Voltage-gated calcium currents in axotomized adult rat cutaneous afferent neurons

The effect of sciatic nerve injury on the somatic expression of voltage-gated calcium currents in adult rat cutaneous afferent dorsal root ganglion (DRG) neurons identified via retrograde Fluoro-gold labeling was studied using whole cell patch-clamp techniques. Two weeks after a unilateral ligation and transection of the sciatic nerve, the L(4)-L(5) DRG were dissociated and barium currents were recorded from cells 3-10 h later. Cutaneous afferents (35-50 microm diam) were classified as type 1 (possessing only high-voltage-activated currents; HVA) or type 2 (having both high- and low-voltage-activated currents). Axotomy did not change the percentage of neurons exhibiting a type 2 phenotype or the properties of low-threshold T-type current found in type 2 neurons. However, in type 1 neurons the peak density of HVA current available at a holding potential of -60 mV was reduced in axotomized neurons (83.9 +/- 5.6 pA/pF, n = 53) as compared with control cells (108.7 +/- 6.9 pA/pF, n = 58, P < 0.01, unpaired t-test). A similar reduction was observed at more negative holding potentials, suggesting differences in steady-state inactivation are not responsible for the effect. Separation of the type 1 cells into different size classes indicates that the reduction in voltage-gated barium current occurs selectively in the larger (capacitance >80 pF) cutaneous afferents (control: 112.4 +/- 10.6 pA/pF, n = 30; ligated: 72.6 +/- 5.0 pA/pF, n = 36; P < 0.001); no change was observed in cells with capacitances of 45-80 pF. Isolation of the N- and P?Q-type components of the HVA current in the large neurons using omega-conotoxin GVIA and omega-agatoxin TK suggests a selective reduction in N-type barium current after nerve injury, as the density of omega-CgTx GVIA-sensitive current decreased from 56.9 +/- 6.6 pA/pF in control cells (n = 13) to 31.3 +/- 4.6 pA/pF in the ligated group (n = 12; P < 0.005). The HVA barium current of large cutaneous afferents also demonstrates a depolarizing shift in the voltage dependence of inactivation after axotomy. Injured type 1 cells exhibited faster inactivation kinetics than control neurons, although the rate of recovery from inactivation was similar in the two groups. The present results indicate that nerve injury leads to a reorganization of the HVA calcium current properties in a subset of cutaneous afferent neurons.     

Developmental changes in hyperpolarization-activated currents I(h) and I(K(IR)) in isolated rat intracardiac neurons.

The hyperpolarization-activated nonselective cation current, I(h), was investigated in neonatal and adult rat intracardiac neurons. I(h) was observed in all neurons studied and displayed slow time-dependent rectification. I(h) was isolated by blockade with external Cs(+) (2 mM) and was inhibited irreversibly by the bradycardic agent, ZD 7288. Current density of I(h) was approximately twofold greater in neurons from neonatal (-4.1 pA/pF at -130 mV) as compared with adult (-2.3 pA/pF) rats; however, the reversal potential and activation parameters were unchanged. The reversal potential and amplitude of I(h) was sensitive to changes in external Na(+) and K(+) concentrations. An inwardly rectifying K(+) current, I(K(IR)), was also present in intracardiac neurons from adult but not neonatal rats and was blocked by extracellular Ba(2+). I(K(IR)) was present in approximately one-third of the adult intracardiac neurons studied, with a current density of -0.6 pA/pF at -130 mV. I(K(IR)) displayed rapid activation kinetics and no time-dependent rectification consistent with the rapidly activating, inward K(+) rectifier described in other mammalian autonomic neurons. I(K(IR)) was sensitive to changes in external K(+), whereby raising the external K(+) concentration from 3 to 15 mM shifted the reversal potential by approximately +36 mV. Substitution of external Na(+) had no effect on the reversal potential or amplitude of I(K(IR)). I(K(IR)) density increases as a function of postnatal development in a population of rat intracardiac neurons, which together with a concomitant decrease in I(h) may contribute to changes in the modulation of neuronal excitability in adult versus neonatal rat intracardiac ganglia.     

A novel mutation L619F in the cardiac Na+ channel SCN5A associated with long-QT syndrome (LQT3): a role for the I-II linker in inactivation gating

Congenital long QT syndrome type 3 (LQT3) is caused by mutations in the gene SCN5A encoding the alpha-subunit of the cardiac Na(+) channel (Nav1.5). Functional studies of SCN5A mutations in the linker between domains III and IV, and more recently the C-terminus, have been shown to alter inactivation gating. Here we report a novel LQT3 mutation, L619F (LF), located in the domain I-II linker. In an infant with prolonged QTc intervals, mutational analysis identified a heterozygous missense mutation (L619F) in the domain I-II linker of the cardiac Na(+) channel. Wild-type (WT) and mutant channels were studied by whole-cell patch-clamp analysis in transiently expressed HEK cells. LF channels increase maintained Na(+) current (0.79 pA/pF for LF; 0.26 pA/pF for WT) during prolonged depolarization. We found a +5.8mV shift in steady state inactivation in LF channels compared to WT (WT, V(1/2)=-64.0 mV; LF, V(1/2)=-58.2 mV). The positive shift of inactivation, without a corresponding shift in activation, increases the overlap window current in LF relative to WT (1.09 vs. 0.58 pA/pF), as measured using a positive voltage ramp protocol (-100 to +50 mV in 2s). The increase in window current, combined with an increase in non-inactivating Na(+) current, may act to prolong the AP plateau and is consistent with the disease phenotype observed in patients. Moreover, the defective inactivation imposed by the L619F mutation implies a role for the I-II linker in the Na(+) channel inactivation process.     

柯萨奇B3病毒对心肌细胞钙平衡的影响

目的 探讨柯萨奇Ｂ３病毒（Ｃｏｘｓａｃｋｉｅｖｉｒｕｓ Ｂ３,ＣＶＢ３）对膜离子通道及离子交换载体的影响,了解病毒感染导致细胞内钙超载的原因。方法 酶灌注法获得单个心肌细胞后用光聚焦显微镜和钙离子荧光探针（Ｆｌｕｏ ３／ＡＭ）检测ＣＶＢ３感染对心肌细胞内游离钙离子浓度的影响及利用模片钳全细胞电流记录技术观察ＣＶＢ３对Ｌ型钙通道电流,钠通道电流和钠钙交换电流的影响。结果 ＣＶＢ３感染使细胞内的游离钙     

Effects of endothelin-1 on calcium and potassium currents in undiseased human ventricular myocytes

Endothelins have been reported to exert a wide range of electrophysiological effects in mammalian cardiac cells. These results are controversial and human data are not available. Our aim was to study the effects of endothelin-1 (ET-1, 8 nmol/l) on the L-type calcium current (ICa-L) and various potassium currents (rapid component of the delayed rectifier, IKr; transient outward current, Ito; and the inward rectifier K current, IK1) in isolated human ventricular cardiomyocytes. Cells were obtained from undiseased donor hearts using collagenase digestion via the segment perfusion technique. The whole-cell configuration of the patch-clamp technique was applied to measure ionic currents at 37 degrees C. ET-1 significantly decreased peak ICa-L from 10.2+/-0.6 to 6.8+/-0.8 pA/pF at +5 mV (66.7% of control, P<0.05, n=5). This reduction of peak current was accompanied by a lengthening of inactivation. The voltage dependence of steady-state activation and inactivation was not altered by ET- 1. IKr, measured as tail current amplitudes at 40 mV, decreased from 0.31+/-0.02 to 0.06+/-0.02 pA/pF (20.3% of control, P<0.05, n=4) after exposure to ET-1. ET-1 failed to change the peak amplitude of Ito, measured at +50 mV (9.3+/-4.6 and 9.0+/-4.4 pA/pF before and after ET-1, respectively), or steady-state IK1 amplitude, measured at the end of a 400-ms hyperpolarization to -100 mV (3.6+/-1.4 and 3.7+/-1.4 pA/pF, n=4). The present results indicate that in undiseased human ventricular myocytes ET-1 inhibits both ICa-L and IKr; however, the degree of suppression of the two currents is different.     

A-type potassium current in myenteric neurons from guinea-pig small intestine

Biophysical properties of A-type K(+) currents (I(A)) in myenteric neurons from guinea-pig small intestine were studied. I(A) was present in both AH- and S-type myenteric neurons. Reduction of external Ca(2+) did not affect the current. Current density was 13.5+/-10.2 pA/pF in 68 AH-type neurons and 23.4+/-8.2 pA/pF in 31 S-type neurons. S-type neurons appeared to be a homogeneous group based on density of I(A). AH-type neurons were subdivided into two groups with current densities of 9.4+/-4.3 and 25.4+/-4.3 pA/pF. All other biophysical properties of the current were not statistically different for AH- and S-type neurons. Steady-state activation and inactivation curves showed half-activation potentials at -7 mV (k=15. 0 mV) and -86 mV (k=11.5 mV). The curves overlapped at potentials near the resting potential of approximately -55 mV. Time constants for activation ranged from 3.6 to 0.52 ms at test potentials between -20 and 50 mV. Inactivation time constants fell between 41.5 and 11 ms at test potentials between -20 and 50 mV. Time constants for recovery from inactivation fit a double-exponential curve with fast and slow recovery times of 11 and 550 ms. 4-Aminopyridine suppressed I(A) when it was activated at -20 mV following a pre-pulse to -110 mV. Addition of Zn(2+) in the external solution resulted in a concentration-dependent shift of the activation and inactivation curves in the depolarized direction. Zn(2+) slowed the activation and inactivation kinetics of I(A) by factors of 3.3- and 1.2-fold over a wide range of potentials. Elevation of external H(+) suppressed the effect of Zn(2+) with a pK of 7.3-7.4. The effects of Zn(2+) were interpreted as not being due to surface charge screening, because the affinity of Zn(2+) for its binding site on the A-channel was estimated to be between 170 and 312 microM, while the background concentration of Mg(2+) was 10 mM.The enteric nervous system is perceived as an independent integrative nervous system (brain-in-the-gut) that is responsible for local organizational control of motility and secretory patterns of gut behavior. AH- and S-type neurons are synaptically interconnected to form the microcircuits of the enteric nervous system. The results suggest that I(A) is a significant determinant of neuronal excitability for both the firing of nerve impulses and the various synaptic events in the two types of neurons.     

雌激素对豚鼠心室肌细胞的非基因组效应研究

目的研究雌激素对豚鼠心室肌细胞的非基因组作用.方法方法采用全细胞膜片钳技术.结果17β-雌二醇1 μmol/L和3 μmol/L在1～2 min内快速抑制INa峰值,抑制率分别13.25%±4.71%,32.46%±4.82%.1 μmol/L 17β-雌二醇亦可影响INa的电流-电压(I-V)曲线,使INa电流密度值在-30mV～+30 mV膜电位下显著降低.在-20 mV处,INa电流密度值由-120.48±7.05 pA/pF降至-101.91±11.00pA/pF(P＜0.01).在+30 mV处,INa电流密度值由-39.55±10.50pA/pF降至-29.88±6.21pA/pF(P＜0.05).17β-雌二醇的抑制效应被DNA转录抑制剂-放线菌素D所阻断.且抑制效应与豚鼠的性别无关.结论雌激素通过推测的非基因组效应快速抑制豚鼠心室肌细胞钠电流(INa),且该效应不被放线菌素D所阻断.     

Inhibitory action of protein kinase Cbeta inhibitor on tetrodotoxin-resistant Na+ current in small dorsal root ganglion neurons in diabetic rats

Experimental evidence has been presented to suggest that protein kinase Cbeta isoform-selective inhibitor LY333531 is effective at alleviating diabetic hyperalgesia. In the present study, we isolated small (< or =25 microm in soma diameter) dorsal root ganglion (DRG) neurons from control and streptozocin (STZ)-induced diabetic rats, and examined the acute action of LY333531 (1-1000 nM) on the tetrodotoxin-resistant Na(+) current (TTX-R I(Na)), which plays an essential role in transmitting nociceptive impulses, using the whole-cell patch-clamp method. TTX-R I(Na) in diabetic DRG neurons was enhanced in amplitude (71.5+/-3.6pA/pF, n=10 versus 41.2+/-3.3pA/pF, n=8) and was activated at more negative potentials (V(1/2), -15.1+/-1.3 mV versus -9.6+/-1.4 mV), compared with that in control neurons. Bath application of LY333531 acutely inhibited TTX-R I(Na) in both control and diabetic DRG neurons, and the degree of inhibition by the drug at concentrations of 1, 10 and 100 nM was significantly greater in diabetic DRG neurons than in control DRG neurons. Thus, TTX-R I(Na), which is upregulated in the diabetic state, is likely to be more potently inhibited by submicromolar concentrations of LY333531. These results suggest that an acute inhibition of TTX-R I(Na) by LY333531 attenuates the exaggerated excitability of DRG neurons in the diabetic state, which appears to be related at least partly to anti-hyperalgesic actions of the drug in diabetic neuropathy.     

Characterization of calcium currents in aortic baroreceptor neurons.

1. Calcium currents in identified rat aortic baroreceptors were characterized with the perforated patch whole-cell voltage-clamp technique. Aortic baroreceptors were distinguished from other neurons by the presence of a fluorescent tracer that was previously applied to the aortic depressor nerve. The diversity of calcium currents in unidentified neurons dissociated from neonatal rat nodose ganglia were also examined. 2. A population of aortic baroreceptors (63%, 7 of 11) possessed a low-threshold, also referred to as a T-type, calcium current. This current was typically less than 100 pA in 2 mM Ca [72.7 +/- 20.9 (SE) pA, n = 7], had a rapid activation and inactivation, and inactivated completely at conditioning voltages positive to -50 mV. 3. All aortic baroreceptors possessed high-threshold calcium currents that were activated at voltages positive to -30 mV, with typical maximum amplitudes of 600-1,000 pA (826 +/- 79 pA, n = 11). 4. The high-threshold current inactivated with three exponential rates of decay of tau = 10.7 +/- 2.2 ms, 138 +/- 14.6 ms, and a third tau greater than 3 s. It was not possible to separate the kinetic components of inactivation with conditioning voltages (voltage-dependent inactivation), activation thresholds, deactivation kinetics, or calcium-channel antagonists. 5. The voltage-dependent inactivation of high-threshold calcium currents began at voltages positive to -70 mV and became steeply voltage dependent between -60 and -10 mV. Unexpectedly, the three decay constants were present after all conditioning voltages. There were no conditioning voltages that excluded any component.(ABSTRACT TRUNCATED AT 250 WORDS)     

An investigation on the inactivation process of sodium currents in single frog ventricular cells

The characteristics of sodium currents (INa) in single frog ventricular cells were studied with the oil gap method. This method improves time- and space-control of the membrane potential under the voltage clamp, thereby making possible accurate analysis of fast events of INa. In this preparation the threshold of INa was about -60 mV and the reversal potential was 58 mV, which is close to the value calculated by the Nernst equation for sodium ions. Because the instantaneous current-voltage (I-V) relationship is linear, the ease of permeation of sodium ions through Na+ channels is well expressed by the chord conductance. The falling phase of INa and the time course of recovery from inactivation follow a time course of single exponential function. The time constants for on- and off-processes of inactivation at the same membrane potential are very close to each other, indicating only a single state of inactivation. Though almost all properties of INa were well described by Hodgkin-Huxley's model, a clear delay of onset of inactivation was demonstrated by two-pulse experiment. In this report the modified kinetics scheme was proposed which can account for both a delay of onset of inactivation development and a falling phase of INa that follows a single exponential time course.     

Voltage-gated sodium currents in isolated retinal ganglion cells of the cat: relation between the inactivation kinetics and the cell type.

Ganglion cells in the cat retina were retrogradely labeled by injecting a fluorescent dye (DiI) into either the lateral geniculate nucleus (LGN) or the superior colliculus (SC). Cells were then dissociated enzymatically from the retinal tissue. LGN-projecting ganglion cells consisted of 2 different populations, one with small and the other with large somata, which were identified as W and X cells, respectively. SC-projecting cells consisted of a single group of cells with small somata, identified as W cells. The voltage-gated sodium current (INa) was recorded from isolated ganglion cells under the voltage-clamp condition using a patch pipette in the whole cell configuration. INa was identified by reversible tetrodotoxin block. INa was activated by depolarization of the cell from the holding potential (Vh) of -95 mV to membrane voltages (Vm) more positive than -45 mV. The maximum INa was recorded at around -15 mV. INa flowed outward at Vm more positive than +65 mV. The reversal potential of INa became more negative voltages with low extracellular Na concentration ([Na+]o) with a relation of 58 mV for a 10-fold change in [Na+]o. INa was inactivated with a few milliseconds. Once inactivated, INa recovered by holding the cell membrane hyperpolarized. While the voltage dependence of INa activation and steady-state inactivation were constant from cell to cell, the time course of recovery was not. Cells with a large soma showed a rapid recovery, while cells with a small soma showed slow recovery. Thus, the rate of recovery is faster for X cells than for W cells. Perhaps this helps to explain the 'sluggish' firing of the latter cell type.     

Chronic high-inspired CO2 decreases excitability of mouse hippocampal neurons

To examine the effect of chronically elevated CO(2) on excitability and function of neurons, we exposed mice to 8 and 12% CO(2) for 4 wk (starting at 2 days of age), and examined the properties of freshly dissociated hippocampal neurons obtained from slices. Chronic CO(2)-treated neurons (CC) had a similar input resistance (R(m)) and resting membrane potential (V(m)) as control (CON). Although treatment with 8% CO(2) did not change the rheobase (64 +/- 11 pA, n = 9 vs. 47 +/- 12 pA, n = 8 for CC 8% vs. CON; means +/- SE), 12% CO(2) treatment increased it significantly (73 +/- 8 pA, n = 9, P = 0.05). Furthermore, the 12% CO(2) but not the 8% CO(2) treatment decreased the Na(+) channel current density (244 +/- 36 pA/pF, n = 17, vs. 436 +/- 56 pA/pF, n = 18, for CC vs. CON, P = 0.005). Recovery from inactivation was also lowered by 12% but not 8% CO(2). Other gating properties of Na(+) current, such as voltage-conductance curve, steady-state inactivation, and time constant for deactivation, were not modified by either treatment. Western blot analysis showed that the expression of Na(+) channel types I-III was not changed by 8% CO(2) treatment, but their expression was significantly decreased by 20-30% (P = 0.03) by the 12% treatment. We conclude from these data and others that neuronal excitability and Na(+) channel expression depend on the duration and level of CO(2) exposure and maturational changes occur in early life regarding neuronal responsiveness to CO(2).     

Sodium currents in isolated rat CA1 pyramidal and dentate granule neurones in the post-status epilepticus model of epilepsy

Status epilepticus (SE) was induced in the rat by long-lasting electrical stimulation of the hippocampus. After a latent period of 1 week, spontaneous seizures occurred which increased in frequency and severity in the following weeks, finally culminating after 3 months in a chronic epileptic state. In these animals we determined the properties of voltage-dependent sodium currents in acutely isolated CA1 pyramidal neurones and dentate granule cells using the whole-cell voltage-clamp technique. The conductance of the fast transient sodium current was larger in SE rats (84+/-7 nS versus 56+/-6 nS) but related to a difference in cell size so that the neurones had a similar specific sodium conductance (control: 7.8+/-0.8 nS/pF, SE: 6.7+/-0.8 nS/pF). Current activation and inactivation were characterised by a Boltzmann function. After SE the voltage dependence of activation was shifted to more negative potentials (control: -45.1+/-1.4 mV, SE: -51.5+/-2.9 mV, P<0.05). In combination with a small shift in the voltage dependence of inactivation to more depolarised potentials (control: -68.8+/-2.3 mV, SE: -66.3+/-2.3 mV), it resulted in a window current that was much increased in the SE neurones (median: 64 pA in control, 217 pA in SE, P<0.05). The peak of this window current shifted to more hyperpolarised potentials (control: -44 mV, SE: -50 mV, P<0.05). No differences were found in the sodium currents analysed in dentate granule cells of control and SE animals. The changes observed in CA1 neurones after SE contribute to enhanced excitability in particular when membrane potential is near firing threshold. They can, at least partly, explain the lower threshold for epileptic activity in SE animals. The comparison of CA1 with DG neurones in the same rats demonstrates a differential response in the two cell types that participated in very similar seizure activity.     

Na(+)-current expression in rat hippocampal astrocytes in vitro: alterations during development

1. With the use of whole-cell patch-clamp recording. Na(+)-current expression was studied in hippocampal astrocytes in vitro, individually identified by filling with Lucifer yellow (LY) and staining for glial fibrillary acidic protein (GFAP) and vimentin. 2. The proportion of astrocytes that express Na+ currents in rat hippocampal cultures changes during development in vitro and decreases from approximately 75% at day 1 to approximately 30% after 10 days in culture. 3. The sodium currents expressed in astrocytes can be differentiated into two types on the basis of kinetics. At early times in culture the time course of Na+ currents is fast in both onset and decay with an average decay time constant of 1.27 ms, whereas after 6 days Na+ currents become comparatively slow and decayed with an average time constant of 1.86 ms. 4. As with the time-course of Na+ currents, the two age groups of astrocytes (i.e., days 1-5 and day 6 and older) differ with respect to their steady-state inactivation characteristics. Early after plating and up to day 5, the midpoint of the steady-state inactivation curve is close to -60 mV, as also observed in hippocampal neurons of various ages; in contrast, after 6 days in culture the curve is shifted by approximately 25 mV toward more hyperpolarized potentials with a midpoint close to -85 mV. 5. In contrast to h infinity-curves, current-voltage (I-V) curves of Na(+)-current activation were identical in all astrocytes studied and did not change with time in culture. 6. In astrocytes expressing Na+ currents, current densities (average of 35 pA/pF on day 1) decreased throughout the first 5 days and were almost abolished around days 4 and 5 in culture. Beginning on day 6, however, current densities increased again and maintained a steady level (average of 14 pA/pF) for the duration of the time period in culture (20 days). This biphasic time course closely correlates with the time course of changes in Na(+)-current kinetics and steady-state inactivation. 7. These data suggest that Na+ currents in cultured hippocampal astrocytes show characteristic changes with increasing time in culture. During the first 4-5 days in culture, hippocampal astrocytes display Na+ currents with properties similar to those of hippocampal neurons. Our data further suggest that Na+ currents with distinctive, "glial-type" characteristics are only expressed in hippocampal astrocytes after 6 days in culture.     

Development of multiple calcium channel types in cultured mouse hippocampal neurons

The development of multiple calcium channel activities was studied in mouse hippocampal neurons in culture, using the patch-clamp technique. A depolarizing pulse (40-50 ms duration) from the holding potential of -80 mV to levels more depolarized than -40 mV produced a low threshold T-type current. The T-type current was observed in 52% of four days in vitro neurons. The number of neurons which expressed T-type current decreased with age of culture, so that the current was detected in only 18% of neurons after 16 days in vitro. The T-type current densities varied between 1.9 pA/pF and 3.29 pA/pF in the mean values during the period studied (4-16 days in vitro). A depolarizing pulse from -80 mV to levels more depolarized than -35 mV evoked a high threshold calcium channel current. The high threshold current density increased in the mean values from 3.9 pA/pF in four days in vitro neurons to 28 pA/pF in 16 days in vitro neurons. We have then examined the effect of nifedipine, omega-Agatoxin IVA and omega-conotoxin GVIA on the high threshold current. Nifedipine (1-5 microM) sensitive current density stayed in the range of 1.9-2.1 pA/pF during 4-16 days in vitro, while omega-Agatoxin IVA (200 nM) sensitive current density increased in the mean values from 1.54 pA/pF in four days in vitro neurons to 21.5 pA/pF in 16 days in vitro neurons. The omega-conotoxin GVIA sensitive N-type channel current was maximum at eight days in vitro (5.44 pA/pF) and it reduced progressively to reach almost half (2.46 pA/pF) in 16 days in vitro neurons. These results showed that diverse subtypes of calcium channels change in density during the early period of culture. We suggest that the temporal expression of each type of channel may be linked to the development of neural activities.     

Block of sodium channels by tyramine and its analogue (N-feruloyl tyramine) in frog ventricular myocytes.

Pharmacological effects of tyramine and its analogue, N-feruloyl tyramine (NFT), on sodium and calcium currents in frog ventricular myocytes were examined using the whole-cell voltage-clamp technique. To improve the temporal and spatial control of the membrane potential, sodium currents (INa) were recorded in 45.5 mM [Na+]o at 10 degrees C. Both tyramine and NFT (1-100 microM) induced a concentration-dependent decrease in INa evoked from a holding potential of -80 mV without affecting a change in either the time to peak or the time constant for the falling phase of INa. Similarly the reversal potential for INa remained unchanged at a value close to that predicted from the Nernst equation. The finding that both tyramine and NFT decreased INa when activated maximally, from a holding potential of -120 mV, indicates that the amplitude of INa can be reduced independently of a change in the kinetics of the current. In addition, tyramine (100 microM) shifted the membrane potential for half maximal inactivation (Vh) of the steady-state inactivation (h infinity)-curve from -74 to -84 mV without affecting its slope. In contrast, NFT failed to affect the h infinity-curve. The calcium current (ICa) recorded in the presence of 0.3 microM TTX was not affected by either 100 microM tyramine or NFT. We concluded that tyramine directly blocks Na channel by shifting h infinity-curve and by suppressing maximum Na channel conductance, while NFT suppresses only maximum Na channel conductance.     

Voltage-dependent K+ currents in rat cardiac dorsal root ganglion neurons

We have assessed the expression and kinetics of voltage-gated K(+) currents in cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of a fluorescent tracer into the pericardial sack. Ninety-nine neurons were labelled: 24% small (diameter<30 microm), 66% medium-sized (diameter 30 microm>.48 microm) and 10% large (>48 microm) neurons. Current recordings were performed in small and medium-sized neurons. The kinetic and pharmacological properties of K(+) currents recorded in these two groups of neurons were identical and the results obtained from these neurons were pooled. Three types of K(+) currents were identified:a) I(As), slowly activating and slowly time-dependently inactivating current, with V(1/2) of activation -18 mV and current density at +30 mV equal to 164 pA/pF, V(1/2) of inactivation at -84 mV. b) I(Af) current, fast activating and fast time-dependently inactivating current, with V(1/2) of activation at two mV and current density at +30 mV equal to 180 pA/pF, V(1/2) of inactivation at -26 mV. At resting membrane potential I(As) was inactivated, whilst I(Af), available for activation. The I(As) currents recovered faster from inactivation than I(Af) current. 4-Aminopiridyne (4-AP) (10 mM) and tetraethylammonium (TEA) (100 mM) produced 98% and 92% reductions of I(Af) current, respectively and 27% and 66% of I(As) current, respectively. c) The I(K) current that did not inactivate over time. Its V(1/2) of activation was -11 mV and its current density equaled 67 pA/pF. This current was inhibited by 95% (100 mM) TEA, whilst 4-AP (10 mM) produced its 23% reduction. All three K(+) current components (I(As), I(Af) and I(K)) were present in every small and medium-sized cardiac DRG neuron.We suggest that at hyperpolarized membrane potentials the fast reactivating I(As) current limits the action potential firing rate of cardiac DRG neurons. At depolarised membrane potentials the I(Af) K(+) current, the reactivation of which is very slow, does not oppose the firing rate of cardiac DRG neurons.     

Electrophysiological properties and chemosensitivity of acutely dissociated trigeminal somata innervating the cornea

Adult rat sensory trigeminal ganglion neurons innervating the cornea (cTGNs) were isolated and identified following retrograde dye labeling with FM1-43. Using standard whole-cell patch clamp recording techniques, cTGNs could be subdivided by their action potential (AP) duration. Fast cTGNs had AP durations <1 ms (40%) while slow cTGNs had AP durations >1 ms and an inflection on the repolarization phase of the AP. With the exception of membrane input resistance, the passive membrane properties of fast cTGNs were different from those of slow cTGNs (capacitance: 61+/-4.5 pF vs. 42+/-2.6 pF, resting membrane potential: -59+/-0.7 mV vs. -53+/-0.9 mV, for fast and slow cTGNs respectively). Active membrane properties also differed between fast and slow cTGNs. Slow cTGNs had a higher AP threshold (-25+/-1.6 mV vs. -38+/-0.8 mV), a larger rheobase (14+/-1.9 pA/pF vs. 6.8+/-1.0 pA/pF), and a smaller AP undershoot (-56+/-1.7 mV vs. -67+/-2.5 mV). The AP overshoot, however was similar between the two types of neurons (46+/-3.1 mV vs. 48+/-4 mV). Slow cTGNs were depolarized by capsaicin (1 microM, 80%) and 60% of their APs were blocked by tetrodotoxin (TTX) (100 nM). Fast cTGNs were unaffected by capsaicin and 100% of their APs were blocked by TTX. Similarly, cTGNs were also heterogeneous with respect to their responses to exogenous ATP and 5-HT. The current work shows that cTGNs have distinctive electrophysiological properties and chemosensitivity profiles. These characteristics may mirror the distinct properties of corneal sensory nerve terminals. The availability of isolated identified cTGNs constitutes a tractable model system to investigate the biophysical and pharmacological properties of corneal sensory nerve terminals.     

Apelin-13对大鼠缺血心室肌细胞瞬时钠电流的影响

 目的：观察apelin对正常和缺血心室肌细胞瞬时钠电流（INa）的影响以及其对室性心律失常和心脏功能的影响。方法：应用Landgendorff逆灌流系统酶解分离大鼠左室心肌细胞,采用全细胞膜片钳技术观察大鼠左室心肌细胞INa,通过改变电极外液的方法来模拟正常/缺血细胞状态,分正常组（N组）、正常apelin组（N+A组）、缺血组（I组）和缺血apelin组（I+A组）（各组n=10）,分别观察apelin-13对正常/模拟缺血心室肌细胞INa的影响;另建立Langendorff灌流离体缺血大鼠心脏模型分为4组（各组n=4）：N组、N+A组、I组、I+A组,观察apelin-13对各组大鼠离体灌流心脏模型心功能和室性心律失常的影响。结果：Apelin-13（100 nmol/L）在N+A 组和I+A组均能增加INa幅度,I-V曲线分析显示N+A组比N组INa幅度增大32%［（-86±13）pA/pF］,I+A组比I组INa幅度增大18% ［（-52±15）pA/pF］;然而I-V曲线分析Apelin-13并未改变最大传导速度,N、N＋A、I和I+A组分别为（3.2±0.2）pS/pF、（3.1±0.3）pS/pF、（2.9±0.1）pS/pF和（2.8±0.4）pS/pF（P＞0.05）;各组的半激活电压（V1/2）分别为（-21.9±0.6）mV、（-28.7±0.3）mV、（-30.5±0.7）mV和（-36.8±0.2）mV;Boltzmann曲线各组斜率分别为5.6±0.3、 5.1±0.4、 4.3±0.3和4.9±0.6（P＞0.05）;V1/2值N+A组比N组改变（-5.9±0.8）mV,I+A组V1/2比I组改变（-7.7±1.3）mV（P＜0.05）。室性心律失常开始时间、室性心动过速持续时间、心室颤动持续时间及心律失常评分情况,N+A组与N组比较、I+A组与I组比较差异无统计学意义（P＞0.05）;N+A组LVEDP低于N组,LVDP高于N组,dp/dtmax和dp/dtmin高于N组;I+A组LVEDP低于I组,LVDP高于I组,dp/dtmax和dp/dtmin高于I组;以β-肌动蛋白（β-actin）作为内参照,4组β-actin表达水平差异无统计学意义,4组Na+通道蛋白Vα亚单位的表达灰度值分别为28.8±3.6、 29.4±4.1、 30.1±2.9和31.3±3.8,组间差异无统计学意义。结论：Apelin-13可能是通过改变Na+通道门控特性增加INa峰值,使正常和缺血组心室肌细胞Na+通道更容易开放。Apelin-13对缺血相关室性心律失常无影响,对正常和缺血心肌均有正性肌力作用。