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.     

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.     

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-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.     

alpha-SNS produces the slow TTX-resistant sodium current in large cutaneous afferent DRG neurons

In this study, we used sensory neuron specific (SNS) sodium channel gene knockout (-/-) mice to ask whether SNS sodium channel produces the slow Na(+) current ("slow") in large (>40 microm diam) cutaneous afferent dorsal root ganglion (DRG) neurons. SNS wild-type (+/+) mice were used as controls. Retrograde Fluoro-Gold labeling permitted the definitive identification of cutaneous afferent neurons. Prepulse inactivation was used to separate the fast and slow Na(+) currents. Fifty-two percent of the large cutaneous afferent neurons isolated from SNS (+/+) mice expressed only fast-inactivating Na(+) currents ("fast"), and 48% expressed both fast and slow Na(+) currents. The fast and slow current densities were 0.90 +/- 0.12 and 0.39 +/- 0.16 nA/pF, respectively. Fast Na(+) currents were blocked completely by 300 nM tetrodotoxin (TTX), while slow Na(+) currents were resistant to 300 nM TTX, confirming that the slow Na(+) currents observed in large cutaneous DRG neurons are TTX-resistant (TTX-R). Slow Na(+) currents could not be detected in large cutaneous afferent neurons from SNS (-/-) mice; these cells expressed only fast Na(+) current, and it was blocked by 300 nM TTX. The fast Na(+) current density in SNS (-/-) neurons was 1.47 +/- 0. 14 nA/pF, approximately 60% higher than the current density observed in SNS (+/+) mice (P < 0.02). A low-voltage-activated TTX-R Na(+) current ("persistent") observed in small C-type neurons is not present in large cutaneous afferent neurons from either SNS (+/+) or SNS (-/-) mice. These results show that the slow TTX-R Na(+) current in large cutaneous afferent DRG is produced by the SNS sodium channel.     

Developmental changes in voltage-activated potassium currents of rat retinal ganglion cells.

Ca2(+)-independent voltage-activated potassium currents were investigated during the differentiation of rat retinal ganglion cells. Whole-cell patch-clamp recordings of Ca2(+)-independent voltage-activated potassium currents and their individual current components, i.e. a sustained, tetraethylammonium-sensitive current, a transient, 4-aminopyridine-sensitive current, and a slowly decaying current that was blocked by Ba2+, revealed distinct ontogenetic modifications in current densities and in activation and inactivation parameters. All three current types were expressed simultaneously at embryonic day 17/18 and were present in all retinal ganglion cells thereafter without showing any significant changes until the end of the first postnatal week. Ca2(+)-independent voltage-activated potassium current densities then increased strongly from postnatal day 8 onwards. Tetraethylammonium-sensitive current density increased about eightfold from 74 pA/pF in embryonic stages to 586 pA/pF in adult cells, whereas the transient potassium currents blocked by 4-aminopyridine increased only about 2.5-fold from 174 pA/pF to 442 pA/pF. The Ba2(+)-sensitive current increased simultaneously from 35 pA/pF to 332 pA/pF. The much higher increase in the sustained current components during retinal ganglion cell differentiation accounted for the changes in decay kinetics of Ca2(+)-independent voltage-activated potassium current observed in later postnatal stages. Alterations in current densities were paralleled by pronounced changes in current kinetics. From postnatal day 8 onwards, activation of Ca2(+)-independent voltage-activated potassium current was right-shifted for about 10 mV owing to a shift in tetraethylammonium-sensitive current-activation, whereas activation of other K+ components remained unaltered. Tetraethylammonium-sensitive current steady-state inactivation was incomplete at all developmental stages. About 50% of the tetraethylammonium-sensitive current elicited by a depolarization to +36 mV did not inactivate after prepulse potentials positive to -10 mV. In contrast, transient potassium current blocked by 4-aminopyridine almost fully inactivated during embryonic stages, whereas in adult retinal ganglion cells about 40% of this current component did not inactivate after prepulse potentials positive to -20 mV. Parallel investigation of the resting membrane potential during retinal ganglion cells differentiation showed an exponential increase from -3 mV at embryonic day 15/16 when no voltage-activated ion currents were expressed to a final value of -58 mV at postnatal day 8. These results show that fundamental potassium current modifications occur relatively late in retinal ganglion cell development and only after the resting potential is at its final value.     

Nitric oxide modulates Ca(2+) channels in dorsal root ganglion neurons innervating rat urinary bladder.

The effect of a nitric oxide (NO) donor on high-voltage-activated Ca(2+) channel currents (I(Ca)) was examined using the whole cell patch-clamp technique in L(6)-S(1) dorsal root ganglion (DRG) neurons innervating the urinary bladder. The neurons were labeled by axonal transport of a fluorescent dye, Fast Blue, injected into the bladder wall. Approximately 70% of bladder afferent neurons exhibited tetrodotoxin (TTX)-resistant action potentials (APs), and 93% of these neurons were sensitive to capsaicin, while the remaining neurons had TTX-sensitive spikes and were insensitive to capsaicin. The peak current density of nimodipine-sensitive L-type Ca(2+) channels activated by depolarizing pulses (0 mV) from a holding potential of -60 mV was greater in bladder afferent neurons with TTX-resistant APs (39.2 pA/pF) than in bladder afferent neurons with TTX-sensitive APs (28.9 pA/pF), while the current density of omega-conotoxin GVIA-sensitive N-type Ca(2+) channels was similar (43-45 pA/pF) in both types of neurons. In both types of neurons, the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) (500 microM), reversibly reduced (23.4-26.6%) the amplitude of I(Ca) elicited by depolarizing pulses to 0 mV from a holding potential of -60 mV. SNAP-induced inhibition of I(Ca) was reduced by 90% in the presence of omega-conotoxin GVIA but was unaffected in the presence of nimodipine, indicating that NO-induced inhibition of I(Ca) is mainly confined to N-type Ca(2+) channels. Exposure of the neurons for 30 min to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), an inhibitor of NO-stimulated guanylyl cyclase, prevented the SNAP-induced reduction in I(Ca). Extracellular application of 8-bromo-cGMP (1 mM) mimicked the effects of NO donors by reducing the peak amplitude of I(Ca) (28.6% of reduction). Action potential configuration and firing frequency during depolarizing current pulses were not altered by the application of SNAP (500 microM) in bladder afferent neurons with TTX-resistant and -sensitive APs. These results indicate that NO acting via a cGMP signaling pathway can modulate N-type Ca(2+) channels in DRG neurons innervating the urinary bladder.     

Nitric oxide is an autocrine regulator of Na(+) currents in axotomized C-type DRG neurons

In this study, we examined whether nitric oxide synthase (NOS) is upregulated in small dorsal root ganglion (DRG) neurons after axotomy and, if so, whether the upregulation of NOS modulates Na(+) currents in these cells. We identified axotomized C-type DRG neurons using a fluorescent label, hydroxystilbamine methanesulfonate and found that sciatic nerve transection upregulates NOS activity in 60% of these neurons. Fast-inactivating tetrodotoxin-sensitive (TTX-S) Na(+) ("fast") current and slowly inactivating tetrodotoxin-resistant (TTX-R) Na(+) ("slow") current were present in control noninjured neurons with current densities of 1.08 +/- 0. 09 nA/pF and 1.03 +/- 0.10 nA/pF, respectively (means +/- SE). In some control neurons, a persistent TTX-R Na(+) current was observed with current amplitude as much as approximately 50% of the TTX-S Na(+) current amplitude and 100% of the TTX-R Na(+) current amplitude. Seven to 10 days after axotomy, current density of the fast and slow Na(+) currents was reduced to 0.58 +/- 0.05 nA/pF (P < 0.01) and 0.2 +/- 0.05 nA/pF (P < 0.001), respectively. Persistent TTX-R Na(+) current was not observed in axotomized neurons. Nitric oxide (NO) produced by the upregulation of NOS can block Na(+) currents. To examine the role of NOS upregulation on the reduction of the three types of Na(+) currents in axotomized neurons, axotomized DRG neurons were incubated with 1 mM N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. The current density of fast and slow Na(+) channels in these neurons increased to 0.82 +/- 0.08 nA/pF (P < 0.01) and 0.34 +/- 0.04 nA/pF (P < 0.05), respectively. However, we did not observe any persistent TTX-R current in axotomized neurons incubated with L-NAME. These results demonstrate that endogenous NO/NO-related species block both fast and slow Na(+) current in DRG neurons and suggest that NO functions as an autocrine regulator of Na(+) currents in injured DRG neurons.     

Effects of caffeine on potassium currents in isolated rat ventricular myocytes

Rapid exposure of cardiac muscle to high concentrations of caffeine releases Ca(2+) from the sarcoplasmic reticulum (SR). This Ca(2+) is then extruded from the cell by the Na(+)/Ca(2+) exchanger. Measurement of the current carried by the exchanger (I(Na/Ca)) can therefore be used to estimate of the Ca(2+) content of the SR. Previous studies have shown that caffeine, however, can also inhibit K(+) currents. We therefore investigated whether the inhibitory effects of caffeine on these currents could contaminate measurements of I(Na/Ca). Caffeine caused partial inhibition of the inward rectifier K(+) current (I(K1)): the outward current at -40 mV was 1.15+/-0.24 pA/pF in control and decreased to 0.34+/-0.15 pA/pF in the presence of 10 mmol/l caffeine (P<0.05, n=15). This was similar to the effect of caffeine on the holding current observed at -40 mV in the absence of K(+) channel block and could therefore account for the contaminating effects of caffeine observed during measurements of I(Na/Ca). Moreover, caffeine also partially inhibited the transient outward ( I(to)) and the delayed rectifier (I(K)) K(+) currents.     

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.     

Reduction in potassium currents in identified cutaneous afferent dorsal root ganglion neurons after axotomy.

Potassium currents have an important role in modulating neuronal excitability. We have investigated the effects of axotomy on three voltage-activated K(+) currents, one sustained and two transient, in cutaneous afferent dorsal root ganglion (DRG) neurons. Fourteen to 21 days after axotomy, L(4) and L(5) DRG neurons were acutely dissociated and were studied 2-8 h after plating. Whole cell patch-clamp recordings were obtained from identified cutaneous afferent neurons (46-50 microm diam); K(+) currents were isolated by blocking Na(+) and Ca(2+) currents with appropriate ion replacement and channel blockers. Separation of the current components was achieved on the basis of sensitivity to dendrotoxin or 4-aminopyridine and by the response to variation in conditioning voltage. Both control and injured neurons displayed qualitatively similar complex K(+) currents composed of distinct kinetic and pharmacological components. Three distinct K(+) current components, a sustained (I(K)) and two transient (I(A) and I(D)), were identified in variable proportions. However, total peak current was reduced by 52% in the axotomized cells when compared with control cells. Two current components were reduced after ligation, I(A) by 60%, I(K) by over 65%, compared with control cells. I(D) appeared unaffected after acute ligation. These results indicate a large reduction in overall K(+) current, resulting from reductions in I(K) and I(A), on large cutaneous afferent neurons after nerve ligation and have implications for excitability changes of injured primary afferent neurons.     

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.     

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.     

On the relationship between\dot V_{\max } of slow responses and Ca-current availability in whole-cell clamped guinea pig heart cells

The relationship between Ca current availability and maximum rate of rise (V max) of slow responses was determined in the same single guinea pig ventricular heart cell under voltage and current clamp conditions (whole-cell clamp technique). The results are as follows. (1) Cell capacitance measured in 32 cells from the current response to a fast ramp voltage-clamp pulse (119.6 +/- 4.6 pF, mean +/- SE) or from Vmax values at a holding potential of -50 or -40 mV (118.6 +/- 5.3 pF) are identical. (2) In control conditions ([Ca]o 1.8, [K]o 4 and [Cs]i 140 mM), voltage-dependence of steady-state inactivation of Ca current (ICa) or Vmax are similar up to -35 mV. However, Vmax significantly (P less than 0.005) underestimates ICa availability at more positive potentials. At -30 mV, ICa and Vmax amplitudes represent respectively 35.6 and 22.4% (n = 14) of their maximum value. (3) In the presence of 50 nM isoprenaline, Vmax and the underlying ICa are respectively increased by 79.2 +/- 13.8% and 71.2 +/- 13.8% (n = 15). No statistically significant deviation from linearity is then observed. (4) When Vmax amplitude is expressed as a function of ICa density, an almost linear relationship is observed for Vmax values between 0 and 25 V/s. Vmax is then best described by the equation: Vmax (V/s) = 1.043 ICa (pA/pF) -0.514 (46 cells). (5) We conclude that, under conditions that minimize outward currents, Vmax of slow responses accurately measures ICa amplitude, except when ICa is decreased to less than 40% of its maximum control amplitude (i.e., below 4 pA/pF). At that point, Vmax underestimates ICa.     

Localized IP3-evoked Ca2+ release activates a K+ current in primary vagal sensory neurons

Electrophysiological and microfluorimetric techniques were used to determine whether intracellular photorelease of caged IP(3), and the consequent release of Ca(2+), could trigger a Ca(2+)-activated K(+) current (I(IP3)). Photorelease of caged IP(3) evoked an I(IP3) that averaged 2.36 +/- 0.35 (SE) pA/pF in 24 of 28 rabbit primary vagal sensory neurons (nodose ganglion neurons, NGNs) voltage-clamped at -50 mV. I(IP3) was abolished by intracellular BAPTA (2 mM), a Ca(2+) chelator. Changing the K(+) equilibrium potential by increasing extracellular K(+) ion concentration caused a predicted Nernstian shift in the reversal potential of I(IP3). These results indicated that I(IP3) was a Ca(2+)-dependent K(+) current. I(IP3) was unaffected by three common antagonists of Ca(2+)-activated K(+) currents: bath-applied iberiotoxin (50 nM) or apamin (100 nM), and intracellular 8-Br-cAMP (100 microM) included in the patch pipette. We have previously demonstrated that both IP(3)-evoked Ca(2+) release and Ca(2+)-induced Ca(2+) release (CICR) are co-expressed in NGNs and that CICR can trigger a Ca(2+)-activated K(+) current. In the present study, using caffeine, a CICR agonist, to selectively attenuate intracellular Ca(2+) stores, we showed that IP(3)-evoked Ca(2+) release occurs independently of CICR, but interestingly, that a component of I(IP3) requires CICR. These data suggest that IP(3)-evoked Ca(2+) release activates a K(+) current that is pharmacologically distinct from other Ca(2+)-activated K(+) currents in NGNs. We describe several models that explain our results based on Ca(2+) signaling microdomains in NGNs.     

Upregulation of the T-type calcium current in small rat sensory neurons after chronic constrictive injury of the sciatic nerve

Recent data indicate that peripheral T-type Ca2+ channels are instrumental in supporting acute pain transmission. However, the function of these channels in chronic pain processing is less clear. To address this issue, we studied the expression of T-type Ca2+ currents in small nociceptive dorsal root ganglion (DRG) cells from L4-5 spinal ganglia of adult rats with neuropathic pain due to chronic constrictive injury (CCI) of the sciatic nerve. In control rats, whole cell recordings revealed that T-type currents, measured in 10 mM Ba2+ as a charge carrier, were present in moderate density (20 +/- 2 pA/pF). In rats with CCI, T-type current density (30 +/- 3 pA/pF) was significantly increased, but voltage- and time-dependent activation and inactivation kinetics were not significantly different from those in controls. CCI-induced neuropathy did not significantly change the pharmacological sensitivity of T-type current in these cells to nickel. Collectively, our results indicate that CCI-induced neuropathy significantly increases T-type current expression in small DRG neurons. Our finding that T-type currents are upregulated in a CCI model of peripheral neuropathy and earlier pharmacological and molecular studies suggest that T-type channels may be potentially useful therapeutic targets for the treatment of neuropathic pain associated with partial mechanical injury to the sciatic nerve.     

Outwards currents in embryonic stem cell-derived cardiomyocytes

The aim of the present study was to investigate the expression and functional role of outwards currents during the early stages of cardiomyogenesis. The predominant repolarizing current in early-stage, embryonic stem (ES) cell-derived cardiomyocytes was a 4-aminopyridine (4-AP) sensitive [concentration for half-maximal inhibition (IC50) 1.7 mM], transient outward current (Ito) with a current density of 10.3+/-2.1 pA/pF (n=72). We observed two additional, rapidly activating, outwardly rectifying current components, I(K),sus and Ires, in early- and late-stage cardiomyocytes. These currents were characterized by slow and no inactivation, respectively, during the depolarizing voltage step. I(K),sus was detected in about 25% of cells investigated and displayed 4-AP hypersensitivity (IC50 29 microM), whereas Ires was found in all cells of both differentiation stages and was 4-AP insensitive. In contrast to early-stage cells, Ires formed the larger portion of the aggregate, whole-cell current in late-stage, ES cell-derived cardiomyocytes. The current densities of all three current components increased during development, however, the most prominent increase was observed for I(res) from 3.6+/-0.8 pA/pF (n=72) to 8+/-1.1 pA/pF (n=35). In current-clamp recordings in early-stage, spontaneously contracting cardiomyocytes, 4-AP depolarized the cells, lengthened the action potential duration (APD) and increased the action potential frequency. In late-stage cells 4-AP had no effect on action potential frequency. We conclude that in early-stage cardiomyocytes I(to) plays an important role in controlling electrical activity.     

Prostaglandin E(2) modulates TTX-R I(Na) in rat colonic sensory neurons

This study was performed to determine the impact of the inflammatory mediator prostaglandin E(2) (PGE(2)) on the biophysical properties of tetrodotoxin resistant voltage-gated Na(+) currents (TTX-R I(Na)) in colonic dorsal root ganglion (DRG) neurons. TTX-R I(Na) was studied in DRG neurons from thoracolumbar (TL: T(13)-L(2)) and lumbosacral (LS: L(6)-S(2)) DRG retrogradely labeled following the injection of DiIC(18) (DiI) into the wall of the descending colon of adult male rats. TTX-R I(Na) in colonic DRG neurons had a high threshold for activation [V(0.5) of conductance-voltage (G-V) curve = -3.1 +/- 1.0 (SE) mV] and steady-state availability (V(0.5) for H-infinity curve = -18.4 +/- 1.4 mV), was slowly inactivating (10.6 +/- 1.4 ms at 0 mV), and recovered rapidly from inactivation (83.5 +/- 5.0% of the current recovered with a time constant of 1.3 +/- 0.1 ms at -80 mV). TTX-R I(Na) was present in every colonic DRG neuron studied (n = 62). PGE(2) induced a rapid (<15 s) increase in TTX-R I(Na) that was associated with a hyperpolarizing shift in the G-V curve (3.4 +/- 0.7 mV), an increase in the rate of inactivation (4.21 +/- 0.7 ms at 0 mV), and no change in steady-state availability. There was no statistically significant difference (P > 0.05) between TL and LS colonic DRG neurons with respect to the biophysical properties of TTX-R I(Na), the current density or the magnitude of PGE(2)-induced changes in the current. However, both the proportion of TL and LS neurons in which TTX-R I(Na) was modulated by PGE(2) (16 of 16 TL neurons and 12 of 14 LS neurons) as well as the magnitude of PGE(2)-induced changes in the current were significantly larger in colonic DRG neurons than in the total population of DRG neurons. These results suggest that changes in nociceptive processing associated with inflammation of the colon does not reflect differences between TL and LS neurons with respect to the properties of TTX-R I(Na), distribution of current, or magnitude of inflammatory mediator-induced changes in the current. However, these results do suggest modulation of TTX-R I(Na) in colonic afferents is an underlying mechanism of hyperalgesia and pain associated with inflammation of the colon and that this current constitutes a novel target for therapeutic relief of visceral inflammatory pain.     

Postnatal development of voltage-gated K currents on rat sympathetic neurons.

1. We have investigated the developmental expression of three voltage-gated K currents on neonatal rat superior cervical ganglion (SCG) neurons in vivo and in culture: a rapidly inactivating current (IAf), a slowly inactivating current (IAs), and a noninactivating current (IK). 2. On postnatal day 1 neurons (P1), mean peak IAs is 67 +/- 4 (SE) pA/pF, peak IAf is 27 +/- 3 pA/pF, and IK is 14 +/- 3 pA/pF. Over the next wk, there is a switch in the expression of these currents: IAs drops by 40%, whereas IAf increases by greater than 100%; there is no change in IK. On P14 neurons, IAs is 38 +/- 2 pA/pF, IAf is 64 +/- 5 pA/pF, and IK is 12 +/- 1 pA/pF. 3. The change in expression of K currents on SCG neurons over the first 2 postnatal wk is unaffected by preganglionic innervation or by innervation of the targets. 4. To learn more about the factors that affect K current expression on these neurons, we grew SCG neurons in culture without other cell types for various times, and we measured the expression of IAf, IAs, and IK. In culture, the currents remained at their P1 levels for the first 4-7 days. Thereafter, both IAs and IAf decreased to low levels over a period of 2-3 wk. These results suggest that an epigenetic factor(s) is necessary for the expression of IAf and IAf in vivo and that this factor is missing in culture. 5. When IAs and IAf decreased on neurons in culture, we observed a compensatory increase in IK. After 4 wk in culture, IK is fourfold greater than on neurons in vivo. This result suggests that these neurons have intrinsic mechanisms that coordinate the expression of different voltage-gated K currents.