Methanethiosulfonate-modification alters local anesthetic block in rNav1.4 cysteine-substituted mutants S1276C and L1280C.

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.

Residue-specific effects on slow inactivation at V787 in D2-S6 of Na(v)1.4 sodium channels.

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.

Increased neuronal excitability after long-term O(2) deprivation is mediated mainly by sodium channels.

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.

A point mutation in domain 4-segment 6 of the skeletal muscle sodium channel produces an atypical inactivation state.

We compared wild-type rat skeletal muscle NaChs (micro1) 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 mu1. After an 8-ms depolarization pulse, Y1586K recovers from inactivation much more slowly than mu1. 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 mu1. 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.

Comparison of slow inactivation in human heart and rat skeletal muscle Na+ channel chimaeras.

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.

Oxygen deprivation inhibits Na+ current in rat hippocampal neurones via protein kinase C.

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.

Comparative effects of UV A and UV B on clonogenic survival and delayed cell death in skin cell lines from humans and fish.

The effects of UV radiation on humans and animals are receiving increasing attention and much interest has recently been focused on the environmental effects of UV A and UV B. This study compares the in vitro effects of UV A and UV B on the clonogenic survival of two human skin keratinocyte cell lines, HaCaT which are immortal but not tumorigenic and HPV-G transfected keratinocytes which form non malignant tumours in nude mice. The effects were also studied on an EPC fish cell line. The aim of the work was to establish if similar initial and delayed survival responses occurred in both species. The cells were exposed to ultraviolet lamps emitting maximally at 365 nm (UV A) and 302 nm (UV B). Clonogenic survival was determined at appropriate times post exposure. Results for the initial survival curves show that the HaCaT and HPV-G cells did not show any appreciable difference in their response to UV A but the EPC cells were more sensitive at doses < 3000 Jm-2. The EPC cells were more sensitive to UV B at doses < 200 Jm-2 in comparison to the human HaCaT and HPV-G cells with the HPV-G cells showing the most sensitivity to UV B at doses > 200 Jm-2. The possible contribution of lethal mutations (delayed cell death) to the UV radiation response in the HaCaT and EPC cell lines was examined. The results showed that lethal mutations were expressed in the HaCaT cells following exposure to UV A and UV B but no lethal mutations were expressed in the EPC cells.

Chronic hypoxia in vivo renders neocortical neurons more vulnerable to subsequent acute hypoxic stress.

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.

Major differences in response to graded hypoxia between hypoglossal and neocortical neurons.

Intracellular electrophysiologic recordings were performed in brain slices from adult rats to compare the response of brain stem hypoglossal neurons (XII) and layer II/III neocortical neurons (NCX) to two levels of oxygen deprivation (hypoxia, pO2 = 15-20 Torr; anoxia, pO2 = 0 Torr). These recordings were also used during re-oxygenation after hypoxia or anoxia to study neuronal recovery. Both groups of neurons showed a greater response to anoxia than hypoxia in terms of membrane potential (Vm) and input resistance (Rm). When the two groups were compared at each level of O2, XII depolarized more and in a shorter period of time than NCX. During anoxia, XII neurons responded with anoxic depolarization (AD) of > 20 mV/min by 3 min, along with a large decrease in Rm. NCX neurons, on the other hand, exhibited AD after a mean latency of approximately 9 min and 18% of NCX neurons did not even show AD. Although all neurons (both XII and NCX) recovered when re-oxygenated before or at AD, XII neurons failed to recover from periods of anoxia that were well tolerated by NCX neurons. We conclude that: (1) there are marked differences in the magnitude and trajectory of membrane depolarization between XII and NCX neurons in response to O2 deprivation, with NCX neurons showing a much longer latency to AD during anoxia than XII; and (2), when exposed to periods of anoxia of similar duration and severity, XII neurons are less likely to recover than NCX neurons and XII neurons may, therefore, be inherently more vulnerable to anoxia-induced injury than NCX neurons.

Predicting to 9-month performance of premature infants.

This article identifies variables found to be predictive of sensorimotor and psychosocial performance in infants at 9 months adjusted age. The subjects were 102 infants with a birth weight of less than 1,500 g, who were participants in an early intervention project. Multiple regression analyses disclosed that poor 9-month performance was related to a lack of special Neonatal Intensive Care Unit (NICU) intervention, to minority group membership, to birth weight, and to performance on selected tests administered in the NICU. We present implications for the early prediction of handicapping conditions and for further research.

Hana kai ii: a 17-day dry saturation dive at 18.6 ATA. VI: Cognitive performance, reaction time, and personality changes.

Measures of spatial orientation, associative memory, general intelligence, arithmetic ability, reaction time, and personal/social perceptions were administered to five subjects during a 31-day saturation exercise. Performance decrements were noted during 17 days of exposure to hyperbaric Hi-O2 at 18.6 ATA. Significant losses in general intellectual ability were noted, as well as trends toward significant losses in other cognitive tests. Reaction time and arithmetic errors increased significantly during the early testing sessions. Performance during a 3-day cold period was equivocal; arithmetic errors increased, but other measures improved or remained constant. Environmental stressors such as fatigue, anxiety, health problems, personal and social adjustment, and aspects of perceptual deprivation were considered to be influential in reducing performance effectiveness.

Hana Kai II: a 17-day dry saturation dive at 18.6 ATA. VII: Auditory, visual, and gustatory sensations.

Five divers were tested for alterations in auditory, visual, and gustatory functioning during a 17-day saturation exposure to He-O2 at 18.6 ATA. No evidence of permanent hearing loss was disclosed. Critical flicker fusion was not affected, but peripheral visual thresholds were significantly increased during the first two weeks at 18.6 ATA; this was interpreted to be evidence of severe psychological and physiological stress. Foveal vision was unaffected across testings. Magnitude estimation techniques disclosed changes in taste sensitivity, with sweet sensitivity increasing over time and sour sensitivity declining over the course of the dive. Subjects were more sensitive to bitter stimuli at maximum pressure than at sea level, and less sensitive to salt at maximum pressure. The results indicate that appreciable alterations in sensory functioning can occur during saturation exposures, although the sense modalities were differentially affected by such environmental stressors as pressure, psycho-social stress, fatigue, and perceptual deprivation.