Rat Brain Type IIA Research Articles

V102862 (Co 102862): a potent, broad‐spectrum state‐dependent blocker of mammalian voltage‐gated sodium channels

1. 4-(4-Fluorophenoxy)benzaldehyde semicarbazone (V102862) was initially described as an orally active anticonvulsant with robust activity in a variety of rodent models of epilepsy. The mechanism of action was not known. We used whole-cell patch-clamp techniques to study the effects of V102862 on native and recombinant mammalian voltage-gated Na+ channels. 2. V102862 blocked Na+ currents (I(Na)) in acutely dissociated cultured rat hippocampal neurons. Potency increased with membrane depolarization, suggesting a state-dependent mechanism of inhibition. There was no significant effect on the voltage dependence of activation of I(Na). 3. The dissociation constant for the inactivated state (K(I)) was approximately 0.6 microM, whereas the dissociation constant for the resting state (K(R)) was >15 microM. 4. The binding to inactivated channels was slow, requiring a few seconds to reach steady state at -80 mV. 5. The mechanism of inhibition was characterized in more detail using human embryonic kidney-293 cells stably expressing rat brain type IIA Na+ (rNa(v)1.2) channels, a major Na+ channel alpha subunit in rat hippocampal neurons. Similar to hippocampal neurons, V102862 was a potent state-dependent blocker of rNa(v)1.2 channels with a K(I) of approximately 0.4 microM and K(R) approximately 30 microM. V102862 binding to inactivated channels was relatively slow (k(+) approximately = 1.7 microM(-1) s(-1)). V102862 shifted the steady-state availability curve in the hyperpolarizing direction and significantly retarded recovery of Na+ channels from inactivation. 6. These results suggest that inhibition of voltage-gated Na+ channels is a major mechanism underlying the anticonvulsant properties of V102862. Moreover, understanding the biophysics of the interaction may prove to be useful in designing a new generation of potent Na+ channel blocker therapeutics.

The conformation and movement of Na channel inactivation gate peptide in linker between domain III and IV during inactivation by NMR spectroscopy and molecular modeling study.

Amodel peptide that their sequence corresponds to the linker part between domain III and IV of rat brain type IIA Na+ channel has been synthesized for the conformational affect study corresponded to different gated states of Na+ channel. Nuclear magnetic resonance spectra of local anesthetic (LA) diphenyl drugs, such as phenytoin, in presence of a model peptide in both phosphate buffer and phospholipid bicelles (dimyristotl phosphocholine/dihexanoyl phospholcholine), which micelles serve to mimic the peptide-lipid interactions, have been measured to obtain information of the interactions between selected drugs and model peptide. Molecular modeling is performed to help to provide possible conformational information about the polypeptide LIII-IV that may be critical for recognition and signal transduction of inactivated Na+ channel. The voltage-sensing mechanism of Na+ channel involves the movement of the inactivation particles (Ile, Phe, and Met) in the LIII-IV while binding to S4-S5 intracellular region within DIII and DIV. The movement of LIII-IV making its C-terminal residues, including Glu1492 and Glu1493, may aligned near and stabilize the LAs bound with their receptors.

Differential interactions of lamotrigine and related drugs with transmembrane segment IVS6 of voltage-gated sodium channels

Voltage-gated sodium channels are blocked by local anesthetic and anticonvulsant drugs. A receptor site for local anesthetics has been defined in transmembrane segment S6 in domain IV (IVS6) of the α subunit, but the anticonvulsant lamotrigine and related compounds have more complex structures than local anesthetics and may interact with additional amino acid residues. Apparent K D values for inactivated-state block of rat brain type IIA sodium channels expressed in Xenopus oocytes were 31.9 μM, 17.3 μM, 3.7 μM and 10.3 μM for lamotrigine and compounds 227c89, 4030w92 and 619c89, respectively. Compound 619c89 was the strongest frequency-dependent blocker, which correlated with higher affinity and a five-fold slower recovery from drug block compared to lamotrigine. Examination of lamotrigine block of mutant sodium channel α subunits, in which alanine had been substituted for each individual amino acid in IVS6, identified mutations I1760A, F1764A and Y1771A as causing the largest reductions in affinity (six-, seven- and 12-fold, respectively). The ratios of effects of these three mutations differed for compounds 227c89, 4030w92, and 619c89. The amino acid residues interacting with these pore-blocking drugs define a surface of IVS6 that is exposed to the pore and may rotate during gating.

Tetrapentylammonium block of chloramine-T and veratridine modified rat brain type IIA sodium channels.

Tetrapentylammonium (TPeA) block of rat brain type IIA sodium channel alpha subunit was studied using whole cell patch clamp. Results indicate that TPeA blocks the inactivating brain sodium channel in a potential and use-dependent manner similar to that of the cardiac sodium channel. Removal of inactivation using chloramine-T (CT) unmasks a time-dependent block by TPeA consistent with slow blocking kinetics. On the other hand, no time dependence is observed when inactivation is abolished by modification with veratridine. TPeA does not bind in a potential-dependent fashion to veratridine-modified channels and does not significantly affect gating of veratridine-modified channels suggesting that high affinity binding of TPeA to the brain sodium channel is lost after veratridine modification.

Solution structures of the inactivation gate particle peptides of rat brain type‐IIA and human heart sodium channels in SDS micelles

The Ile-Phe-Met (IFM) motif located in the Ill-IV linker of voltage-gated sodium channels has been identified as a major component of the fast inactivation gate. If Gln was substituted for Phe, the role in the gate was disrupted completely. If Ile was replaced by Gln inactivation became slightly incomplete and if the Thr, which is adjacent to the IFM motif (-IFMT-), was replaced by Met, inactivation became much more incomplete than in the I/Q mutation, but not as vigorous as in the F/Q mutation. Previously, we studied the structures of the inactivation gate-related peptide (K1480-K1496 in rat brain type-IIA, MP-3A) and its F1489/Q substituted one (MP-4A) in SDS micelles and found that the conformational change of the IFM hydrophobic cluster due to the F/Q substitution may be a reason for disrupting the gate. In this study, in order to obtain supporting evidence for this view and also to further knowledge of the effect of I/Q and T/M mutations on the structure of the IFM cluster, we studied the structures of 11488Q [MP(rb)-3QFMT] and T1491M [MP(rb)-31FMM] substituted peptides. The fragment peptide K1477-K1493 [MP(hh)-3A] and its T1488M substituted peptide [MP(hh)-3IFMM] in the human heart sodium channel were also studied. It was found that the backbone structures around the IMF motif of MP-3A, MP(hh)-3A and MP(rb)-3QFMT resemble one another in such a manner that the residues Ile(Gln) and Thr are brought so close together that they form a unique type of lid to occlude the pore. In contrast, the residues between Ile and M1491 of MP(rb)-3IFMM or M1488 of MP(hh)-3IFMM were fairly far apart from each other. We conclude that Thr plays an important role in forming a structure of the IFM hydrophobic cluster for inactivation.

Solution structure of the cytoplasmic linker between domain III-S6 and domain IV-S1 (III-IV linker) of the rat brain sodium channel in SDS micelles.

The solution structure of the 36-mer peptide MP-5A in SDS micelles was investigated by CD and (1)H-NMR spectroscopies. The MP-5A was dissected from the cytoplasmic linker (K1482-A1517) connecting domain III-segment 6 (IIIS6) and domain IV-segment 1 (IVS1; III-IV linker) of the rat brain type IIA sodium channel. The molecular energy calculations including nuclear Overhauser effect and dihedral angle restraints gave a well-converged set of the structures of MP-5A for the region between I1488 and S1506. It was found that a large hydrophobic cluster is formed by I1488-F1489-M1490 (IFM motif), Y1497-Y1498, and M1501, which may be related to the fast inactivation process of the sodium channel. The solvent-accessible surface area of the IFM motif (195 A(2)), which is known to work essentially as an inactivation gate particle to occlude the ion permeation pore, gave the free energy (DeltaG) of stabilization of -3.9 kcal mol(-1) as a result of the hydrophobic interactions with its receptor. This value agreed well with the free energy of binding (inactivation) of -4.1 kcal mol(-1) calculated for the equilibrium between the open and the inactivated states of the sodium channels. It is concluded that the fast inactivation of the sodium channel is achieved by the environmental polarity-dependent conformational switching at the IFM motif, in response to the voltage-dependent activation and the movement of the S4 segments of the sodium channel.

Molecular Determinants of Voltage-dependent Gating and Binding of Pore-blocking Drugs in Transmembrane Segment IIIS6 of the Na+ Channel α Subunit

Mutations of amino acid residues in the inner two-thirds of the S6 segment in domain III of the rat brain type IIA Na(+) channel (G1460A to I1473A) caused periodic positive and negative shifts in the voltage dependence of activation, consistent with an alpha-helix having one face on which mutations to alanine oppose activation. Mutations in the outer one-third of the IIIS6 segment all favored activation. Mutations in the inner half of IIIS6 had strong effects on the voltage dependence of inactivation from closed states without effect on open-state inactivation. Only three mutations had strong effects on block by local anesthetics and anticonvulsants. Mutations L1465A and I1469A decreased affinity of inactivated Na(+) channels up to 8-fold for the anticonvulsant lamotrigine and its congeners 227c89, 4030w92, and 619c89 as well as for the local anesthetic etidocaine. N1466A decreased affinity of inactivated Na(+) channels for the anticonvulsant 4030w92 and etidocaine by 3- and 8-fold, respectively, but had no effect on affinity of the other tested compounds. Leu-1465, Asn-1466, and Ile-1469 are located on one side of the IIIS6 helix, and mutation of each caused a positive shift in the voltage dependence of activation. Evidently, these amino acid residues face the lumen of the pore, contribute to formation of the high-affinity receptor site for pore-blocking drugs, and are involved in voltage-dependent activation and coupling to closed-state inactivation.

Structural study of the sodium channel inactivation gate peptide including an isoleucine-phenylalanine-methionine motif and its analogous peptide (phenylalanine/glutamine) in trifluoroethanol solutions and SDS micelles

In order to gain insight into the gating mechanisms of Na+ channels, in particular their inactivation mechanisms, we studied the structures of the Na+ channel inactivation gate related peptide which includes the IFM (Ile-Phe-Met) motif (Ac-KKKFGGQDIFMTEEQKK-NH2; K1480-K1496 in rat brain type-IIA Na+ channels, MP-3A) and its F/Q(Gln) substituted one (MP-4A) in trifluoroethanol (TFE) solutions and sodium dodecyl sulfate (SDS) micelles using circular dichroism (CD) and 1H-NMR spectroscopies. Based on observed nuclear Overhauser effect constraints, three-dimensional structures of MP-3A and MP-4A were determined using simulated annealing molecular dynamics/ energy minimization calculations. In TFE solutions, no appreciable differences in the structure were observed using either CD or NMR spectra. In SDS micelles, however, the two peptides exhibited definitely different structures from each other. It was found that in MP-3A, residues 11488 and T1491 were spatially proximate with each other owing to hydrogen bonding between the amide proton of 11488 and the hydroxyl oxygen atom of T1491, whereas in MP-4A, F/Q substitution separated them owing to conformational changes. The solvent-accessible surfaces calculated for the structures of MP-3A and MP-4A showed that the former has a smoother interaction surface to the hydrophobic docking site than the latter. In conclusion, the conformational changes, as well as decreased hydrophobicity around the IFM motif owing to the F/Q mutation, may be one reason why F1489Q mutated channels cannot inactivate almost completely.

Interactions between local anesthetics and Na+ channel inactivation gate peptides in phosphatidylserine suspensions as studied by 1H-NMR spectroscopy.

Interactions between local anesthetics and a sodium channel inactivation gate peptide (Ac-GGQDIFMTEEQK-NH2, MP-1A), which was dissected from the cytoplasmic linker between domains III and IV of the sodium channel alpha-subunit (G1484-K1495 in rat brain type IIA), have been studied by 1H-NMR spectroscopy. Changes in 1H-NMR chemical shifts of the aromatic proton resonances of dibucaine (pH 7.0) and lidocaine (pH 6.0 and 9.0) in phosphatidylserine (PS) suspensions were observed. The effects of substitution of glutamine (F1489Q; MP-2A) or D-phenylalanine (MP-1A') for L-phenylalanine (F1489) in MP-1A and the effects of substitution of neutral amino acid residues for the corresponding acidic amino acid residues (D1487N, MP-1NA; E1492Q, MP-IQEA; E1493Q, MP-IEQA) in MP-1A, on the aromatic 1H-NMR chemical shift changes of dibucaine and lidocaine were also investigated. From these results it was concluded that: the aromatic ring of phenylalanine of MP-1A and the aromatic ring of the cationic form of dibucaine or lidocaine are interacting by pi-pi stacking; the tertiary amine nitrogen of dibucaine is interacting electrostatically with D1487, whereas that of lidocaine is interacting with E1492.

Functional Roles of the Extracellular Segments of the Sodium Channel α Subunit in Voltage-dependent Gating and Modulation by β1 Subunits

Voltage-gated sodium channels consist of a pore-forming alpha subunit associated with beta1 subunits and, for brain sodium channels, beta2 subunits. Although much is known about the structure and function of the alpha subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) alpha subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) alpha subunit. In comparison with rH1, wild-type rIIA alpha subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of beta1 subunits in Xenopus oocytes. When alpha subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA alpha subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA alpha subunits, all of the chimeric rIIA alpha subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with beta1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of beta1 subunits in shifting rIIA alpha subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by beta1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation.

Modification of alpha subunit of RIIA sodium channels by aconitine.

The effect of aconitine (AC), an alkaloid toxin, on the electrophysiological properties of the rat brain type IIA alpha subunit expressed heterologously in the Chinese Hamster Ovarian (CHO) cell line was studied under the whole-cell patch-clamp configuration. The activation threshold of modified channels shifted by about -40 mV. As the number of depolarizations increased, the transient current at 0 mV decreased and, in proportion, the AC-modified current at -50 mV increased. This suggests a transition of channels to an AC-modified state. The rate of modification was nearly four times faster when 50 microM AC was applied internally than when applied in the bath solution. This supports the idea that the AC-binding site is located close to the cytoplasmic mouth of the channel pore. The AC-modified sodium currents inactivated completely, although with slower kinetics. The steady-state inactivation followed a simple Boltzmann function. AC-modified currents activated without a sigmoidal delay. The permeability of the NH4+ ion was enhanced such that its permeability ratio increased from an initial value of 0. 18 to 0.95 and for Cs+ it was enhanced from 0.03 to 0.07. These studies show that the AC-binding site resides at the pore region of the alpha subunit of the Na+ channel, and that the presence of beta subunit/s is not essential for AC binding.

Shear Stress-mediated Extracellular Signal-regulated Kinase Activation Is Regulated by Sodium in Endothelial Cells: POTENTIAL ROLE FOR A VOLTAGE-DEPENDENT SODIUM CHANNEL

Fluid shear stress is an important regulator of endothelial cell (EC) function. To determine whether mechanosensitive ion channels participate in the EC response to shear stress, we characterized the role of ion transport in shear stress-mediated extracellular signal-regulated kinase (ERK1/2) stimulation. Replacement of all extracellular Na+ with either N-methyl-D-glucamine or choline chloride increased the ERK1/2 stimulation in response to shear stress by 1.89 +/- 0.1-fold. The Na+ effect was concentration-dependent (maximal effect, </=12.5 mM) and was specific for shear stress-mediated ERK1/2 activation as epidermal growth factor-stimulated ERK1/2 activation was unaffected by removal of extracellular Na+. Shear stress-mediated ERK1/2 activation was potentiated by the voltage-gated sodium channel antagonist, tetrodotoxin (100 nM), to a magnitude similar to that achieved with extracellular Na+ withdrawal. Transfection of Chinese hamster ovary cells with a rat brain type IIa voltage-gated sodium channel completely inhibited shear stress-mediated ERK1/2 activation in these cells. Inhibition was reversed by performing the experiment in sodium-free buffer or by including tetrodotoxin in the buffer. Western blotting of bovine and human EC lysates with SP19 antibody detected a 250-kDa protein consistent with the voltage-gated sodium channel. Degenerate polymerase chain reaction of cDNA from primary human EC yielded transcripts whose sequences were identical to the sodium channel SCN4a and SCN8a alpha subunit genes. These results indicate that shear stress-mediated ERK1/2 activation is regulated by extracellular sodium and demonstrate that ion transport via Na+ channels modulates EC responses to shear stress.

Fast inactivation of a brain K+ channel composed of Kv1.1 and Kvbeta1.1 subunits modulated by G protein beta gamma subunits.

Modulation of A-type voltage-gated K+ channels can produce plastic changes in neuronal signaling. It was shown that the delayed-rectifier Kv1.1 channel can be converted to A-type upon association with Kvbeta1.1 subunits; the conversion is only partial and is modulated by phosphorylation and microfilaments. Here we show that, in Xenopus oocytes, expression of Gbeta1gamma2 subunits concomitantly with the channel (composed of Kv1.1 and Kvbeta1.1 subunits), but not after the channel's expression in the plasma membrane, increases the extent of conversion to A-type. Conversely, scavenging endogenous Gbetagamma by co-expression of the C-terminal fragment of the beta-adrenergic receptor kinase reduces the extent of conversion to A-type. The effect of Gbetagamma co-expression is occluded by treatment with dihydrocytochalasin B, a microfilament-disrupting agent shown previously by us to enhance the extent of conversion to A-type, and by overexpression of Kvbeta1.1. Gbeta1gamma2 subunits interact directly with GST fusion fragments of Kv1.1 and Kvbeta1.1. Co-expression of Gbeta1gamma2 causes co-immunoprecipitation with Kv1.1 of more Kvbeta1.1 subunits. Thus, we suggest that Gbeta1gamma2 directly affects the interaction between Kv1.1 and Kvbeta1.1 during channel assembly which, in turn, disrupts the ability of the channel to interact with microfilaments, resulting in an increased extent of A-type conversion.

Competition for binding between veratridine and KIFMK: an open channel blocking peptide of the RIIA sodium channel.

Veratridine, an alkaloid isolated from the rhizome of V. album, binds and slows the inactivation of the brain sodium channels. The synthetic pentapeptide KIFMK causes a voltage- and use-dependent open-channel block of the RIIA (rat brain type IIA) sodium channel (Eaholtz, Scheuer & Catterall, 1994). Our studies on the RIIA sodium channel expressed in CHO cells reveal that the fraction of veratridine modified sodium channels decreases linearly with increasing KIFMK concentration. However, the time constant for dissociation of veratridine from the channel remains unchanged in the presence of a high concentration of KIFMK, as opposed to that in the presence of QX314 where the dissociation appears to be more complex. These data are consistent with mutually exclusive binding of the open channel blocking peptide and veratridine to the brain sodium channel.

Functional expression of Drosophila para sodium channels. Modulation by the membrane protein TipE and toxin pharmacology.

The Drosophila para sodium channel alpha subunit was expressed in Xenopus oocytes alone and in combination with tipE, a putative Drosophila sodium channel accessory subunit. Coexpression of tipE with para results in elevated levels of sodium currents and accelerated current decay. Para/TipE sodium channels have biophysical and pharmacological properties similar to those of native channels. However, the pharmacology of these channels differs from that of vertebrate sodium channels: (a) toxin II from Anemonia sulcata, which slows inactivation, binds to Para and some mammalian sodium channels with similar affinity (Kd congruent with 10 nM), but this toxin causes a 100-fold greater decrease in the rate of inactivation of Para/TipE than of mammalian channels; (b) Para sodium channels are >10-fold more sensitive to block by tetrodotoxin; and (c) modification by the pyrethroid insecticide permethrin is >100-fold more potent for Para than for rat brain type IIA sodium channels. Our results suggest that the selective toxicity of pyrethroid insecticides is due at least in part to the greater affinity of pyrethroids for insect sodium channels than for mammalian sodium channels.

Persistent Sodium Currents through Brain Sodium Channels Induced by G Protein βγ Subunits

Persistent Na+currents are thought to be important for integration of neuronal responses. Here, we show that βγ subunits of G proteins can induce persistent Na+ currents. Coexpression of Gβ2γ3, Gβ1γ3, or Gβ5γ3, but not Gβ1γ1 subunits with rat brain type IIA Na+ channel α subunits in tsA-201 cells greatly enhances a component of Na+ current with a normal voltage dependence of activation but with dramatically slowed and incomplete inactivation and with steady-state inactivation shifted +37 mV. Synthetic peptides containing the proposed Gβγ-binding motif, Gln-X-X-Glu-Arg, from either adenylyl cyclase 2 or the Na+ channel α subunit C-terminal domain reversed the effect of Gβ2γ3 subunits. These results are consistent with direct binding of Gβγ subunits to the C-terminal domain of the Na+ channel, stabilizing a gating mode responsible for slowed and persistent Na+ current. Modulation of Na+ channel gating by Gβγ subunits is expected to have profound effects on neuronal excitability.

Molecular analysis of the putative inactivation particle in the inactivation gate of brain type IIA Na+ channels.

Fast Na+ channel inactivation is thought to involve binding of phenylalanine 1489 in the hydrophobic cluster IFM in L(III-IV) of the rat brain type IIA Na+ channel. We have analyzed macroscopic and single channel currents from Na+ channels with mutations within and adjacent to hydrophobic clusters in L(III-IV). Substitution of F1489 by a series of amino acids disrupted inactivation to different extents. The degree of disruption was closely correlated with the hydrophilicity of the amino acid at position 1489. These mutations dramatically destabilized the inactivated state and also significantly slowed the entry into the inactivated state, consistent with the idea that F1489 forms a hydrophobic interaction with a putative receptor during the fast inactivation process. Substitution of a phe residue at position 1488 or 1490 in mutants lacking F1489 did not restore normal inactivation, indicating that precise location of F1489 is critical for its function. Mutations of T1491 disrupted inactivation substantially, with large effects on the stability of the inactivated state and smaller effects on the rate of entry into the inactivated state. Mutations of several other hydrophobic residues did not destabilize the inactivated state at depolarized potentials, indicating that the effects of mutations at F1489 and T1491 are specific. The double mutant YY1497/8QQ slowed macroscopic inactivation at all potentials and accelerated recovery from inactivation at negative membrane potentials. Some of these mutations in L(III-IV) also affected the latency to first opening, indicating coupling between L(III-IV) and channel activation. Our results show that the amino acid residues of the IFM hydrophobic cluster and the adjacent T1491 are unique in contributing to the stability of the inactivated state, consistent with the designation of these residues as components of the inactivation particle responsible for fast inactivation of Na+ channels.

Locations of local anesthetic dibucaine in model membranes and the interaction between dibucaine and a Na+ channel inactivation gate peptide as studied by 2H- and 1H-NMR spectroscopies

To study the molecular mechanisms of local anesthesia, locations of local anesthetic dibucaine in model membranes and the interactions of dibucaine with a Na+ channel inactivation gate peptide have been studied by 2H- and 1H-NMR spectroscopies. The 2H-NMR spectra of dibucaine-d9 and dibucaine-d1, which are deuterated at the butoxy group and at the 3 position in its quinoline ring, respectively, have been observed in multilamellar dispersions of the lipid mixture composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. 2H-NMR spectra of deuterated palmitic acids incorporated, as a probe, into the lipid mixture containing cholesterol have also been observed. An order parameter, SCD, for each carbon segment was calculated from the observed quadrupole splittings. Combining these results, we concluded that first, the butoxy group of dibucaine is penetrating between the acyl chains of lipids in the model membranes, and second, the quinoline ring of dibucaine is located at the polar region of lipids but not at the hydrophobic acyl chain moiety. These results mean that dibucaine is situated in a favorable position that permits it to interact with a cluster of hydrophobic amino acids (Ile-Phe-Met) within the intracellular linker between domains III and IV of Na+ channel protein, which functions as an inactivation gate. To confirm whether the dibucaine molecule at the surface region of lipids can really interact with the hydrophobic amino acids, we synthesized a model peptide that includes the hydrophobic amino acids (Ac-GGQDIFMTEEQK-OH, MP-1), the amino acid sequence of which corresponds to the linker part of rat brain type IIA Na+ channel, and the one in which Phe has been substituted by Gln (MP-2), and measured 1H-NMR spectra in both phosphate buffer and phosphatidylserine liposomes. It was found that the quinoline ring of dibucaine can interact with the aromatic ring of Phe by stacking of the rings; moreover, the interaction can be reinforced by the presence of lipids. In conclusion, we wish to propose that local anesthesia originates from the pi-stacking interaction between aromatic rings of an anesthetic molecule located at the polar headgroup region of the so-called boundary lipids and of the Phe in the intracellular linker between domains III and IV of the Na+ channel protein, prolonging the inactivated state and consequently making it impossible to proceed to the resting state.

State-dependent inhibition of Na+ currents by the neuroprotective agent 619C89 in rat hippocampal neurons and in a mammalian cell line expressing rat brain type IIA Na+ channels.

The compound 619C89 [4-amino-2-(4-methyl-1-piperazinyl)-5-(2,3,5-tricholorophenyl)-pyr imidine] is an effective neuroprotective agent in in vivo models of cerebral ischaemia. It has been suggested to act by inhibiting voltage-gated Na+ channels. To test this hypothesis, the action of 619C89 on recombinant rat brain type IIA Na+ channels expressed in Chinese hamster ovary cells and on native Na+ channels in acutely dissociated rat hippocampal neurons has been studied using whole-cell voltage-clamp recording techniques. In the cell line expressing type IIA Na+ channels, 619C89 caused a reversible inhibition of Na+ currents in a concentration- and voltage-dependent manner. A half-maximal inhibitory concentration (IC50) of approximately 50 microM was obtained at a holding potential of -90 mV whereas, with a conditioning prepulse to -60 mV for 30 s, the IC50 was reduced to 8 microM. Furthermore, the inhibition was markedly enhanced by a use-dependent action, which was dependent not only on the frequency of stimulation, but also on the duration (3.5-40 ms) of the pulses. Trains (10-50 Hz) of up to 60 depolarizing pulses of 0.7 ms duration did not evoke any use-dependent inhibition in the presence of 619C89, suggesting that this compound is not an open channel blocker. The voltage- and use-dependent inhibition by 619C89 was also observed on native Na+ channels in hippocampal neurons. 619C89 (10 microM) produced a small hyperpolarizing shift in the fast inactivation curve and a substantial (13 mV) hyperpolarizing shift in slow inactivation. The compound dramatically delayed the recovery from inactivation without affecting the development of inactivation. Moreover, 619C89 has no effect on the shape of the current-voltage relationship or on the voltage activation curve. These data indicate that 619C89 interacts selectively with the inactivated state of the Na+ channel with an estimated affinity of 3 microM. This primary action of 619C89 may underlie its neuroprotective effects.

Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line.

1. The rat brain type IIA Na+ channel alpha-subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole-cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell-shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range -30 to + 40 mV, with a dominating fast component and a small slower component. 3. The steady-state inactivation, h infinity, was related to potential by a Boltzmann distribution, underlying three states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range -70 to -120 mV were characterized by an initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin-Huxley model, a three-state scheme of the form O<-->I1<-->I2 was proposed, replacing the original two-state scheme of the Hodgkin-Huxley model, and the rate constants are reported. 6. The instantaneous current-voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant-field equation. 7. The time course of activation was described by an m chi model. However, the best-fitted value of chi varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha m and beta m, were determined. Their functional dependence on the membrane potential was investigated.