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Slow inactivation in voltage-gated Na+ channels (Navs) plays an important physiological role in excitable tissues (muscle, heart, nerves) and mutations that disrupt Nav slow inactivation can result in pathophysiologies (myotonia, arrhythmias, epilepsy). While the molecular mechanisms responsible for slow inactivation remain elusive, previous studies have suggested a role for the pore-lining D1-S6 helix. The goals of this research were to determine if (1) cysteine substitutions in D1-S6 affect gating kinetics and (2) methanethiosulfonate ethylammonium (MTSEA) accessibility changes in different kinetic states. Site-directed mutagenesis in the human skeletal muscle isoform hNav1.4 was used to substitute cysteine for eleven amino acids in D1-S6 from L433 to L443. Mutants were expressed in HEK cells and recorded from with whole-cell patch clamp. All mutations affected one or more baseline kinetics of the sodium channel, including activation, fast inactivation, and slow inactivation. Substitution of cysteine (for nonpolar residues) adjacent to polar residues destabilized slow inactivation in G434C, F436C, I439C, and L441C. Cysteine substitution without adjacent polar residues enhanced slow inactivation in L438C and N440C, and disrupted possible H-bonds involving Y437:D4 S4-S5 and N440:D4-S6. MTSEA exposure in closed, fast-inactivated, or slow-inactivated states in most mutants had little-to-no effect. In I439C, MTSEA application in closed, fast-inactivated, and slow-inactivated states produced irreversible reduction in current, suggesting I439C accessibility to MTSEA in all three kinetic states. D1-S6 is important for Nav gating kinetics, stability of slow-inactivated state, structural contacts, and state-dependent positioning. However, prominent reconfiguration of D1-S6 may not occur in slow inactivation.

The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na(+) channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na(+) current (I(Na)). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in I(Na). Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (4min), there is little effect on I(Na). However, with longer exposures (4-8min), there is a large decrease in I(Na), but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) - MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na(+) channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.

To examine conformational changes during slow inactivation involving domain 2-segment 6 (D2-S6) of human cardiac Na(+) channel (hNav1.5), we applied the substituted-cysteine accessibility method (SCAM) using methanethiosulfonate ethylammonium (MTSEA). We substituted cysteine (C) for native valine (V) at position 930 of D2-S6 in the MTSEA-resistant hNav1.5 mutant C373Y to produce the double mutant C373Y-V930C. Whole-cell Na(+) currents were recorded using patch-clamp techniques in transiently transfected HEK cells. In C373Y-V930C, we find that MTSEA (1.5 mM) applied in the closed state (-160 mV) has no significant effect on whole-cell Na(+) current, while MTSEA applied in the slow-inactivated state (prolonged depolarization at 0 mV) decreases current. We propose that D2-S6 in hNav1.5 undergoes molecular rearrangement during slow inactivation exposing the side chain of residue 930 such that it becomes accessible to modification by MTSEA.

We previously showed that lysine substitutions at two residues in segment 6 of domain 3 in voltage-gated Na(+) channel rNav1.4 (S1276K, L1280K) reduced steady-state inactivated local anesthetic block. Here we studied cysteine substitutions at the same residues (S1276C, L1280C). We used whole-cell recordings to determine local anesthetic block (100 microM bupivacaine) before and after cysteine modification with 1.5 mM 2-aminoethyl methanethiosulfonate (MTSEA). Compared with rNav1.4, steady-state resting bupivacaine block at -180 mV was increased in S1276C, while inactivated block at -50 mV was not different in the mutants. After application of MTSEA at -160 mV, rNav1.4 showed enhanced bupivacaine block and a negative shift in V(1/2) of the bupivacaine affinity curve, while L1280C and S1276C showed a decrease in inactivated bupivacaine block after MTSEA. Application of MTSEA at 0 mV produced similar results in rNav1.4 and L1280C, but an opposite effect in S1276C, i.e., enhancement of bupivacaine block, with a large negative shift in V(1/2) of the bupivacaine affinity curve similar to that found in rNav1.4. We conclude that 1) MTSEA modification of 1276C or 1280C decreases inactivated bupivacaine block similar to that found in L1280K and S1276K, 2) residue 1276C is only accessible to MTS-modification in the resting state, and 3) MTSEA may modify a native cysteine in rNav1.4 that produces an allosteric, indirect effect on bupivacaine affinity.

The steady-state rate of glucose oxidation through the mitochondrial TCA cycle (V(TCA)) was measured in acid extracts of 10- and 30-day-old cerebral cortex of rats receiving [1-13C]glucose intravenously and in neocortical slices superfused in vitro with the same isotope. TCA cycle flux was determined for each age group based on metabolic modeling analysis of the isotopic turnover of cortical glutamate and lactate. The sensitivity of the calculated rates to assumed parameters in the model were also assessed. Between 10 and 30 postnatal days, V(TCA) increased by 4.3-fold (from 0.46 to 2.0 micromol g(-1) min(-1)) in the cortex in vivo, whereas only a 2-fold (from 0.17 to 0.34 micromol g(-1) min(-1)) increase was observed in neocortical slices. The much greater increase in glucose oxidative metabolism of the cortex measured in vivo over that measured in vitro as the cortex matures suggests that function-related energy demands increase during development, a process that is deficient in the slice as a result of deafferentiation and other mechanisms.

We have previously observed that prolonged O(2) deprivation alters membrane protein expression and membrane properties in the central nervous system. In this work, we studied the effect of prolonged O(2) deprivation on the electrical activity of rat cortical and hippocampal neurons during postnatal development and its relationship to Na(+) channels. Rats were raised in low O(2) environment (inspired O(2) concentration = 9.5+/-0.5%) for 3-4 weeks, starting at an early age (2-3 days old). Using electrophysiologic recordings in brain slices, RNA analysis (northern and slot blots) and saxitoxin (a specific ligand for Na(+) channels) binding autoradiography, we addressed two questions: (1) does long-term O(2) deprivation alter neuronal excitability in the neocortical and hippocampal neurons during postnatal development? and (2) if so, what are the main mechanisms responsible for the change in excitability in the exposed brain? Our results show that (i) baseline membrane properties of cortical and hippocampal CA1 neurons from rats chronically exposed to hypoxia were not substantially different from those of naive neurons; (ii) acute stress (e.g., hypoxia) elicited a markedly exaggerated response in the exposed neurons as compared to naive ones; (iii) chronic hypoxia tended to increase Na(+) channel mRNA and saxitoxin binding density in the cortex and hippocampus as compared to control ones; and (iv) the enhanced neuronal response to acute hypoxia in the exposed cortical and CA1 neurons was considerably attenuated by applying tetrodotoxin, a voltage-sensitive Na(+) channel blocker, in a dose-dependent manner. We conclude that prolonged O(2) deprivation can lead to major electrophysiological disturbances, especially when exposed neurons are stressed acutely, which renders the chronically exposed neurons more vulnerable to subsequent micro-environmental stress. We suggest that this Na(+) channel-related over-excitability is likely to constitute a molecular mechanism for some neurological sequelae, such as epilepsy, resulting from perinatal hypoxic encephalopathy.

We studied the neurophysiology of neurons from the central nervous system (CNS) of rats that were exposed to a long-term (3-4 weeks) low oxygen (FiO2 = 9.5 +/- 0.5%) environment (exposed). Age-matched normoxic animals served as controls (naive). We measured membrane potential (Vm) and input resistance (Rm) at rest and in response to two levels (20% and 0% O2) of acute in vitro hypoxia using intracellular recordings in the brain slice from two areas of the CNS, layer 2/3 of the neocortex (NCX) and the hypoglossal nucleus in the brainstem (XII). Resting Vm and Rm were not different between exposed and naive neurons. However, acute hypoxia elicited dramatic differences between exposed and naive NCX neurons. Exposed NCX depolarized 5 x more (delta Vm = 53.2 +/- 7.0 mV; n = 13; mean +/- S.E.M.) than naive NCX (delta Vm = 10.6 +/- 2.0; n = 8) in response to 20% O2. In 0% O2, naive NCX showed anoxic depolarization (delta Vm > 20 mV/min) much sooner (mean latency of 4.8 +/- 0.4 min; n = 18) than naive NCX (8.8 +/- 1.0 min; n = 19). Rm decreased 2-4 times more in exposed NCX compared to naive NCX in response to O2 deprivation. In addition, while all naive NCX recovered to baseline Vm and Rm when re-oxygenated, exposed NCX exhibited a much slower recovery compared to naive NCX, and almost 20% of the exposed NCX failed to recover Vm and Rm following in vitro hypoxia. In contrast to NCX, there was little difference between exposed XII and naive XII. We conclude that chronic hypoxia renders neurons in the neocortex more vulnerable to subsequent acute stress such as O2 deprivation.

Transmembrane segment 6 is implicated in slow inactivation (SI) of voltage-gated Na+ channels (Navs). To further study its role and understand differences between SI phenotypes of different Nav isoforms, we analyzed several domain 2-segment 6 (D2-S6) mutants of the human cardiac hNav1.5, which is relatively resistant to SI. Mutants were examined by transient HEK cell transfection and patch-clamp recording of whole cell Na+ currents. Substitutions with lysine (K) included N927K, V930K, and L931K. We show recovery from short (100 ms) depolarization to 0 mV in N927K and L931K is comparable to wild type, whereas recovery in V930K is delayed and biexponential, suggesting rapid entry into a slow-inactivated state. SI protocols confirm enhanced SI phenotype (rapid development, hyperpolarized steady state, slowed recovery) for V930K, contrasting with the resistant phenotype of wild-type hNav1.5. This enhancement, not found in N927K or L931K, suggests that the effect in V930K is site specific. Glutamine (Q) substituted at V930 also exhibits an enhanced SI phenotype similar to that of V930K. Therefore, K or Q substitution eliminates hNav1.5 resistance to SI. Alanine (A) or cysteine (C) substitution at V930 shows no enhancement of SI, and in fact, V930A and V930C, as well as L931K, exhibit a resistance to SI, demonstrating that characteristics of specific amino acids (e.g., size, hydrophobicity) differentially affect SI gating. Thus V930 in D2-S6 appears to be an important structural determinant of SI gating in hNav1.5. We suggest that conformational change involving D2-S6 is a critical component of SI in Navs, which may be differentially regulated between isoforms by other isoform-specific determinants of SI phenotype.

Slow inactivation in voltage-gated sodium channels (NaChs) occurs in response to depolarizations of seconds to minutes and is thought to play an important role in regulating membrane excitability and action potential firing patterns. However, the molecular mechanisms of slow inactivation are not well understood. To test the hypothesis that transmembrane segment 6 of domain 2 (D2-S6) plays a role in NaCh slow inactivation, we substituted different amino acids at position V787 (valine) in D2-S6 of rat skeletal muscle NaCh mu(1) (Na(v)1.4). Whole-cell recordings from transiently expressed NaChs in HEK cells were used to study and compare slow inactivation phenotypes between mutants and wild type. V787K (lysine substitution) showed a marked enhancement of slow inactivation. V787K enters the slow-inactivated state approximately 100x faster than wild type (tau(1) approximately 30 ms vs. approximately 3 s), and occurs at much more hyperpolarized potentials than wild type (V(1/2) of s(infinity) curve approximately -130 mV vs. approximately -75 mV). V787C (cysteine substitution) showed a resistance to slow inactivation, i.e., opposite to that of V787K. Entry into the slow inactivation state in V787C was slower (tau(1) approximately 5 s), less complete, and less voltage-dependent (V(1/2) of s(infinity) curve approximately -50 mV) than in wild type. Application of the cysteine modification agent methanethiosulfonate ethylammonium (MTSEA) to V787C demonstrated that the 787 position undergoes a relative change in molecular conformation that is associated with the slow inactivation state. Our results suggest that the V787 position in Na(v)1.4 plays an important role in slow inactivation gating and that molecular rearrangement occurs at or near residue V787 in D2-S6 during NaCh slow inactivation.

1. Voltage-gated Na+ channels undergo two types of inactivation in response to depolarization. One type, fast inactivation, occurs with a time scale of milliseconds. The other, slow inactivation, occurs over seconds to minutes. In addition, these two processes appear to be distinct at the molecular level. However, the molecular mechanism of Na+ channel slow inactivation is unknown. 2. We used patch clamp techniques to study slow inactivation, activation and fast inactivation in alpha-subunit cDNA clones for wild-type human heart Na+ channels (hH1) and rat skeletal muscle Na+ channels (mu1) transiently expressed in human embryonic kidney (HEK) cells. Our experiments showed that the Na+ channel slow inactivation phenotype (development, steady state and recovery) differed dramatically between hH1 and mu1. Slow inactivation in mu1 had a faster onset, a steeper voltage dependence, and was more complete compared with hH1. In addition, recovery from slow inactivation was much slower for mu1 than for hH1. Activation and fast inactivation kinetics were also different in hH1 and mu1. In hH1, fast inactivation was slower and V values of activation and steady-state fast inactivation (hthorn ) were more negative than in mu1. 3. To better understand the molecular basis of Na+ channel slow inactivation, Na+ channel chimaeras were constructed with domains from hH1 and mu1. The slow inactivation phenotype in the chimaeras (domains denoted by subscripts) mu1(1)hH1(2,3,4), mu1(1,2)hH1(3,4) and mu1(1,2,3)hH1(4) was intermediate compared with that of wild-type. However, the chimaera mu1(1)hH1(2,3,4) was more like wild-type hH1, while the chimaeras mu1(1,2)hH1(3,4) and mu1(1,2,3)hH1(4) were more similar to wild-type mu1. In the chimaeras, activation resembled that of mu1, fast inactivation resembled that of hH1, and steady-state fast inactivation fell between that of hH1 and mu1. 4. The data demonstrate that all four domains can modulate the Na+ channel slow inactivation phenotype. However, domains D1 and D2 may play a more prominent role in determining Na+ channel slow inactivation phenotype than D3 and D4. The results also support previous conclusions that D3 and D4 (and the D3-D4 linker) play an important role in Na+ channel fast inactivation, and that activation may require non-equivalent contributions from all four domains.

1. Hippocampal neurones respond to acute oxygen deprivation (hypoxia) with an inhibition of whole-cell Na+ current (INa), although the mechanism of the inhibition is unknown. Kinases can modulate INa and kinases are activated during hypoxia. We hypothesized that kinase activation may play a role in the hypoxia-induced inhibition of INa. 2. Single electrode patch clamp techniques were used in dissociated hippocampal CA1 neurones from the rat. INa was recorded at baseline, during exposure to kinase activators (with and without kinase inhibitors), and during acute hypoxia (with and without kinase inhibitors). 3. Hypoxia (3 min) reduced INa to 38.1 +/- 4.5% of initial values, and shifted steady-state inactivation in the negative direction. Hypoxia produced no effect on activation or fast inactivation. 4. Protein kinase A (PKA) activation with 2.5 mM adenosine 3',5'-cyclic adenosine monophosphate, N6,O2-dibutyryl, sodium salt (db-cAMP) resulted in reduction of INa to 62.8 +/- 5.5% without an effect on activation or steady-state inactivation. INa was also reduced by activation of protein kinase C (PKC) with 5 nM phorbol 12-myristate 13-acetate (PMA; to 40.0 +/- 3.7%) or 50 microM 1-oleoyl-2-acetyl-sn-glycerol (OAG; to 46.1 +/- 2.8%). In addition, steady-state inactivation was shifted in the negative direction by PKC activation. Neither the activation curve nor the kinetics of fast inactivation was altered by PKC activation. 5. The response to PKA activation was blocked by the PKA inhibitor N-[2-p-bromocinnamyl-amino) ethyl]-5-isoquinolinesulphonamide (H-89; 30 microM) and by 30 microM of PKA inhibitory peptide PKA5-24 (PKAi). PKC activation was blocked by the kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7; 100 microM), by the PKC inhibitor calphostin C (10 microM) and by 20 microM of the inhibitory peptide PKC19-31 (PKCi). 6. The hypoxia-induced inhibition of INa and shift in steady-state inactivation were greatly attenuated with H-7, calphostin C, or PKCi, but not with H-89 or PKAi. 7. We conclude that hypoxia activates PKC in rat CA1 neurones, and that PKC activation leads to the hypoxia-induced inhibition of INa.

An approximate model (D) for the calculation of lanthanide-induced shifts or some trans -fused benzylidene acetals which chelate to shift reagents is described. Shifts calculated for the model system 4,5-dimethylveratrole using model D were in good agreement with the reported data. With the aid of model D, rotamer conformations of the methoxyl group on C-1 of methyl 4,6- O -benzylidene-3- O -methyl-α- d -mannopyranoside, methyl 3- O -benzyl-4,6- O -benzylidene-α- d -mannopyranoside, and methyl 4,6- O -benzylidene-α- d -mannopyranoside were investigated.

We compared wild-type rat skeletal muscle NaChs ( μ 1) and a mutant NaCh (Y1586K) that has a single amino acid substitution, lysine (K) for tyrosine (Y), at position 1586 in the S6 transmembrane segment of domain 4. In Y1586K, macroscopic current decay is faster, the V 1/2 of the activation curve is shifted in the depolarized direction, and the fast-inactivation curve is less steep compared with μ 1. After an 8-ms depolarization pulse, Y1586K recovers from inactivation much more slowly than μ 1. The recovery is double exponential, suggesting recovery from two inactivation states. Varying the depolarization protocols isolates entry into an additional, "atypical" inactivation state in Y1586K that is distinct from typical fast or slow inactivation. Substitution of positively charged arginine (R) at Y1586 produces an inactivation phenotype similar to that of Y1586K. Substitution by negatively charged aspartic acid (D) or uncharged alanine (A) at Y1586 produces an inactivation phenotype similar to μ 1. Our results suggest that the positive charge of lysine (K) produces the atypical inactivation state in Y1586K. We propose that a conformational change during depolarization alters the relative position of the 1586K residue in the D4-S6 segment and that atypical inactivation in Y1586K occurs via an electrostatic interaction in or near the inner pore region.