Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Ca(2+) channels

Limited information is available regarding the effects of protein kinase C (PKC) isozyme(s) in the regulation of L-type Ca(2+) channels due to lack of isozyme-selective modulators. To dissect the role of individual PKC isozymes in the regulation of cardiac Ca(2+) channels, we used the recently developed novel peptide activator of the epsilonPKC, epsilonV1-7, to assess the role of epsilonPKC in the modulation of L-type Ca(2+) current (I(Ca,L)). Whole cell I(Ca,L) was recorded using patch-clamp technique from rat ventricular myocytes. Intracellular application of epsilonV1-7 (0.1 microM) resulted in a significant inhibition of I(Ca,L) by 27.9 +/- 2.2% (P < 0.01, n = 8) in a voltage-independent manner. The inhibitory effect of epsilonV1-7 on I(Ca,L) was completely prevented by the peptide inhibitor of epsilonPKC, epsilonV1-2 [5.2 +/- 1.7%, not significant (NS), n = 5] but not by the peptide inhibitors of cPKC, alphaC2-4 (31.3 +/- 2.9%, P < 0.01, n = 6) or betaC2-2 plus betaC2-4 (26.1 +/- 2.9%, P < 0.01, n = 5). In addition, the use of a general inhibitor (GF-109203X, 10 microM) of the catalytic activity of PKC also prevented the inhibitory effect of epsilonV1-7 on I(Ca,L) (7.5 +/- 2.1%, NS, n = 6). In conclusion, we show that selective activation of epsilonPKC inhibits the L-type Ca channel in the heart.     

Block of inactivation-deficient cardiac Na(+) channels by acetyl-KIFMK-amide

The Na(+) channel alpha-subunit contains an IFM motif that is critical for the fast inactivation process. In this study, we sought to determine whether an IFM-containing peptide, acetyl-KIFMK-amide, blocks open cardiac Na(+) channels via the inner cavity. Intracellular acetyl-KIFMK-amide at 2mM elicited a rapid time-dependent block (tau=0.24 ms) of inactivation-deficient human heart Na(+) channels (hNav1.5-L409C/A410W) at +50 mV. In addition, a peptide-induced tail current appeared conspicuously upon repolarization, suggesting that the activation gate cannot close until acetyl-KIFMK-amide is cleared from the open pore. Repetitive pulses (+50 mV for 20 ms at 1Hz) produced a substantial use-dependent block of both peak and tail currents by approximately 65%. A F1760K mutation (hNav1.5-L409C/A410W/F1760K) abolished the use-dependent block by acetyl-KIFMK-amide and hindered the time-dependent block. Competition experiments showed that acetyl-KIFMK-amide antagonized bupivacaine binding. These results are consistent with a model that two acetyl-KIFMK-amide receptors exist in proximity within the Na(+) channel inner cavity.     

Na(+) current through KATP channels: consequences for Na(+) and K(+) fluxes during early myocardial ischemia

During early myocardial ischemia, the myocytes are loaded with Na(+), which in turn leads to Ca(2+) overload and cell death. The pathway of the Na(+) influx has not been fully elucidated. The aim of the study was to quantify the Na(+) inward current through sarcolemmal KATP channels (IKATP,Na) in anoxic isolated cardiomyocytes at the actual reversal potential (Vrev) and to estimate the contribution of this current to the Na(+) influx in the ischemic myocardium. IKATP,Na was determined in excised single channel patches of mouse ventricular myocytes and macropatches of Xenopus laevis oocytes expressing SUR2A/Kir6.2 channels. In the presence of K+ ions, the respective permeability ratios for Na(+) to K(+) ions, PNa/PK, were close to 0.01. Only in the presence of Na(+) ions on both sides of the membrane was IKATP,Na similarly large to that calculated from the permeability ratio PNa/PK, indicative of a Na(+) influx that is largely independent of the K+ efflux at Vrev. With the use of a peak KATP channel conductance in anoxic cardiomyocytes of 410 nS, model simulations for a myocyte within the ischemic myocardium showed that the amplitude of the Na(+) influx and K(+) efflux is even larger than the respective fluxes by the Na(+) - K(+) pump and all other background fluxes. These results suggest that during early ischemia the Na(+) influx through KATP channels essentially contributes to the total Na+ influx and that it also balances the K(+) efflux through KATP channels.     

Effect of Na(+) flow on Cd(2+) block of tetrodotoxin-resistant Na(+) channels

Tetrodotoxin-resistant (TTX-R) Na(+) channels are 1,000-fold less sensitive to TTX than TTX-sensitive (TTX-S) Na(+) channels. On the other hand, TTX-R channels are much more susceptible to external Cd(2+) block than TTX-S channels. A cysteine (or serine) residue situated just next to the aspartate residue of the presumable selectivity filter "DEKA" ring of the TTX-R channel has been identified as the key ligand determining the binding affinity of both TTX and Cd(2+). In this study we demonstrate that the binding affinity of Cd(2+) to the TTX-R channels in neurons from dorsal root ganglia has little intrinsic voltage dependence, but is significantly influenced by the direction of Na(+) current flow. In the presence of inward Na(+) current, the apparent dissociation constant of Cd(2+) ( approximately 200 microM) is approximately 9 times smaller than that in the presence of outward Na(+) current. The Na(+) flow-dependent binding affinity change of Cd(2+) block is true no matter whether the direction of Na(+) current is secured by asymmetrical chemical gradient (e.g., 150 mM Na(+) vs. 150 mM Cs(+) on different sides of the membrane, 0 mV) or by asymmetrical electrical gradient (e.g., 150 mM Na(+) on both sides of the membrane, -20 mV vs. 20 mV). These findings suggest that Cd(2+) is a pore blocker of TTX-R channels with its binding site located in a multiion, single-file region near the external pore mouth. Quantitative analysis of the flow dependence with the flux-coupling equation reveals that at least two Na(+) ions coexist with the blocking Cd(2+) ion in this pore region in the presence of 150 mM ambient Na(+). Thus, the selectivity filter of the TTX-R Na(+) channels in dorsal root ganglion neurons might be located in or close to a multiion single-file pore segment connected externally to a wide vestibule, a molecular feature probably shared by other voltage-gated cationic channels, such as some Ca(2+) and K(+) channels.     

The interaction of Na(+) and K(+) in the pore of cyclic nucleotide-gated channels

The permeability ratio between K(+) and Na(+) ions in cyclic nucleotide-gated channels is close to 1, and the single channel conductance has almost the same value in the presence of K(+) or Na(+). Therefore, K(+) and Na(+) ions are thought to permeate with identical properties. In the alpha-subunit from bovine rods there is a loop of three prolines at positions 365 to 367. When proline 365 is mutated to a threonine, a cysteine, or an alanine, mutant channels exhibit a complex interaction between K(+) and Na(+) ions. Indeed K(+), Rb(+) and Cs(+) ions do not carry any significant macroscopic current through mutant channels P365T, P365C and P365A and block the current carried by Na(+) ions. Moreover in mutant P365T the presence of K(+) in the intracellular (or extracellular) medium caused the appearance of a large transient inward (or outward) current carried by Na(+) when the voltage command was quickly stepped to large negative (or positive) membrane voltages. This transient current is caused by a transient potentiation, i.e., an increase of the open probability. The permeation of organic cations through these mutant channels is almost identical to that through the wild type (w.t.) channel. Also in the w.t. channel a similar but smaller transient current is observed, associated to a slowing down of the channel gating evident when intracellular Na(+) is replaced with K(+). As a consequence, a rather simple mechanism can explain the complex behavior here described: when a K(+) ion is occupying the pore there is a profound blockage of the channel and a potentiation of gating immediately after the K(+) ion is driven out. Potentiation occurs because K(+) ions slow down the rate constant K(off) controlling channel closure. These results indicate that K(+) and Na(+) ions do not permeate through CNG channels in the same way and that K(+) ions influence the channel gating.     

Effects of channel cytoplasmic regions on the activation mechanisms of cardiac versus skeletal muscle Na(+) channels

Functional comparison of skeletal muscle (rSkM1) and cardiac (hH1) voltage-gated sodium channel isoforms expressed in Chinese hamster ovary cells showed rSkM1 half-activation (V(a)) and inactivation (V(i)) voltages 7 and 10 mV more depolarized than hH1 V(a) and V(i), respectively. Internal papain perfusion removed fast inactivation from each isoform and caused a 20-mV hyperpolarizing shift in hH1 V(a), with an insignificant change in rSkM1 V(a). Activation voltage of the inactivation-deficient hH1 mutant, hH1Q3, was nearly identical to wild-type hH1 V(a), both before and after papain treatment, with hH1Q3 V(a) also shifted by nearly 20 mV after internal papain perfusion. These data indicate that while papain removes both hH1 and rSkM1 inactivation, it has a second effect only on hH1 that causes a shift in activation voltage. Internal treatment with an antibody directed against the III-IV linker essentially mimicked papain treatment by removing some inactivation from each isoform and causing a 12-mV shift in hH1 V(a), while rSkM1 V(a) remained constant. This suggests that some channel segment within, near, or interacting with the III-IV linker is involved in establishing hH1 activation voltage. Together the data show that rSkM1 and hH1 activation mechanisms are different and are the first to suggest a role for a cytoplasmic structure in the voltage-dependent activation of cardiac sodium channels.     

Differential regulation of Na(+),K(+)-ATPase and the Na(+)-coupled glucose transporter in hypertensive rat kidney

Several Na(+) transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na(+),K(+)-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na(+),K(+)-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na(+),K(+)-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K(m)) for ATP, but not with a change of maximum velocity (V(max)). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V(max) for alpha-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na(+),K(+)-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na(+),K(+)-ATPase and SGLT1 activity occurs via protein phosphorylation.     

S-adenosyl-L-homocysteine hydrolase is necessary for aldosterone-induced activity of epithelial Na(+) channels

The A6 cell line was used to study the role ofS-adenosyl-L-homocysteine hydrolase (SAHHase) inthe aldosterone-induced activation of the epithelial Na⁺channel (ENaC). Because aldosterone increases methylation of severaldifferent molecules, and because this methylation is associated withincreased Na⁺ reabsorption, we tested the hypothesis thataldosterone increases the expression and activity of SAHHase protein.The rationale for this work is that general methylation may be promotedby activation of SAHHase, the only enzyme known to metabolize SAH, apotent end-product inhibitor of methylation. Although aldosteroneincreased SAHHase activity, steroid did not affect SAHHase expression.Antisense SAHHase oligonucleotide decreased SAHHaseexpression and activity. Moreover, this oligonucleotide, as well as apharmacological inhibitor of SAHHase, decreased aldosterone-inducedactivity of ENaC via a decrease in ENaC open probability. The kineticsof ENaC in cells treated with antisense plus aldosterone were similarto those reported previously for the channel in the absence of steroid. This is the first report showing that active SAHHase, in part, increases ENaC open probability by reducing the transition rate fromopen states in response to aldosterone. Thus aldosterone-induced SAHHase activity plays a critical role in shifting ENaC from a gatingmode with short open and closed times to one with longer open andclosed times.

     

Voltage-dependent stimulation of the Na(+)-K(+) pump by insulin in rabbit cardiac myocytes

Insulin enhancesNa⁺-K⁺ pump activity in various noncardiactissues. We examined whether insulin exposure in vitro regulates Na⁺-K⁺ pump function in rabbit ventricularmyocytes. Pump current (I_p) was measured using thewhole-cell patch-clamp technique at test potentials(V_ms) from 100 to +60 mV. When theNa⁺ concentration in the patch pipette([Na]_pip) was 10 mM, insulin caused aV_m-dependent increase in I_p.The increase was ~70% when V_m was at nearphysiological diastolic potentials. This effect persisted afterelimination of extracellular voltage-dependent steps and whenK⁺ and K⁺-congeners were excluded from thepatch pipettes. When [Na]_pip was 80 mM, causingnear-maximal pump stimulation, insulin had no effect, suggesting thatit did not cause an increase in membrane pump density. Effects oftyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I inpipette solutions suggested that the insulin-induced increase inI_p involved activation of tyrosine kinase,phosphatidylinositol 3-kinase, and protein phosphatase 1, whereasprotein phosphatase 2A and protein kinase C were not involved.

     

Gating in iodate-modified single cardiac Na+ channels

Summary Elementary Na⁺ currents were recorded at 19°C during 220-msec lasting step depolarizations in cell-attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the modifying influence of iodate, bromate and glutaraldehyde on single cardiac Na⁺ channels.Iodate (10 mmol/liter) removed Na⁺ inactivation and caused repetitive, burst-like channel activity after treating the cytoplasmic channel surface. In contrast to normal Na⁺ channels under control conditions, iodate-modified Na⁺ channels attain two conducting states, a short-lasting one with a voltage-independent lifetime close to 1 msec and, likewise tested between –50 and +10 mV, a long-lasting one being apparently exponentially dependent on voltage. Channel modification by bromate (10 mmol/liter) and glutaraldehyde (0.5 mmol/liter) also included the occurrence of two open states. Also, burst duration depended apparently exponentially on voltage and increased when shifting the membrane in the positive direction, but there was no evidence for two bursting states. Chemically modified Na⁺ channels retain an apparently normal unitary conductance (12.8±0.5 pS). Of the two substates observed, one of them is remarkable in that it is mostly attained from full-state openings and is very short living in nature; the voltage-independent lifetime was close to 2 msec. Despite removal of inactivation, open probability progressively declined during membrane depolarization. The underlying deactivation process is strongly voltage sensitive but, in contrast to slow Na⁺ inactivation, responds to a voltage shift in the positive direction with a retardation in kinetics. Chemically modified Na⁺ channels exhibit a characteristic bursting state much shorter than in DPI-modified Na⁺ channels, a difference not consistent with the hypothesis of common kinetic properties in noninactivating Na⁺ channels.     

I(Ca(TTX)) channels are distinct from those generating the classical cardiac Na(+) current

The Na(+) current component I(Ca(TTX)) is functionally distinct from the main body of Na(+) current, I(Na). It was proposed that I(Ca(TTX)) channels are I(Na) channels that were altered by bathing media containing Ca(2+), but no, or very little, Na(+). It is known that Na(+)-free conditions are not required to demonstrate I(Ca(TTX).) We show here that Ca(2+) is also not required. Whole-cell, tetrodotoxin-blockable currents from fresh adult rat ventricular cells in 65 mm Cs(+) and no Ca(2+) were compared to those in 3 mM Ca(2+) and no Cs(+) (i.e., I(Ca(TTX))). I(Ca(TTX)) parameters were shifted to more positive voltages than those for Cs(+). The Cs(+) conductance-voltage curve slope factor (mean, -4.68 mV; range, -3.63 to -5.72 mV, eight cells) is indistinguishable from that reported for I(Ca(TTX)) (mean, -4.49 mV; range, -3.95 to -5.49 mV). Cs(+) current and I(Ca(TTX)) time courses were superimposable after accounting for the voltage shift. Inactivation time constants as functions of potential for the Cs(+) current and I(Ca(TTX)) also superimposed after voltage shifting, as did the inactivation curves. Neither of the proposed conditions for conversion of I(Na) into I(Ca(TTX)) channels is required to demonstrate I(Ca(TTX)). Moreover, we find that cardiac Na(+) (H1) channels expressed heterologously in HEK 293 cells are not converted to I(Ca(TTX)) channels by Na(+)-free, Ca(2+)-containing bathing media. The gating properties of the Na(+) current through H1 and those of Ca(2+) current through H1 are identical. All observations are consistent with two non-interconvertable Na(+) channel populations: a larger that expresses little Ca(2+) permeability and a smaller that is appreciably Ca(2+)-permeable.     

Protein kinase Cepsilon contributes to regulation of the sarcolemmal Na(+)-K(+) pump

A reduction in angiotensin II (ANG II) in vivo by treatment of rabbits with the angiotensin-converting enzyme inhibitor, captopril, increases Na(+)-K(+) pump current (I(p)) of cardiac myocytes. This increase is abolished by exposure of myocytes to ANG II in vitro. Because ANG II induces translocation of the epsilon-isoform of protein kinase C (PKCepsilon), we examined whether this isozyme regulates the pump. We treated rabbits with captopril, isolated myocytes, and measured I(p) of myocytes voltage clamped with wide-tipped patch pipettes. I(p) of myocytes from captopril-treated rabbits was larger than I(p) of myocytes from controls. ANG II superfusion of myocytes from captopril-treated rabbits decreased I(p) to levels similar to controls. Inclusion of PKCepsilon-specific blocking peptide in pipette solutions used to perfuse the intracellular compartment abolished the effect of ANG II. Inclusion of psiepsilonRACK, a PKCepsilon-specific activating peptide, in pipette solutions had an effect on I(p) that was similar to that of ANG II. There was no additive effect of ANG II and psiepsilonRACK. We conclude that PKCepsilon regulates the sarcolemmal Na(+)-K(+) pump.     

Responsiveness of cardiac Na+ channels to antiarrhythmic drugs: The role of inactivation

Summary Elementary Na⁺ currents were recorded at 9°C in inside-out patches from cultured neonatal rat heart myocytes. In characterizing the sensitivity of cooled, slowly inactivating cardiac Na⁺ channels to several antiarrhythmic drugs including propafenone, lidocaine and quinidine, the study aimed to define the role of Na⁺ inactivation for open channel blockade.In concentrations (1–10 mol/liter) effective to depressNP _o significantly, propafenone completely failed to influence the open state of slowly inactivating Na⁺ channels. With 1 mol/liter, _open changed insignificantly to 96±7% of the control. Even a small number of ultralong openings of 6 msec or longer exceeding _open of the whole ensemble several-fold and attaining _open (at –45 mV) in cooled, (-)-DPI-modified, noninactivating Na⁺ channels proved to be drug resistant and could not be flicker-blocked by 10 mol/liter propafenone. The same drug concentration induced in(-)-DPI-modified Na⁺ channels a discrete block with association and dissociation rate constants of 16.1 ± 5.3 × 10⁶ mol^–1 sec^–1 and 675 ± 25 sec^–1, respectively. Quinidine, known to have a considerable affinity for activated Na⁺ channels, in lower concentrations (5 mol/liter) left _open unchanged or reduced, in higher concentrations (10 mol/liter) _open only slightly to 81% of the predrug value whereasNP _o declined to 30%, but repetitive blocking events during the conducting state could never be observed. Basically the same drug resistance of the open state was seen in cardiac Na⁺ channels whose open-state kinetics had been modulated by the cytoplasmic presence of F^– ions. But in this case, propafenone reduced reopening and selectively abolished a long-lasting open state. This drug action is unlikely related to the inhibitory effect onNP _o since hyperpolarization and the accompanying block attenuation did not restore the channel kinetics. It is concluded that cardiac Na⁺ channels cannot be flicker-blocked by antiarrhythmic drugs unless Na⁺ inactivation is removed.     

Evidence for Na(+) influx via the NtpJ protein of the KtrII K(+) uptake system in Enterococcus hirae

The ntpJ gene, a cistron located at the tail end of the vacuolar-type Na(+)-ATPase (ntp) operon of Enterococcus hirae, encodes a transporter of the KtrII K(+) uptake system. We found that K(+) accumulation in the ntpJ-disrupted mutant JEM2 was markedly enhanced by addition of valinomycin at pH 10. Studies of the membrane potential (DeltaPsi; inside negative) by 3, 3'-dihexyloxacarbocyanine iodide fluorescence revealed that the DeltaPsi was hyperpolarized at pH 10 in JEM2; the DeltaPsi values of the parent strain ATCC 9790 and JEM2, estimated by determining the equilibrium distribution of K(+) or Rb(+) in the presence of valinomycin, were -118 and -160 mV, respectively. DeltaPsi generation at pH 10 was accomplished by an electrogenic Na(+) efflux via the Na(+)-ATPase, whose levels in the two strains were quite similar. Na(+) uptake driven by an artificially imposed DeltaPsi (inside negative) was missing in JEM2, suggesting that NtpJ mediates Na(+) movement in addition to K(+) movement. Finally, the growth of JEM2 arrested in K(+)-limited high-Na(+) medium at pH 10 was restored by addition of valinomycin. These results suggest that NtpJ mediates electrogenic transport of K(+) as well as Na(+), that it likely mediates K(+) and Na(+) cotransport, and that Na(+) movement via NtpJ is the major Na(+) reentry pathway at high pH values.     

A pore segment in DEG/ENaC Na(+) channels.

DEG/ENaC Na(+) channels have diverse functions, including Na(+) absorption, neurotransmission, and sensory transduction. The ability of these channels to discriminate between different ions is critical for their normal function. Several findings suggest that DEG/ENaC channels have a pore structure similar to K(+) channels. To test this hypothesis, we examined the accessibility of native and introduced cysteines in the putative P loop of ENaC. We identified residues that span a barrier that excludes amiloride as well as anionic and large methanethiosulfonate reagents from the pore. This segment contains a structural element ((S/G)CS) involved in selectivity of ENaC. The results are not consistent with predictions from the K(+) channel pore, suggesting that DEG/ENaC Na(+) channels have a novel pore structure.     

Modulation of cardiac Na channels by angiotensin II

The modulation of Na channels by the vasoactive peptide angiotensin II (AT II) has been studied in isolated ventricular cells of guinea pigs using the patch clamp technique. In cell-attached patches the maximal probability of the channel being open was increased in a concentration range between 0.05 and 1 microM, but decreased at higher concentrations. A maximal increased of 2.5 +/- 0.86 was found at 1 microM AT II. The increase in the probability of the channel being open was due to a decrease in the number of nulls. In all affected cells (n = 17) we observed a delayed inactivation after application of AT II at concentrations between 0.05 and 10 microM. At -30 mV, the time constant of inactivation increased from 1.1 +/- 0.1 ms (controls) to 5.6 +/- 1.6 ms (10 microM AT II). This effect was due to an increased number of openings per sweeps. No significant effect on the mean open time and the first latency were observed. However, due to pronounced bursting, the averaged closed time was significantly increased from 0.8 +/- 0.1 ms to 1.3 +/- 0.1 ms in the presence of 1 microM AT II at -30 mV. An effect of AT II on cardiac Na channels via protein kinase C is discussed.     

Location of a ligand recognition site of FMRFamide-gated Na(+) channels

The second FMRFamide-gated Na(+) channel (HtFaNaC), from Helisoma trivolvis, has been cloned. HtFaNaC has some different pharmacological properties to HaFaNaC, from Helix aspersa, which has enabled a rational approach to be made to start to identify the FMRFamide recognition site. Several chimeras were made by switching sections between the channels. The differences in sensitivity to FMRFamide, and amiloride, were assessed after expression in Xenopus oocytes. The data suggest that a recognition site for FMRFamide, and the potentiating action of amiloride, resides in a sequence of about 120 amino acids in the extracellular loop proximal to the first transmembrane segment.     

Na(+) interaction with the pore of Shaker B K(+) channels: zero and low K(+) conditions.

The Shaker B K(+) conductance (G(K)) 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. 499:3-15; Gómez-Lagunas, F. 1999. Biophys. J. 77:2988-2998). In this work, the role of Na(+) ions in the collapse of G(K) 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 G(K). Second, external Na(+) fosters the drop of G(K) by binding to a site with a K(d) = 3.3 mM. External K(+) competes, in a mutually exclusive manner, with Na(o)(+) for binding to this site, with an estimated K(d) = 80 microM. Third, NMG and choline are relatively inert regarding the stability of G(K); fourth, with [K(o)(+)] = 0, the energy required to relieve Na(i)(+) block of Shaker (French, R.J., and J.B. Wells. 1977. J. Gen. Physiol. 70:707-724; Starkus, J.G., L. Kuschel, M. Rayner, and S. Heinemann. 2000. J. Gen. Physiol. 110:539-550) decreases with the molar fraction of Na(i)(+) (X(Na,i)), in an extent not accounted for by the change in Delta(mu)(Na). Finally, when X(Na,i) = 1, G(K) collapses by the binding of Na(i)(+) to two sites, with apparent K(d)s of 2 and 14.3 mM.