Functional roles of Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of mouse atrioventricular cells: insights into the atrioventricular pacemaker mechanism

The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Ca(v)3.1 (Ca(v)3.1(-/-)), Ca(v)1.3 (Ca(v)1.3(-/-)), channels or both (Ca(v)1.3(-/-)/Ca(v)3.1(-/-)). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Ca(v)3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Ca(v)3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Ca(v)1.3 channels were required for spontaneous automaticity, because Ca(v)1.3(-/-) and Ca(v)1.3(-/-)/Ca(v)3.1(-/-) AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Ca(v)1.3 channels are required for automaticity, Ca(v)3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for β-adrenergic regulation.     

Functional roles of Cav1.3, Cav3.1 and HCN channels in automaticity of mouse atrioventricular cells

The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Ca_v1.3, Ca_v3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Ca_v3.1 (Ca_v3.1^-/-), Ca_v1.3 (Ca_v1.3^-/-), channels or both (Ca_v1.3^-/-/Ca_v3.1^-/-). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Ca_v3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Ca_v3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Ca_v1.3 channels were required for spontaneous automaticity, because Ca_v1.3^-/- and Ca_v1.3^-/-/Ca_v3.1^-/- AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Ca_v1.3 channels are required for automaticity, Ca_v3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for β-adrenergic regulation.     

Two blocking sites of amino-adamantane derivatives in open N-methyl-D-aspartate channels.

Using whole-cell patch-clamp techniques, we studied the blockade of open N-methyl-D-aspartate (NMDA) channels by amino-adamantane derivatives (AADs) in rat hippocampal neurons acutely isolated by the vibrodissociation method. The rapid concentration-jump technique was used to replace superfusion solutions. A kinetic analysis of the interaction of AAD with open NMDA channels revealed fast and slow components of their blockade and recovery. Mathematical modeling showed that these kinetic components are evidence for two distinct blocking sites of AADs in open NMDA channels. A comparative analysis of different simplest models led us to conclude that these AAD blocking sites can be simultaneously occupied by two blocker molecules. The voltage dependence of the AAD block suggested that both sites were located deep in the channel pore.     

Electrophysiological basis for antiarrhythmic drug action

Arrhythmias result from abnormalities of impulse initiation or impulse conduction or a combination of both. Abnormal impulse initiation results from either automaticity or triggered activity. Automaticity can further be subdivided into (1) automaticity caused by the normal automatic mechanism (a normal property of cardiac cells in the sinus node, in some parts of the atria, in the atrioventricular junctional region, and in the His-Purkinje system) and (2) automaticity caused by an abnormal mechanism (resulting from a decrease in membrane potential of cardiac fibers, which normally have a high level of membrane potential). Triggered activity is caused by afterdepolarizations, which are second depolarizations that occur either during repolarization (referred to as early afterdepolarizations) or after repolarization is complete or nearly complete (referred to as delayed afterdepolarizations). Abnormal impulse conduction results in reentrant excitation. Usually a combination of slowed conduction and unidirectional conduction block provides the conditions necessary for reentry to occur.     

Contribution of Nav1.1 to cellular synchronization and automaticity in spontaneous beating cultured neonatal rat ventricular cells

Two factors of cell coupling influence cellular synchronization and automaticity: gap junction coupling and ion channels activity. However, the role of Na(+) channel isoforms underlying cell-to-cell interaction and cellular automaticity is not well understood. To address these questions, we studied mRNA expression of Na(+) channel isoforms and the effects of TTX on spontaneously beating cultured ventricle cells. Using RT-PCR technique we demonstrated the presence of Na(v)1.1 and Na(v)1.5 channels. The reduction of Na(v)1.1 channel activity disturbed cell-to-cell interaction and changed beating rates. Thus, Na(v)1.1 channel is involved in cellular synchronization and automaticity.     

Opposite change with ischaemia in the antifibrillatory effects of class I and class IV antiarrhythmic drugs resulting from the alteration in ion transmembrane exchanges related to depolarization

It is known that class I antiarrhythmic drugs lose their antifibrillatory activity with severe ischaemia, whereas class IV antiarrhythmic drugs acquire such activity. Tachycardia, which is also a depolarizing factor, has recently been shown to give rise to an alteration of ion transmembrane exchanges which is particularly marked in the case of calcium. This leads one to wonder if the change in antifibrillatory activity of antiarrhythmic drugs caused by ischaemia depends on the same process. The change in antifibrillatory activity was studied in normal conditions ranging to those of severe ischaemia with a class I antiarrhythmic drug, flecainide (1.00 mg x kg(-1) plus 0.04 mg x kg(-1)x min(-1), a sodium channel blocker, and a class IV antiarrhythmic drug, verapamil (50 microg x kg(-1) plus 2 microg x kg(-1) x min(-1)), a calcium channel blocker. The experiments were performed in anaesthetized, open-chest pigs. The resulting blockade of each of these channels was assessed at the end of ischaemic periods of increasing duration (30, 60, 120, 180, 300, and 420 s) by determining the ventricular fibrillation threshold (VFT). VFT was determined by means of trains of diastolic stimuli of 100 ms duration delivered by a subepicardial electrode introduced into the myocardium (heart rate 180 beats per min). Ischaemia was induced by completely occluding the left anterior descending coronary artery. The monophasic action potential was recorded concurrently for the measurement of ventricular conduction time (VCT). The monophasic action potential duration (MAPD) varied with membrane polarization of the fibres. The blockade of sodium channels by flecainide, which normally raises VFT (7.0 +/- 0.4 to 13.8 +/- 0.8 mA, p < 0.001) and lengthens VCT (28 +/- 3 to 44 +/- 5 ms, p < 0.001), lost its effects in the course of ischaemia. This resulted in decreased counteraction of the ischaemia-induced fall of VFT and decreased aggravation of the ischaemia-induced lengthening of VCT. The blockade of calcium channels, which normally does not alter VFT (between 7.2 +/- 0.6 and 8.4 +/- 0.7 mA, n.s.) or VCT (between 30 +/- 2 and 34 +/- 3 ms, n.s.), slowed the ischaemia-induced fall of VFT. VFT required more time to reach 0 mA, thus delaying the onset of fibrillation. Membrane depolarization itself was opposed as the shortening of MAPD and the lengthening of VCT were also delayed. Consequently there is a progressive decrease in the role played by sodium channels during ischaemia in the rhythmic systolic depolarization of the ventricular fibres. This reduces or suppresses the ability of sodium channel blockers to act on excitability or conduction, and increases the role of calcium channel blockers in attenuating ischaemia-induced disorders.     

Oscillating contractions in protoplasmic strands of Physarum: effects of external Ca++-depletion and Ca++-antagonistic drugs on intrinsic contraction automaticity

1. The minimal requirement of external Ca-concentration for continuance of contraction activity lies in the range of 10(-4) M. 2. As in mammalian smooth muscle, the Ca antagonistic drugs verapamil and D 600 (5.10(-4) M) suppress the minute-rhythms. The action of the drugs is inhibited by 5mM Ca, La or Mn. De novo generation of contraction automaticity is not inhibited by external Ca depletion or by Ca antagonists. 3. It is concluded that rhythmical Ca fluxes across the cortical plasmalemma are not a precondition for triggering continuance or de novo generation of contraction automaticity.     

Functional Reconstitution of Purified Human Hv1 H Channels

Voltage-dependent H⁺ (Hv) channels mediate proton conduction into and out of cells under the control of membrane voltage. Hv channels are unusual compared to voltage-dependent K⁺, Na⁺, and Ca²⁺ channels in that Hv channel genes encode a voltage sensor domain (VSD) without a pore domain. The H⁺ currents observed when Hv channels are expressed heterologously suggest that the VSD itself provides the pathway for proton conduction. In order to exclude the possibility that the Hv channel VSD assembles with an as yet unknown protein in the cell membrane as a requirement for H⁺ conduction, we have purified Hv channels to homogeneity and reconstituted them into synthetic lipid liposomes. The Hv channel VSD by itself supports H⁺ flux.     

Effect of thioridazine on gap junction intercellular communication in connexin 43-expressing cells

Propagation of electrical activity between myocytes in the heart requires gap junction channels, which contribute to coordinated conduction of the heartbeat. Some antipsychotic drugs, such as thioridazine and its active metabolite, mesoridazine, have known cardiac conduction side-effects, which have resulted in fatal or nearly fatal clinical consequences in patients. The physiological mechanisms responsible for these cardiac side-effects are unknown. We tested the effect of thioridazine and mesoridazine on gap junction-mediated intercellular communication between cells that express the major cardiac gap junction subtype connexin 43. Micromolar concentrations of thioridazine and mesoridazine inhibited gap junction-mediated intercellular communication between WB-F344 epithelial cells in a dose-dependent manner, as measured by fluorescent dye transfer. Kinetic analyses demonstrated that inhibition by 10 μmol/L thioridazine occurred within 5 min, achieved its maximal effect within 1 h, and was maintained for at least 24 h. Inhibition was reversible within 1 h upon removal of the drug. Western blot analysis of connexin 43 in a membrane-enriched fraction of WB-F344 cells treated with thioridazine revealed decreased amounts of unphosphorylated connexin 43, and appearance of a phosphorylated connexin 43 band that co-migrated with a “hyperphosphorylated” connexin 43 band present in TPA-inhibited cells. When tested for its effects on cardiomyocytes isolated from neonatal rats, thioridazine decreased fluorescent dye transfer between colonies of beating myocytes. Microinjection of individual cells with fluorescent dye also showed inhibition of dye transfer in thioridazine-treated cells compared to vehicle-treated cells. In addition, thioridazine, like TPA, inhibited rhythmic beating of myocytes within 15 min of application. In light of the fact that the thioridazine and mesoridazine concentrations used in these experiments are in the range of those used clinically in patients, our results suggest that inhibition of gap junction intercellular communication may be one factor contributing to the cardiac side-effects observed in some patients taking these medications.     

Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume

Large-conductance Ca²⁺- and voltage-activated K⁺ (BK) channels have the largest conductance (250–300 pS) of all K⁺-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA⁻ increased currents and MTSET⁺ decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high [K⁺]_i. Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance ∼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger.     

Myocardial gap junctions: targets for novel approaches in the prevention of life-threatening cardiac arrhythmias

Direct cell-to-cell communication in the heart is maintained via gap junction channels composed of proteins termed connexins. Connexin channels ensure molecular and electrical signals propagation and hence are crucial in myocardial synchronization and heart function. Disease-induced gap junctions remodeling and/or an impairment or even block of intercellular communication due to acute pathological conditions results in derangements of myocardial conduction and synchronization. This is critical in the development of both ventricular fibrillation, which is a major cause of sudden cardiac death and persistent atrial fibrillation, most common arrhythmia in clinical practice often resulting in stroke. Many studies suggest that alterations in topology (remodeling), expression, phosphorylation and particularly function of connexin channels due to age or disease are implicated in the development of these life-threatening arrhythmias. It seems therefore challenging to examine whether compounds that could prevent or attenuate gap junctions remodeling and connexin channels dysfunction can protect the heart against arrhythmias that cause sudden death in humans. This assumption is supported by very recent findings showing that an increase of gap junctional conductance by specific peptides can prevents atrial conduction slowing or re-entrant ventricular tachycardia in ischemic heart. Suppression of ischemia-induced dephosphorylation of connexin seems to be one of the mechanisms involved. Another approach for identifying novel treatments is based on the hypothesis that even non-antiarrhythmic drugs with antiarrhythmic ability can modulate gap junctional communication and hence attenuate arrhythmogenic substrates.     

Conduction barriers and pathways of the sinoatrial pacemaker complex: their role in normal rhythm and atrial arrhythmias

Since Keith and Flack's anatomical discovery of the sinoatrial node (SAN), the primary pacemaker of the heart, the question of how such a small SAN structure can pace the entire heart has remained for a large part unanswered. Recent advances in optical mapping technology have made it possible to unambiguously resolve the origin of excitation and conduction within the animal and human SAN. The combination of high-resolution optical mapping and histological structural analysis reveals that the canine and human SANs are functionally insulated from the surrounding atrial myocardium, except for several critical conduction pathways. Indeed, the SAN as a leading pacemaker requires anatomical (fibrosis, fat, and blood vessels) and/or functional barriers (paucity of connexins) to protect it from the hyperpolarizing influence of the surrounding atrium. The presence of conduction barriers and pathways may help explain how a small cluster of pacemaker cells in the SAN pacemaker complex manages to depolarize different, widely distributed areas of the right atria as evidenced functionally by exit points and breakthroughs. The autonomic nervous system and humoral factors can further regulate conduction through these pathways, affecting pacemaker automaticity and ultimately heart rate. Moreover, the conduction barriers and multiple pathways can form substrates for reentrant activity and thus lead to atrial flutter and fibrillation. This review aims to provide new insight into the function of the SAN pacemaker complex and the interaction between the atrial pacemakers and the surrounding atrial myocardium not only in animal models but also human hearts.     

Molecular restraints in the permeation pathway of ion channels

Ion channels assist and control the diffusion of ions through biological membranes. The conduction process depends on the structural characteristics of these nanopores, among which are the hydrophobicity and the afforded diameter of the conduction pathway. In this contribution, we use full atomistic free-energy molecular dynamics simulations to estimate the effect of such characteristics on the energetics of ion conduction through the activation gate of voltage-gated potassium (Kv) channels. We consider specifically the ionic translocation through three different permeation pathways, corresponding to the activation gate of an atomistic model of Shaker channels in closed and partially opened conformations, and that of the open conformation of the Kv1.2 channel. In agreement with experiments, we find that the region of Val(478) constitutes the main gate. The conduction is unfavorable through this gate when the constriction is smaller than an estimated threshold of 4.5-5.0 A, mainly due to incomplete coordination-hydration of the ion. Above this critical size, e.g., for the Kv1.2, the valine gate is wide enough to allow fully coordination of the ion and therefore its diffusion on a flat energy surface. Similar to other ion channels, Kv channels appear therefore to regulate diffusion by constricting hydrophobic regions of the permeation pathway.     

Influence of frequency and temperature on the mechanisms of nerve conduction block induced by high-frequency biphasic electrical current

The influences of stimulation frequency and temperature on mechanisms of nerve conduction block induced by high-frequency biphasic electrical current were investigated using a lumped circuit model of the myelinated axon based on Schwarz and Eikhof (SE) equations. The simulation analysis showed that a temperature-frequency relationship was determined by the axonal membrane dynamics (i.e. how fast the ion channels can open or close.). At a certain temperature, the axonal conduction block always occurred when the period of biphasic stimulation was smaller than the action potential duration (APD). When the temperature decreased from 37 to 15 degrees C, the membrane dynamics slowed down resulting in an APD increase from 0.4 to 2.4 ms accompanied by a decrease in the minimal blocking frequency from 4 to 0.5 kHz. The simulation results also indicated that as the stimulation frequency increased the mechanism of conduction block changed from a cathodal/anodal block to a block dependent upon continuous activation of potassium channels. Understanding the interaction between the minimal blocking frequency and temperature could promote a better understanding of the mechanisms of high frequency induced axonal conduction block and the clinical application of this method for blocking nerve conduction.     

The role of stretch-activated channels in atrial fibrillation and the impact of intracellular acidosis

The incidence of atrial fibrillation correlates with increasing atrial size. The electrical consequences of atrial stretch contribute to both the initiation and maintenance of atrial fibrillation. It is suggested that altered calcium handling and stretch-activated channel activity could explain the experimental findings of stretch-induced depolarisation, shortened refractoriness, slowed conduction and increased heterogeneity of refractoriness and conduction. Stretch-activated channel blocking agents protect against these pro-arrhythmic effects. Gadolinium, GsMTx-4 toxin and streptomycin prevent the stretch-related vulnerability to atrial fibrillation without altering the drop in refractory period associated with stretch. Changes the activity of two-pore K+ channels, which are sensitive to stretch and pH but not gadolinium, could underlie the drop in refractoriness. Intracellular acidosis induced with propionate amplified the change in refractoriness with stretch in the isolated rabbit heart model in keeping with the clinical observation of increased propensity to atrial fibrillation with acidosis. We propose that activation of non-specific cation stretch-activated channels provides the triggers for acute atrial fibrillation with high atrial pressure while activation of atrial two-pore K+ channels shortens atrial refractory period and increases heterogeneity of refractoriness, providing the substrate for atrial fibrillation to be sustained. Stretch-activated channel blockade represents an exciting target for future antiarrhythmic drugs.     

Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium

The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)- gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single- channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single- ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.     

Acetylcholine inhibition in rabbit sinoatrial node is prevented by pertussis toxin

The effects of acetylcholine (ACh) were examined on the naturally occurring slow action potentials (APs) of the isolated, organ-cultured, spontaneously beating sinoatrial (SA) node of the rabbit, in the presence or absence of pertussis toxin. The sensitivity of the SA-node preparations to ACh was not altered after 24 h incubation in organ culture medium. Activation of the muscarinic receptor hyperpolarized the cells and reduced the frequency of spontaneous activity at low concentrations (1 X 10(-6) and 3 X 10(-6) M), and completely abolished automaticity at higher concentrations (1 X 10(-5) M). However, stimulated activity was maintained. Increased concentrations (1 X 10(-4) M) of ACh completely abolished excitability. When the SA-node preparations were cultured in the presence of 0.5 micrograms/mL pertussis toxin, concentrations of ACh as high as 1 X 10(-4) M had no effect on the AP parameters and frequency of spontaneous activity. The results indicate that inactivation of G proteins by pertussis toxin caused inhibition of the ACh effects on the automaticity of the SA node. In addition, the blocking effect of ACh to the naturally occurring slow APs was also inhibited by pertussis toxin. We conclude that in the rabbit SA node, the effects of ACh on automaticity and on the slow channels are mediated by G protein.     

Multi-ion versus single-ion conduction mechanisms can yield current rectification in biological ion channels

There is clear evidence that the net magnitude of negative charge at the intracellular end of inwardly rectifying potassium channels helps to generate an asymmetry in the magnitude of the current that will pass in each direction. However, a complete understanding of the physical mechanism that links these charges to current rectification has yet to be obtained. Using Brownian dynamics, we compare the conduction mechanism and binding sites in rectifying and non-rectifying channel models. We find that in our models, rectification is a consequence of asymmetry in the hydrophobicity and charge of the pore lining. As a consequence, inward conduction can occur by a multi-ion conduction mechanism. However, outward conduction is restricted, since there are fewer ions at the intracellular entrance and outwardly moving ions must cross the pore on their own. We pose the question as to whether the same mechanism could be at play in inwardly rectifying potassium channels.     

Fast- or Slow-inactivated State Preference of Na+ Channel Inhibitors: A Simulation and Experimental Study

Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the “steady-state slow inactivation curve”), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated.