Correspondence to: Froylán Gómez-Lagunas, Departamento de Fisiología, Facultad de Medicina, UNAM, Universitaria, México City 04510, México. Fax:52-73-172388 E-mail:froylan{at}ibt.unam.mx.
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Abstract |
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The Shaker B K+ conductance (GK) collapses (in a reversible manner) if the membrane is depolarized and then repolarized in, 0 K+, Na+-containing solutions (Gómez-Lagunas, F. 1997. J. Physiol. 499:315; Gómez-Lagunas, F. 1999. Biophys. J. 77:29882998). In this work, the role of Na+ ions in the collapse of GK in 0-K+ solutions, and in the behavior of the channels in low K+, was studied. The main findings are as follows. First, in 0-K+ solutions, the presence of Na+ ions is an important factor that speeds the collapse of GK. Second, external Na+ fosters the drop of GK by binding to a site with a Kd = 3.3 mM. External K+ competes, in a mutually exclusive manner, with Nao+ for binding to this site, with an estimated Kd = 80 µM. Third, NMG and choline are relatively inert regarding the stability of GK; fourth, with [Ko+] = 0, the energy required to relieve Nai+ block of Shaker (French, R.J., and J.B. Wells. 1977. J. Gen. Physiol. 70:707724; Starkus, J.G., L. Kuschel, M. Rayner, and S. Heinemann. 2000. J. Gen. Physiol. 110:539550) decreases with the molar fraction of Nai+ (XNa,i), in an extent not accounted for by the change in µNa. Finally, when XNa,i = 1, GK collapses by the binding of Nai+ to two sites, with apparent Kds of 2 and 14.3 mM.
Key Words: K+ affinity , Na+ block, conductance, selectivity, zero K+
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INTRODUCTION |
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In addition to permeate through voltage-dependent K channels (Kv channels)* and modulate their gating, K+ ions are an essential factor needed to keep these proteins in their normal, functional, state. It is now becoming clear however, that the K+ requirements of Kv channels are highly variable, as revealed by the diversity of effects that K+ depletion exerts on different kinds of channels. For example, in the absence of K+ on both sides of the membrane, the activity of the delayed rectifier (DR) squid K channel is irreversibly lost (
Recently, it was shown that in the absence of K+ on both sides of the membrane, the Shaker B K+ conductance (GK) collapses when the membrane is depolarized and then repolarized. Briefly,
The above observations were interpreted as meaning that closing without K+ sinks the channels into a stable nonconducting, noninactivated, closed state(s) (
Here, it is shown that Na+ ions foster the collapse of GK in the absence of K+. External K+ and Na+ compete, in a mutually exclusive manner, for binding to a externally located site (probably external to the selectivity filter), where GK is modulated (available versus reluctant). On the other hand, to see if the collapse of GK occurs as a discontinuous change in the properties of the channels in the limit of zero K+, experiments with internal solutions containing both K+ and Na+ ions were done. The results show that as the molar fraction of Nai+ (XNa,i) increases, the energy required to relieve Nai+ block changes in an extent not accounted for by the change in the driving force of Na+. This suggests that, as XNa,i increases, there is either an increased electrostatic repulsion between the blocking Na+ and a neighbor ion, or, more likely, a deformation (change of the energy profile) of the pore, that could be maximal in the limit XNa,i = 1. Under the latter conditions, GK collapses, and Nai+ interacts with the channels with a kinetics described by the noncooperative binding to two, kinetically distinguishable, sites.
A preliminary account of this work has been presented in abstract form (
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MATERIALS AND METHODS |
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Cell Culture and Channel Expression
Insect Sf9 cells kept in culture in Grace's media (GIBCO BRL) at 27°C were infected, with a multiplicity of infection of 10, with a recombinant baculovirus, Autographa californica nuclear polyhedrosis virus, containing the cDNA of Shaker B, as previously reported (
Electrophysiology
Macroscopic currents were recorded under whole-cell patch clamp, with an Axopatch-1D (Axon Instruments). The currents were filtered at 5 KHz with the built in filter of the amplifier, and sampled at 100 µs/point, with a TL1 interface (Axon Instruments). Electrodes were pulled from borosilicate glass (KIMAX 51) to a final resistance of 12 M;
80% of the series resistance was electronically compensated. Activating pulses (unless otherwise indicated = +20 mV/30 ms) in 0-K+ solutions, were delivered at 1 Hz. This procedure will be referred to as pulsing.
Solutions
Solutions will be named by their main cation and represented as external/internal (e.g., Ko/NMGi). Their composition is listed in Table 1. All other solutions in which the concentration of the test cation exceeded 5 mM, were made by the appropriate mixing of the listed solutions, keeping the osmolarity constant. Total exchange of the external solution was achieved in at most 15 s. Pulsing in 0 K+ was performed under continuous perfusion, beginning 1 min after the start of the perfusion.
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Data Analysis
Where necessary, the t test was used to evaluate statistical significance. The results are expressed as mean ± SEM of at least four cells. Curve fitting was performed with Sigmaplot 3.0 (Jandel).
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RESULTS |
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Na+ Ions Speed the Collapse of GK in 0 K+ Conditions
With Na+-containing, 0-K+ solutions on both sides of the membrane (Nao/Nai; MATERIALS AND METHODS), the delivery of depolarizing pulses that activate the channels followed by repolarization to a negative potential (usually the HP = -80 mV) collapse the Shaker B K+ conductance (GK; INTRODUCTION). Fig 1 shows that Na+ ions foster the collapse of GK. Fig 1 A illustrates the collapse-recovery cycle of GK in Nao/Nai solutions. The traces show inward K+ currents (IK), evoked by +20 mV/30-ms activating pulses, in Ko/Nai. The left trace (Fig 1 A, before) is a control IK, recorded at the beginning of the experiment. The middle trace (Fig 1 A, After) is the current left after the delivery of 20 activating pulses (a procedure hereafter referred to as pulsing) while the cell was bathed in the test Nao/Nai solutions (not shown). After pulsing in 0 K+, Na+containing solutions, the channels become reluctant to conduct K+. The right trace (Fig 1 A, Recovery) shows the recovery of IK brought about by a 3-min depolarization to 0 mV (
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In contrast to the dramatic effect of pulsing in Nao/Nai, the delivery of the same number of pulses in NMG-containing, 0 K+, and 0 Na+ (not added) solutions scarcely affects GK. This is shown in Fig 1 B, which presents two K+ currents recorded in Ko/NMGi, before (left trace) and after (right trace) pulsing in NMGo/NMGi (not shown). There was only a 13% reduction of IK.
Fig 1 C compares the reduction of GK after pulsing in a variety of solutions. Notice that, whereas in Nao/Nai (last bar) GK is basically erased (99 ± 1% reduction, n = 4), in NMGo/NMGi (second bar) GK drops only 20 ± 2% (n = 16). Thus, pulsing in 0 Na+, NMG-containing, solutions collapses GK, but far less than in the presence of Na+ ions (in fact, in Nao/Nai, only 1015 pulses are needed to completely eliminate GK, see 80%) remain in a state from which they readily collapse upon the addition of Na+ (not shown). The third bar in Fig 1 C, shows that external choline, another impermeant and nonblocking cation, is as inert as NMG (GK drop = 21 ± 6%, n = 5; see DISCUSSION).
In summary, the combined condition, presence of Na+ and lack of K+, makes GK more liable to collapse. Finally, notice that pulsing with Na+ ions present (added) in only the external solution (in Nao/NMGi, first bar) drops GK in about the same extent (90 ± 4%, n = 9) as it does with Na+ on both sides of the membrane (99 ± 1%, last bar). So, even when under physiological conditions, external Na+ neither permeates nor blocks Kv channels (but see
External Na+ Interaction with Shaker Channels
The interaction of external Na+ with Shaker was further studied by looking at the extent of GK collapse produced by pulsing in solutions of variable [Nao+], with NMGi as the internal solution (in (NMGo + [Nao])/NMGi, see MATERIALS AND METHODS). Fig 2 A shows that as [Nao+] increases, GK drops following a Hill saturation curve (line through the points, labeled 0 K+), with a Kd = 3.3 mM, and maximal extent of collapse = 1.05 (Hill number n = 0.97). When the same measurements are done in the presence of either 0.08 or 0.3 mM Ko+, it is seen that the apparent Kd for Na+ increases to either 4.7 or 5.3 mM, respectively, without a significant change in the maximal extent of collapse (0.98 or 0.91 with 0.08 or 0.3 mm K+, respectively). This indicates that K+ protection (reduction of GK drop) is more efficient at the lower [Nao+], and thus shows that K+ inhibits in a competitive manner the binding of Na+, to the site where GK is modulated. The inset shows the plot in an expanded [Nao+] scale.
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The competitive, mutually exclusive, binding of Na+ and K+ is best seen in Fig 2 B, that presents the double reciprocal plot of the points in A. The presence of either 0.08 or 0.3 mM Ko+ increases the slope of the least-square lines (i.e., the apparent Kd for Nao+), without significantly changing the (1/F.lost) - axis intercept (0.95 in 0 K+ vs. 1.02 or 1.1 with 0.08 or 0.3 mM K+, respectively). The above results, allow the actual affinity for Ko+ (i.e., Kd(Ko+), in the absence of the competing Na+ ions) to be estimated, with the use of the known equation for the apparent Kd(Kapp) of a ligand (K+) in the presence of a competitive inhibitor I (Na+) of known Ki = Kd(Nao+), by taken Kapp(Ko+) = 2.9 mM, the previously reported Ko+ affinity, that had been obtained in the presence of saturating [Na+] = 140 mM ( Kapp(Ko+)/(1 + ([I]/Ki)) = 80 µM.
However, it is pertinent to mention that considering the possibility of ion permeation through the channels, deviations from the equilibrium conditions, on which the saturation Michaelis-Menten or Hill equations are based (
If the site where Nao+ (and Ko+) binds was located within the electric field of the membrane, it would be expected that pulsing from a hyperpolarized HP should increase the drop of GK, by favoring the Nao+ occupancy of the site.
In contrast to the above prediction, Fig 2 C shows that, with a nonsaturating [Nao+] = 2.5 mM, the drop of GK is slightly, although significantly, reduced (P < 0.05), instead of increased, by pulsing from a more negative HP (from 49 ± 4% [n = 7] at HP -80 mV; to 37 ± 1% [n = 6] at HP = -120 mV). On the other hand, Fig 2 D shows that within the range of 0 to +60 mV, the pulse potential (Vp) does not play a significant role on the extent of collapse. The dependence on holding potential of Nao+ action is qualitatively equivalent to that of Bao2+ protection (
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Internal Na+ Interaction with Shaker Channels
It is known that internal Na+ interacts with the pore of K channels blocking IK (
For a reference, Fig 3 A shows a typical I-V relationship obtained under standard conditions (Nao/Ki). Above 0 mV, where the probability of opening is 1, IK increases linearly with the voltage. With the XNa,i = 0, this is always observed. An I-V obtained with a low XNa,i = 0.21 is shown in Fig 3 B. First IK increases with the voltage, but at about +40 mV, the current starts to deviate from the linearity and after that a region of negative conductance develops, as Nai+ blocks the channels. The departure from linearity, at positive voltages (+40 mV), is more clearly seen by comparing the experimental points with the straight line in the plot. The latter is the least-square fit of the points between 0 and +30 mV, and therefore gives an estimate of the expected Ik if XNa,i was zero. Thus, the difference between the line and the points in the graph is an estimate of the extent of block at each voltage, the latter is plotted in Fig 3 C, which presents the mean ± SEM of four cells. The line is the fit of the points with a Woodhull equation (Fig 3 legend), with electrical distance
= 0.7, and Kd(0 mV) = 538.3 mM (
In contrast to the behavior with XNa,i 0.42, Fig 4 A shows that with XNa,i = 0.71, the I-V relationship acquires an N shape (
+160 mV. IK first increases, but starts to deviate from the linearity as Nai+ blocks the channels, and thereafter a region of negative conductance develops, until a "critical" voltage (Vc) is reached (in this case Vc = +70 mV) where Nai+ block is relieved and the current increases again with the voltage (pointed out by the arrow), giving the I-V its N shape. The traces in the right panel of Fig 4 A illustrate the N shape of the I-V relationship. See that the peak currents are in the following order: I (+110 mV) > I (+30 mV) > I (+70 mV). Finally, Fig 4 B shows an I-V obtained with XNa,i = 0.83. Notice that although its overall shape is like that observed with XNa,i = 0.71, Vc is smaller (Vc = +50 mV). The traces in the right panel illustrate the N shape of the I-V relationship.
In the N-shaped I-Vs, there is a voltage (Vc) where Nai+ block is overcome (
Fig 5 A compares BNa at the two XNa,i where a corresponding Vc could be reached within the range Vp +160 mV. In going from a XNa,i of 0.71 to 0.83, BNa changes from 8.4 ± 0.3 (n = 5) to 5.5 ± 0.5 kJ/mol (n = 5). On the other hand, the electrochemical gradient of Na+ (
µNa), evaluated at Vc,
µNa = F(Vc VNa), where VNa is the Nernst potential of Na+, changes from 5.5 (XNa,i = 0.71) to 4.0 kJ/mol (XNa,i = 0.83). So, whereas BNa decreases 2.9 kJ/mol,
µNa decreases only 1.5 kJ/mol, this indicates that the change in Vc, with XNa,i, cannot be entirely accounted for by the change in the driving force of Na+.
The lack of coincidence between the change of BNa and µNa is also seen when Fig 3 and Fig 4 are compared. For example, with XNa,i = 0.42 the I-V is not N-shaped, so Vc
+160 mV, then when XNa,i increases to for example 0.83, BNa changes from at least 15.5 (XNa,i = 0.42, underestimating Vc as +160 mV) to 5.5 ± 0.5 kJ/mol (XNa,i = 0.83) that is 10 kJ/mol, whereas
µNa changes from at least 12.9 to 4.0 kJ/mol, that is 8.9 kJ/mol.
By looking at Nai+ block of the squid K channel, N-shaped I-Vs were first observed by French and Wells (
Following this simplifying approach, and taking BK = 0, and BNa as in Fig 5 A, it is seen that when XNa,i increases from 0.71 to 0.83 (1.2 times), PNa/PK increases about four times (Fig 5 B). The results in Fig 3 Fig 4 Fig 5 show that as XNa,i increases there is an actual reduction (i.e., a reduction not entirely accounted for by the change in µNa) of the energy required to relieve Nai+ block, yielding a substantial change of the PNa/PK ratio.
The above effect could be explained as the result of an increase in the electrostatic repulsion between the blocking Na+ and a neighbor ion, as XNa increases. However, it is not easy to see why this repulsion should be bigger as the molar fraction of the permeant K+ ion decreases. Alternatively, it could be that as XNa,i increases, there is a deformation of the pore, measured as a reduction of the energy required to allow Na+ permeation. If that were the case, it could be that this deformation were maximal in the limit where XNa,i = 1. Pulsing under the latter conditions (Nao/Nai) collapses GK (
Fig 6 A shows that pulsing with Na+ present (added) in only the internal solution (NMGo/Nai), drops GK in about the same extent (93 ± 5%, n = 6) as it does with Na+ present on both sides of the membrane (last bar, 99 ± 1%, n = 4). For comparison, the figure also shows the collapse in NMGo/NMGi, as in Fig 1. The above observation suggests that Na+ could possibly act on the same site regardless of the side of the membrane from where it comes. To further explore this point the collapse of GK as a function of [Nai+] (in NMGo/(NMGi-[Nai])) was determined. Fig 6 B shows that as [Nai+] increases GK drops following a Hill curve (line through the points) with n = 1.16 (which indicates that there is, at least, one site where Nai+ binds) plus an offset, that accounts for the collapse in NMGo/NMGi (0.20, Fig 6 A). However, Fig 6 C shows that the double-reciprocal plot of the points in B is best fitted by two straight lines, which indicates that Nai+ binds to two sites: one with a maximal collapse (m.c) of 60% and Kd = 2 mM, and a second, lower affinity site, with m.c = 36% and Kd = 14.3 mM. The presence of two sites is also clearly distinguished in the inset, which presents the Eadie-Scatchard plot of the points in B (
It is pertinent to mention that by contrast to the qualitative similarity of the voltage dependence of the Nao+ and Bao2+ effects, the voltage dependence of Nai+ and Bai2+ actions are different. The latter decreases markedly with hyperpolarized HPs, and it is not dependent on Vp (
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DISCUSSION |
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The Shaker B GK collapses when the channels close in 0-K+ solutions (
Here, it was shown that the combined condition 0 K+ and presence of Na+ makes GK more liable to collapse, as Na+ ions speed (in number of pulses) the drop of GK.
Nonetheless, Na+ is not necessary for GK to collapse, as it also falls in NMG or choline solutions. Considering that Na+ binds with millimolar affinity from both sides of the membrane, it seems unlikely that the drop of GK in the 0-Na+ solutions, could be produced by contaminant Na+ ions. More likely, the binding of Na+, instead of K+, or just the absence of K+ in the pertinent site(s) when the channels close, elicits the drop of GK, although with significantly different speeds that might be accounted for by a smaller dwelling time of K+, in the pertinent site(s), in the presence of Na+ ions than in their absence, in agreement with the observed mutually exclusive binding of K+ and Na+ ions to the externally located site.
Extracellular Na+ and the Drop of GK
Under physiological conditions, Nao+ neither permeates nor blocks Kv channels (but see
Could the site where Ko+ and Nao+ bind modulating GK be located within the selectivity filter? The protection exerted by TEAo suggests that the site is externally located (
However, considering that in multi-ion pores, the voltage dependence of ligand binding depends on ion occupancy (
Internal Na+ Interaction with Shaker
For voltages up to +160 mV, I-V relationships obtained with the Nao solution, and with a variable XNa,i, have a shape that depends on XNa,i. With XNa,i 0.71, the I-Vs are N-shaped. In contrast to the behavior of Shaker channels,
Recently, by looking at the onset of the slow inactivation of Shaker channels lacking the NH2 terminus domain,
It has been shown that mammalian Kv2.1 channels as well as DR channels of bullfrog neurons although remain stable in 0 K+, conducting Na+, undergo a conformational change of the pore region, which is observed as a reduction of the capability of TEA to block the channels (
When XK,i = 0, pulsing with Na+ ions added to the internal solution alone drops GK in about the same extent as it does with Na+ on both sides of the membrane, suggesting that Nai+ might be able to reach the externally located site where GK is modulated, in agreement with the observation that Vc decreases as XNa,i approaches one, and with reports showing Na+ currents through Kv channels in 0 K+ (
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Footnotes |
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* Abbreviations used in this paper: DR, delayed rectifier; HP, holding potential; Kv channel, voltage-dependent K channel; m.c, maximal collapse.
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Acknowledgements |
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The author thanks Dr. L. Possani for allowing the use of his laboratory for the realization of this work.
This work was supported by Dirección General de Asuntos del Personal Academico (IN-216900) and Consejo Nacional de Ciencia y Tecnología grants 26525N and Z-005.
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References |
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