17beta-estradiol potentiates the cardiac cystic fibrosis transmembrane conductance regulator chloride current in guinea-pig ventricular myocytes

There is a well-characterized membrane chloride current (ICl,cAMP) in the heart that can be activated by beta-adrenergic agonists and is due to expression of the cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR). We have investigated whether 17beta-estradiol (E2) modulates ICl,cAMP in single ventricular myocytes. Under whole-cell tight-seal voltage-clamp conditions, ICl,cAMP was evoked by exposing cells to 20 nM isoprenaline. On the addition of 30 microM E2, membrane slope conductance, measured at potentials near 0 mV, increased over that induced by isoprenaline alone by 2.46 +/- 0.16 (p < 0.001). The effects of E2 were concentration-dependent and described by a Hill Plot with an EC50 of 8.2 microM and a Hill coefficient of 1.63. The application of membrane-impermeant E2 conjugated to bovine serum albumin (E2-BSA) potentiated isoprenaline-evoked ICl,cAMP by approximately the same degree as that for the equivalent level of free E2. Cell surface binding was observed with confocal microscopy by using BSA-FITC tagged E2. This binding was inhibited by nonlabeled, nonconjugate E2, the specific E2 antagonist ICl 182,780, and incubation of E2coBSA with a specific anti-E2 antibody (E2885). ICl 182,780 (100 microM) significantly reduced the increase in ICl,cAMP evoked by 10 microM E2 to 1.46 +/- 0.10 (p < 0.02). The preincubation of myocytes with the NOS inhibitor N-omega-nitro-arginine (L-NNA, 1 mM) reduced the potentiation of ICl,cAMP by 30 microM E2, to 1.93 +/- 0.06 (p < 0.02), and for 10 microM E2, to 1.32 +/- 0.05 (p < 0.002). E2 also increased ICl,cAMP evoked by bath application of 0.5 microM Forskolin. These experiments demonstrate that, under our experimental conditions, E2 dramatically increases ICl,cAMP in ventricular myocytes by mechanisms involving a contribution by NOS, but that can be only partially accounted for through binding to classical plasma membrane estrogen receptor sites. This potentiation of ICl,cAMP by E2 may play a significant role in the observed clinical actions of E2 on the incidence of cardiac arrhythmias and hypertrophy.     

Exogenous lysophosphatidylcholine increases non-selective cation current in guinea-pig ventricular myocytes

Whole cell, patch-clamp studies were performed to examine the effect of lysophosphatidylcholine (LPC) on the membrane current in guinea-pig ventricular myocytes. The addition of 10 μM LPC to the external solution induced a membrane current which had a reversal potential of 0 mV. When Na⁺, the main cation in the external solution, was replaced by either K⁺, N-methyl-D-glucamine (NMG) or 90 mM Ca²⁺, LPC induced a current with the reversal potential near 0 mV, indicating that the current passed through a Ca²⁺-permeable non-selective cation channel. The order of the cationic permeability calculated from the reversal potential of the current was Cs⁺ > K⁺ > NMG > Na⁺ > Ca²⁺. Cl⁻ did not pass through the LPC-induced channel. The LPC-induced current was not blocked by Gd³⁺ in the external solution, nor by the absence of Ca²⁺ in the pipette solution. In conclusion, LPC induces a Ca²⁺-permeable non-selective cation channel in guinea-pig ventricular myocytes. Received: 11 September 1995/Received after revision: 3 January 1996/Accepted: 12 February 1996     

Volume-sensitive chloride current activated by hyposmotic swelling in antral gastric myocytes of the guinea-pig

 The characteristics of volume-sensitive chloride current (I _Cl) induced by osmotic cell swelling were studied using the whole-cell patch-clamp technique and cell diameters of antral circular guinea-pig myocytes were simultaneously measured under isosmotic and hyposmotic conditions by using a video image analysis system. At –60 mV, osmotic cell swelling (200 mosmol/l) activated a sustained inward current. Instantaneous current/voltage (I-V) relations obtained by step voltage pulses showed an outward rectification. At potentials above +40 mV, the current exhibited time-dependent decay. The outward current amplitude was decreased and the reversal potential was shifted to more positive potentials by replacement of external Cl^– with gluconate^–, while the current amplitude and the I/V relation were not affected by replacing extracellular Na⁺ with N-methyl-D-glucamine. The anion permeability sequence of the swelling-induced current was I^– (1.80) > Br^– (1.31) > Cl^– (1) > F^– (0.85) > gluconate^– (0.18). The I _Cl was effectively inhibited by the Cl^– channel blockers, 4,4′-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS, 100 μM), and niflumic acid (10 μM). DIDS suppressed outward current more effectively than inward current. Also, the I _Cl was dose-dependently inhibited by arachidonic acid, an unsaturated fatty acid and also inhibited by other unsaturated fatty acids (linoleic acid and oleic acid) but not by stearic acid, a saturated fatty acid. The inhibitory effect of arachidonic acid on I _Cl was not prevented by indomethacin, a cyclo-oxygenase inhibitor and chelerythrine, a protein kinase C inhibitor. Under whole-cell patch-clamp conditions, the cell diameter was continuously measured using video image analysis, which reflects the change in cell volume. A hyposmotic-stimulation-induced increase of cell diameter was followed by I _Cl activation. In intact single gastric myocytes, relatively severe hyposmotic (176 mosmol/l) superfusing solution increased the cell diameter and the pretreatment with DIDS or with niflumic acid significantly potentiated the above effect of hyposmotic superfusion. These results suggest that volume-sensitive outwardly rectifying chloride current (I _Cl) is present in guinea-pig gastric myocyte and the I _Cl may play a role in smooth muscle cell volume regulation. Received: 6 June 1997 / Received after revision and accepted: 24 July 1997     

A sodium-activated potassium current in intact ventricular myocytes isolated from the guinea-pig heart

A current generated by the Na-activated K channel has been identified in whole cell currents recorded from isolated guinea-pig ventricular myocytes. A partial activation of this current can be achieved near to the physiological range of intracellular sodium concentration [( Na+]i) when it contributes significantly to the global outward current. The decline of the Na-activated K current, the lengthening of the action potential duration and the recovery of [Na+]i occur with a similar time course during recovery from Na loading.     

Ultra-slow voltage-dependent inactivation of the calcium current in guinea-pig and ferret ventricular myocytes

L-type Ca²⁺ current, i _Ca, has been recorded in guinea-pig ventricular myocytes at 36° C using the whole cell patch clamp technique. Intracellular Ca²⁺ was buffered with ethylenebis(oxonitrilo)tetraacetate (EGTA). An increase in the rate of stimulation from 0.5 to 3 Hz resulted in an abrupt decrease in i _Ca in the first beat at the high rate, followed by a progressive decrease ( approx. 7 s) over the next 30 s. The changes were not the result of Ca²⁺-dependent inactivation, because similar changes occurred with either Ba²⁺ or Na⁺ as the charge carrier. During 20-s voltage clamp pulses there was an ultra-slow phase of inactivation of Ba²⁺ or Na⁺ current through the Ca²⁺ channel ( approx. 6 s at 0 mV). This was confirmed by applying test pulses after conditioning pulses of different duration: the Ba²⁺ current during the test pulse decreased progressively when the duration of the conditioning pulse was increased progressively to 20 s. Ultra-slow inactivation of Ba²⁺ current was voltage dependent and increased monotonically at more positive potentials. Recovery of Ba²⁺ current from ultra-slow inactivation occurred with a time constant of 3.7 s at –40 mV and 0.7 s at –80 mV. The gradual decrease in i _Ca on increasing the rate to 3 Hz may have been the result of the development of ultra-slow voltage-dependent inactivation.     

Inactivation of Ca current during the action potential in guinea-pig ventricular myocytes.

The inactivation of Ca channels during the action potential plateau of guinea-pig ventricular myocytes was investigated by interrupting action potentials with voltage clamp pulses to assess Ca channel availability. The influence of the bulk cytosolic calcium [( Ca]i) transient on Ca channel inactivation was also studied by impaling cells with microelectrodes containing the Ca chelator BAPTA (1,2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; 125-200 mM). Ca channel availability decreased progressively with action potential duration, reaching approximately 20% of maximum availability after 100 ms and falling close to zero at the end of the plateau. When membrane potential became more negative than -40 mV Ca channel availability increased. Elevation of the action potential plateau to more positive levels increased Ca channel availability (even though this was expected to increase peak [Ca]i). When the cytosol was loaded with BAPTA Ca channel availability during the plateau increased. Inactivation of Ca channels was not, however, abolished. The observations are consistent with the hypothesis that in guinea-pig ventricular myocytes the majority of Ca channels are inactivated during the plateau and recovery does not occur until repolarization is almost complete. It may be that while the cytosolic Ca transient (that is generated in part by release of Ca from the intracellular Ca stores) modulates Ca channel availability, significant inactivation of the Ca channel during the action potential plateau is due to voltage dependent inactivation and to Ca-induced inactivation resulting from the Ca which enters the myocyte via Ca channels.     

花生四烯酸抑制豚鼠胃窦环行肌细胞容积敏感氯电流

利用全细胞膜片钳技术 ,在用胶原酶急性分离的豚鼠胃窦环行肌细胞上观察了外源性花生四烯酸 (arachidonicacid ,AA)及其它不饱和脂肪酸对容积敏感氯电流 (volume sensitivechloridecurrent,VSCC)的影响。细胞膜电位钳制在- 6 0mV条件下 ,低渗性细胞膨胀激活VSCC。当给予按 2 0mV间隔的去极化阶跃性方波刺激时 ,VSCC的电流 电压曲线具有外向性整流的特点 ,并表现出去极化到 +40mV以上时呈现时间依赖性衰减的特点。AA呈齐量依赖性地抑制VSCC ,而对VSCC的随时间衰减部分的抑制效应更明显。其它不饱和脂肪酸 ,如含有 2个双键的亚油酸 (Linoleicacid,LA)、含有 1个双键的油酸 (Oleicacid ,OA)也抑制VSCC ,抑制强度顺序为AA >LA >OA ,但饱和脂肪酸 (stearicacid,SA)对VSCC无抑制作用。提示外源性不饱和脂肪酸抑制VSCC ,其抑制效应在VSCC的随时间衰减部分更明显。外源性不饱和脂肪酸对VSCC的抑制作用可能与脂肪酸链中的双键数目有关     

Sodium current block caused by group IIb cations in calf Purkinje fibres and in guinea-pig ventricular myocytes

The action of group IIb cations [Cadmium (Cd²⁺), Zinc (Zn²⁺), Mercury (Hg²⁺)] on the cardiac fast sodium current (I _Na) was investigated in calf Purkinje fibres and in ventricular cells isolated from guinea-pig hearts. In calf Purkinje fibres, I _Na was depressed by submillimolar concentrations of Zn²⁺ and Hg²⁺. With both cations, the current reduction occurred at all voltages in the range of current activation and the voltage dependence of peak current was unchanged. The degree of peak current inhibition depended on the cation concentration but not on voltage. The position of the inactivation curve on the voltage axis was unaltered at cation concentrations giving substantial current inhibition, and moved to the right only with concentration exceeding 1–1.5 mM. These effects can be interpreted as due to I _Na channel blockade. The action of Zn²⁺ and Hg²⁺ was similar to that described earlier of Cd²⁺ on Purkinje fibres (DiFrancesco et al. 1985b). I _Na was also inhibited by group IIb cations in isolated guinea-pig ventricular cells. Depression of I _Na by Cd²⁺, Zn²⁺ and Hg²⁺ was essentially voltage-independent, in agreement with its being caused by channel block. The dependence of I _Na block by Cd²⁺ upon external Na concentration [Na⁺ ₀] was investigated in ventricular myocytes. The fraction of I _Na block by 0.1 mM CdCl₂ was 0.50 at 140 mM, 0.81 at 70 mM and 0.83 at 35 mM [Na⁺]₀. A similar increase of block efficiency at low [Na⁺ ₀] was observed with 0.05 mM CdCl₂. In both the Purkinje fibre and the ventricular cell, the order of potency of I _Na block by group IIb cations was Hg²⁺> Zn²⁺> Cd²⁺. Manganese (Mn²⁺, 2–5 mM), an ion of group VIIa, also depressed the I _Na in Purkinje fibres and ventricular myocytes. This effect was however due mainly to a positive shift on the voltage dependence of current kinetics rather than to a reduction of the conductance of the channel (G _Na), and can be accounted for by an ion-screening action of Mn²⁺ on the external membrane surface. The block by group IIb cations is a typical property of cardiac Na⁺ channels and characterizes the cardiac as opposed to other types of Na⁺ channel. Offprint request to: D. DiFrancesco     

Tyrosine kinase and phosphatase regulation of slow delayed-rectifier K+ current in guinea-pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the regulation of the cardiac slowly activating delayed-rectifier K⁺ current ( I _Ks) that is important for action potential repolarization. Constitutive I _Ks recorded from guinea-pig ventricular myocytes was suppressed by broad-spectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC₅₀, 4.1 ± 0.6 μ m ), tyrphostin A25 (IC₅₀, 12.1 ± 2.1 μ m ) and genistein (IC₅₀, 64 ± 4 μ m ), but was relatively insensitive to the inactive analogues tyrphostin A1, tyrphostin A63, daidzein and genistin. I _Ks was unaffected by AG1478 (10 μ m ), an inhibitor of epidermal growth factor receptor TK, and was strongly suppressed by the Src TK inhibitor PP2 (10 μ m ) but not by the inactive analogue PP3 (10 μ m ). The results of experiments with forskolin, H89 and bisindolylmaleimide I indicate that the suppression of I _Ks by TK inhibitors was not mediated via inhibition of ( I _Ks-stimulatory) protein kinases A and C. To evaluate whether the suppression was related to lowered tyrosine phosphorylation, myocytes were pretreated with TK inhibitors and then exposed to the phosphotyrosyl phosphatase inhibitor orthovanadate (1 m m ). Orthovanadate almost completely reversed the suppression of I _Ks induced by broad-spectrum TK inhibitors at concentrations around their IC₅₀ values. We conclude that basal I _Ks is strongly dependent on tyrosine phosphorylation of Ks channel (or channel-regulatory) protein.     

The action of strophanthidin on calcium-activated current and contraction in single guinea-pig ventricular myocytes

Calcium-activated tail current was used as a qualitative indicator of changes in [Ca]i in order to investigate the mechanism of strophanthidin-induced inotropy in single guinea-pig ventricular myocytes. Action potentials were interrupted (by application of a voltage clamp to -40 mV) in order to evoke Ca-activated current. Exposure to 10 microM-strophanthidin for 2 min resulted in an increase in contractions associated with complete action potentials and in an increase in Ca-activated current. Strophanthidin appeared not to substantially modify the time course of the envelope of tail currents (recorded by interrupting action potentials at different durations), which is thought to reflect the time course of the systolic [Ca]i transient. Exposure to 1 microM-ryanodine slowed the development of the Ca-activated current envelopes and abolished the above effects of strophanthidin. Exposure to strophanthidin led to reduction in Ca current in a majority of cells (measured by a voltage-clamp step from -40 to 0 mV). These results are consistent with the hypothesis that in single guinea-pig ventricular myocytes strophanthidin causes an increased loading of ryanodine-sensitive intracellular stores of Ca, possibly through reduced extrusion of Ca from the cell by Na-Ca exchange during the action potential plateau.     

Inactivation of outward Na(+)-Ca2+ exchange current in guinea-pig ventricular myocytes.

1. Outward Na(+)-Ca2+ exchange currents were measured in freshly dissociated guinea-pig myocytes to probe in intact cells the functional status of exchanger inactivation reactions, described previously in giant excised cardiac membranes patches. 2. When the cytoplasmic (pipette) solution contained 40 mM Na+ and 0.1 microM free Ca2+ (50 mM EGTA), the outward exchange current activated by extracellular Ca2+ decayed with time (time constant, 13.1 +/- 2.6 s; n = 6), and an inward current transient was observed upon removal of extracellular Ca2+. Both the current decay and the subsequent inward current transient were remarkably diminished with a saturating (100 mM) pipette Na+ concentration. 3. With 100 mM cytoplasmic Na+ and 140 mM extracellular Na+, a significant fraction of the exchanger population is predicted to be in an inactive state. Intracellular application of 2 mg ml-1 chymotrypsin and 5 microM sodium tetradecylsulphate, both of which decrease Na(+)-dependent inactivation in giant membrane patches, increased the outward exchange current by about 160-170%, suggesting that about 60-70% of exchangers might be inactivated. 4. With 100 mM cytoplasmic Na+ and no extracellular Na+ (replaced with 140 mM Li+), application of extracellular Ca+ was predicted to reorient exchanger binding sites from the extracellular side to the cytoplasmic side and thereby favour inactivation. During such protocols, the outward exchange current decayed by 60-80% when activated by extracellular Ca2+. The current decayed similarly when extracellular Ca2+ and Na+ were applied together, whereby current magnitudes were about 3-fold smaller. 5. The decay of outward exchange current usually followed a biexponential time course (5.8 +/- 3.5 and 27.3 +/- 16.3 s, means +/- S.D., n = 11). Intracellular application of 0.5-2 mg ml-1 trypsin attenuated the fast component more than the slow component, suggesting that the fast component reflects an inactivation process. 6. Current-voltage (I-V) relations of the outward exchange current became less steep during the inactivation protocols, but this flattening could not be correlated with inactivation. 7. Replacement of extracellular Li+ with N-methyl-D-glucamine (NMG), tetraethylammonium (TEA), sucrose or Cs+ resulted in a flattening of I-V relations and a decrease of the outward exchange current amplitude by approximately 3-fold, but the kinetics and extent of inactivation were not remarkably changed. Thus, the mechanism of inactivation appears to be independent of the mechanism(s) of activation by extracellular monovalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of oximes on the dihydropyridine-sensitive Ca current of isolated guinea-pig ventricular myocytes

Exposure of isolated guinea-pig ventricular myocytes to the uncharged oximes 2,3 butanedionemonoxine (BDM) and norPAM (but not by the charged PAM) results in a dose-dependent reduction of the duration of the action potential. The nifedipine-sensitive Ca current is fully inhibited by BDM (IC₅₀5.8±0.4 mM) and nor PAM but is little affected by PAM. This inhibition is unaltered by the presence of BAY K 8644 but is antagonized by isoprenaline. The effect of isoprenaline is more pronounced when the solution in the patch pipette contains the non-hydrolysable analogue of adenosine 5-triphosphate, ATPS (the IC₅₀ is increased to 44.0±5.2 mM). A hastening of the inactivation of the L-type Ca current persists when either 10 mM 1,2-bis(2-aminophenoxy)-ethane-N, N, N, N-tetraacetic acid (BAPTA) or 3 mM ATPS is present in the pipette solution or when BAY K 8644 or isoprenaline are present in the bathing fluid. These results suggest that the inhibition of the Ca current is due to the phosphatase-like activity of the oximes but differs in some respects from previous work where a reduced level of phosphorylation is achieved by the introduction of protein kinase inhibitors or protein phosphatases into the sarcoplasm in guinea-pig myocytes. These differences could be explained if Ca channel availability is regulated by at least two sites of cAMP-dependent phosphorylation with oximes able to rapidly dephosphorylate both sites, while one of these sites is not readily dephosphorylated by the endogenous phosphatases.     

Endothelin-1 inhibitsl-type Ca currents enhanced by isoproterenol in guinea-pig ventricular myocytes

To investigate the action of Endothelin-1 (ET-1) on L-type Ca currents (I _Ca,L) in guinea-pig ventricular cells, whole-cell currents were recorded at 36-37° C in enzymatically isolated myocytes. ET-1 ( 10 nM) suppressed the basal I_ca,L to 79 ± 8% of control at 20 nM. Bath application of isoproterenol (ISO; 10 nM) enhanced I_Ca,L to 192±28% with about a – 10-mV shift of its relationship with membrane potential. ET-1 concentration dependently inhibited this ISO-enhanced I_Ca,L with a half-maximally inhibitory concentration (IC₅₀) of 168 pM. The inhibitory actions of ET-1 were antagonised by BQ-123 (300 nM), cyclo(d-Asp-l-Pro-d-Val-l-Leu-d-Trp), a specific ETA receptor antagonist. Histamine-enhancedI _Ca,L was also suppressed by ET-1, butI _Ca,L potentiated by internal adenosine 3,5-cyclic monophosphate (cAMP) was unaffected. Preincubation of myocytes with pertussis toxin (PTX, at 5 g/ml for >60 min at 36° C) completely occluded the ET-1 action. Thus, stimulation of ET_A receptors by subnanomolar ET-1 inhibitsI _Ca,L via PTX-sensitive G-proteins.     

1-磷酸鞘氨醇延长豚鼠心室肌细胞动作电位及抑制电压依赖性钾电流

目的研究1-磷酸鞘氨醇(S1P)对豚鼠心室肌细胞动作电位(AP)及电压依赖性钾电流(KV)的作用及其机制。方法利用Langendorff离体心脏逆向灌流法分离豚鼠心室肌细胞,采用全细胞膜片钳技术记录心室肌细胞AP和KV。结果S1P可延长动作电位复极50%时程(APD50)和动作电位复极90%时程(APD90),降低KV,其作用可被百日咳毒素(PTX)和卡弗他丁C(Calphostin C)阻断。结论S1P可能通过PKC途径抑制KV及延长AP时程。     

On the mechanism of the enhancement of delayed rectifier K+ current by extracellular ATP in guinea-pig ventricular myocytes

 The effects of extracellular adenosine 5′-triphosphate (ATP) on the delayed rectifier K⁺ current (I _K) were studied in guinea-pig ventricular myocytes using the whole-cell voltage-clamp technique. ATP increased I _K concentration dependently with a concentration eliciting a half-maximal response of 1.86 μM and a maximal increase of about 1.8-fold. The enhancement of I _K developed slowly, the effect reaching a maximum in about 1.6 min after application of ATP. The rank order of agonist potency in enhancing I _K was 2-methylthio-ATP≥ ATP>>α,β-methylene-ATP. The ATP response was attenuated in guanosine 5′-O-(2-thiodiphosphate) (GDPβS)- loaded cells, but was not affected by pertussis toxin (PTX)-pre-treatment, indicating that a PTX-insensitive G protein is involved in the response. These features are consistent with operation of P_2Y-type purinoceptors. ATP produced a further increase in I _K stimulated maximally either by isoprenaline (1 μM) through protein kinase A (PKA) or by 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nM) through protein kinase C (PKC), while 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 10 μM) did not affect the ATP response, suggesting that PKA and PKC do not mediate the response. ATP irreversibly enhanced I _K in cells loaded with adenosine 5′-O-(3-thiotriphosphate) (ATPγS, 5 mM) or okadaic acid (10 μM), a phosphatase inhibitor, suggesting that a phosphorylation step is present after the receptor stimulation. Genistein, an inhibitor of tyrosine phosphorylation, suppressed the ATP response significantly, while daidzein, an inactive analogue of genistein, had little effect on it, although both genistein or daidzein alone decreased I _K. It is hypothesized that tyrosine phosphorylation plays a role in the signalling pathway involved in the enhancement of cardiac I _K by P_2Y-purinergic stimulation. Received: 10 August 1998 / Received after revision: 14 November 1998 / Accepted: 4 December 1998     

The mechanism of the inactivation of the inward-rectifying K current during hyperpolarizing steps in guinea-pig ventricular myocytes

The time course of the inward-rectifying K current during hyperpolarizing clamp steps was investigated in single myocytes isolated from guinea-pig ventricles. The experiments were done using a two-electrode voltage-clamp technique with two patch pipettes in the whole-cell configuration. Hyperpolarizations to potentials negative to –100 mV, induced large inward-rectifying K currents (i _K1), which showed a marked decay. The current-voltage relation of the peak inward current was almost linear, but the steadystate current-voltage relation had a region of negative slope at potentials negative to –140 mV. These findings indicate that the channel inactivates during hyperpolarizing steps. When Na ions in the extracellular solution were replaced by choline, Tris, TMA or sucrose, the decay of the inward currents was largely reduced, and the negative slope in the steady-state current-voltage relation was absent. When divalent ions were removed from the Na-free bathing solution, a marked increase ini _K1 was found, and the currents became time-independent. These experiments demonstrate that the inactivation during hyperpolarization is largely due to a block of the channel by external Na ions. The block by Na is most pronounced at very negative potentials, and is strongly voltage-dependent. External Ca and Mg ions also cause a marked block of the channel. The block by these divalent ions is however much less voltage-dependent than the one by Na, but is already present at the cell's resting potential.