, and the blocking effect occurred only at negative potentials.

The relative density of K_Na channels in the nodal as compared to the paranodal membrane is demonstrated by experiments in which many patches were obtained from a single axon. The highest number of simultaneously open channels recorded with 20 MΩ pipettes was four within the nodal area, and the probability of finding a channel was 0.8. Because the patch area corresponds to approximately 1 to 4 μm² (Sakmann and Neher, 1983 ), the minimum density of this channel at the node of Ranvier could be estimated at about 0.25 to 1 K_Na channel per μm². The patches made within paranodal regions revealed a density of K_Na channels at least 10 times lower.

Cytoplasmic sodium concentration is increased during repetitive activity (Bergman, 1970 ) and may reach the range of about 30 mM where K_Na channels change their activity steeply. This gives a hint as to the functional role of these potassium channels. During periods of high sodium channel activity and depending on activities of the Na K pumps, sodium-activated potassium channels may contribute to the nodal membrane potential. A detailed description of the sodium-activated potassium channel is given elsewhere (Koh et al., 1994 ).

Flicker Potassium Channel.

A potential-independent (“leakage”) potassium conductance that cannot be blocked by the classical potassium channel blocker TEA has been suggested to generate the resting potential in sympathetic ganglia (Jones, 1989 ), in invertebrate axons (Chang, 1986 ), and in vertebrate myelinated axons (Schmidt and Stämpfli, 1966 ). All potassium channels described so far except the S channel and the K_Na channel have a low open probability around the resting potential under physiological conditions. Moreover, they are all blocked by less than 10 mM external TEA. Therefore, these channels are not likely to provide a major contribution to the resting potential of the peripheral myelinated axon, which is almost completely TEA resistant (Schmidt and Stämpfli, 1966 ).

Recordings of potassium channels often are disturbed by background activity of voltage-insensitive potassium channels. The activity of the so-called flicker channel is shown in Figure 13-15A with symmetrical potassium concentrations on both sides of the membrane (105 mM). Flickering dominates the gating of this potassium channel type. The apparent single-channel conductance is 49 pS in high-potassium solution. The i − E curve with Ringer’s solution on the outer side of the membrane shows a single-channel conductance of 17 pS for outward currents. The reversal potential was E _rev = −70 mV in 2.5 mM K⁺ _o as opposed to 0 mV in external high-K _o solutions, indicating that the channel is fairly selective for K⁺ ions. In addition, the flicker channel is weakly potential dependent.

The effect of external TEA at negative membrane potential is illustrated in Figure 13-15B . High concentrations of TEA (50 mM) reduce the mean current during bursts without noticeably changing the channel activity. The IC₅₀ is 19 mM TEA. Compared to other potassium channels in this preparation, the flicker channel (p. 271 )

fig. 13-15. TEA block of flicker potassium channel. A, Outside-out patch recordings with 105 mM K⁺ _o (control) and with 105 mM K⁺ _o containing 50 mM TEA in the bath and wash pipette; 105 mM K⁺ _i , E = −90 mV, low-pass filtered at 1 kHz (−3 dB, four-pole Bessel). B, Fractional block of mean current during bursts versus concentration of TEA at E = −90 mV; three outside-out patches. IC₅₀ = 19.0 mM and Hill coefficient a = 1.0 as obtained by least-squares fit (see Figure 13-14 ). (From Koh et al., 1992 .)

The functional role of the flicker channel was investigated by recording ionic currents from “macropatches” containing several channels under voltage-clamp conditions in the range of possible resting membrane potentials. At E_m = −90 mV, an inward current with 105 mM K⁺ _o was observed (Figure 13-17A ), which at higher time resolution shows the rapid kinetics typical for the flicker channel (not shown). This current could not be blocked by 10 mM TEA, but was inhibited by barium and cesium. Its pharmacological features are thus similar to (p. 272 )

fig. 13-16. Bupivacaine block of flicker potassium channel. A, Outside-out patch recordings with 105 mM K⁺ _o (control) and with 105 mM K⁺ _o containing 1 μM bupivacaine in the bath and wash pipette; 105 mM K⁺ _i , E_m = −100 mV. Three channels were open at most times in the control recordings. B, Fractional block of mean total current versus concentration of bupivacaine at the same potential; six outside-out patches. IC₅₀ = 0.165 μM and a = 1.0, obtained as in Figure 13-14 . (From Koh et al., 1992 .)

The flicker channel has been observed in high density in nodal, paranodal, and internodal regions of the axonal membrane of thin myelinated nerve fibers. It has been suggested that paranodal and internodal potassium channels contribute to the resting potential (Chiu and Ritchie, 1984 ), and therefore it is likely that not only the nodal but also the paranodal and internodal flicker channels contribute to the resting potential of the myelinated nerve fiber. A more detailed description of this channel is given elsewhere (Koh et al., 1992 ).

Other Channel Types.

In rat and human demyelinated axons, a chloride channel has been observed with a conductance of 33 to 65 pS (high symmetrical CsCl solutions, 22°C). Its selectivity for sodium and cesium is P _Na/P _Cs/P _Cl = 0.1/0.2/1 (Strupp and Grafe, 1991 ). Chloride channels were observed much more often in patches from nodal regions than in those from paranodal regions.

Calcium channels, although obviously present in the soma membrane and at the presynaptic terminal of the peripheral neuron, have not been found so far in the nodal or paranodal demyelinated membrane (C. Nau, personal communication).

There are nonspecific leakage currents of considerable amplitude in macroscopic voltage-clamp measurements from the node of Ranvier, especially from mammalian nerve fibers, as mentioned above. In the patch-clamp recordings, however, leakage current is minimal or absent. This supports the hypothesis of Barrett and Barrett ( 1982 ) that the leakage current originates from the internodal membrane segments and reenters the external (p. 273 )

fig. 13-17. Pharmacological blockade of currents recorded at E = −90 mV (A) and E = −60 mV (B). Currents from an outside-out macropatch with 105 mM K⁺ _i in the pipette were recorded in different external solutions: 105 mM K⁺ _o (control), 105 mM K⁺ _o + 10 mM TEA (TEA), 105 mMK⁺ _o + 1 mM barium + 10 mM TEA (Ba + TEA), and 105 mM K⁺ _o + 10 mM cesium + 10 mM TEA (Cs + TEA). Record at −60 mV was obtained more than 1 minute after the voltage step to this potential, when the current was stationary. Pipette resistance: 18 MΩ. (From Koh et al., 1992 .)