Functional modulation of human brain Nav1.3 sodium channels,expressed in mammalian cells,by auxiliary beta 1, beta 2 and beta 3 subunits

Voltage-gated sodium channels consist of a pore-forming alpha subunit and two auxiliary beta subunits. Excitable cells express multiple alpha subtypes, designated Na(v)1.1-Na(v)1.9, and three beta subunits, designated beta1, beta2 and beta3. Understanding how the different alpha subtypes, in combination with the various beta subunits, determine sodium channel behavior is important for elucidating the molecular basis of sodium channel functional diversity. In this study, we used whole-cell electrophysiological recording to examine the properties of the human Na(v)1.3 alpha subtype, stably expressed in Chinese hamster ovary cells, and to investigate modulation of Na(v)1.3 function by beta1, beta2 and beta3 subunits. In the absence of beta subunits, human Na(v)1.3 formed channels that inactivated rapidly (tau(inactivation) approximately equals 0.5 ms at 0 mV) and almost completely by the end of 190-ms-long depolarizations. Using an intracellular solution with aspartate as the main anion, the midpoint for channel activation was approximately -12 mV. The midpoint for inactivation, determined using 100-ms conditioning pulses, was approximately -47 mV. The time constant for repriming of inactivated channels at -80 mV was approximately 6 ms. Coexpression of beta1 or beta3 did not affect inactivation time course or the voltage dependence of activation, but shifted the inactivation curve approximately 10 mV negative, and slowed the repriming rate ca. three-fold. beta2 did not affect channel properties, either by itself or in combination with beta1 or beta3. Na(v)1.3 expression is increased in damaged nociceptive peripheral afferents. This change in channel expression levels is correlated with the emergence of a rapidly inactivating and rapidly repriming sodium current, which has been proposed to contribute to the pathophysiology of neuropathic pain. The results of this study support the hypothesis that Na(v)1.3 may mediate this fast sodium current.     

The modulation effects of BmK I, an alpha-like scorpion neurotoxin, on voltage-gated Na(+) currents in rat dorsal root ganglion neurons

The present study investigated the effects of BmK I, a Na(+) channel receptor site 3 modulator purified from the Buthus martensi Karsch (BmK) venom, on the voltage-gated sodium currents in dorsal root ganglion (DRG) neurons. Whole-cell patch-clamping was used to record the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) components of voltage-gated Na(+) currents in small DRG neurons. It was found that the inhibitory effect of BmK I on open-state inactivation of TTX-S Na(+) currents was stronger than that of TTX-R Na(+) currents. In addition, BmK I exhibited a selective enhancing effect on voltage-dependent activation of TTX-S currents, and an opposite effect on time-dependent activation of TTX-S and TTX-R Na(+) currents. The results suggested that the inhibitory effect of BmK I on open-state inactivation might contribute to the increase of peak TTX-S and TTX-R currents, and the enhancing effect of BmK I on time-dependent activation might also contribute to the increase of peak TTX-S currents. It was further suggested that a combined effect of BmK I including inhibiting the inactivation of TTX-S and TTX-R channels, accelerating activation and decreasing the activation threshold of TTX-S channels, might produce a hyperexcitability of small DRG neurons, and thus contribute to the BmK I-induced hyperalgesia.     

Voltage-dependent Na(+) currents in mammalian retinal cone bipolar cells

Voltage-dependent Na(+) channels are usually expressed in neurons that use spikes as a means of signal coding. Retinal bipolar cells are commonly thought to be nonspiking neurons, a category of neurons in the CNS that uses graded potential for signal transmission. Here we report for the first time voltage-dependent Na(+) currents in acutely isolated mammalian retinal bipolar cells with whole cell patch-clamp recordings. Na(+) currents were observed in approximately 45% of recorded cone bipolar cells but not in rod bipolar cells. Both ON and OFF cone bipolar cells were found to express Na(+) channels. The Na(+) currents were activated at membrane potentials around -50 to -40 mV and reached their peak around -20 to 0 mV. The half-maximal activation and steady-state inactivation potentials were -24.7 and -68.0 mV, respectively. The time course of recovery from inactivation could be fitted by two time constants of 6.2 and 81 ms. The amplitude of the Na(+) currents ranged from a few to >300 pA with the current density in some cells close or comparable to that of retinal third neurons. In current-clamp recordings, Na(+)-dependent action potentials were evoked in Na(+)-current-bearing bipolar cells by current injections. These findings raise the possibility that voltage-dependent Na(+) currents may play a role in bipolar cell function.     

Characteristics of late Na(+) current in adult rat small sensory neurons

Na(+) currents were recorded using patch-clamp techniques from small-diameter (<25 micrometers) dorsal root ganglion neurons, cultured from adult rats (>150 g). Late Na(+) currents maintained throughout long-duration voltage-clamp steps (>/=200 ms) were of two types: a low-threshold, tetrodotoxin-sensitive (TTX-s) current that was largely blocked by 200 nM TTX, and a high-threshold, TTX-resistant (TTX-r) current. TTX-s late current was found in approximately 28% (10/36) of small-diameter neurons and was recorded only in neurons exhibiting TTX-s transient current. TTX-s transient current activation/inactivation gating overlap existed over a narrow potential range, centered between -30 and -40 mV, whereas late current operated over a wider range. The kinetics associated with de-inactivation of TTX-s late current were slow (tau approximately 37 ms at -50 mV), strongly suggesting that different subpopulations of TTX-s channel generate transient and late current. High-threshold TTX-r late current was only present in neurons generating TTX-r transient current. TTX-r late current operated over the same potential range as that for TTX-r transient current activation/inactivation gating overlap, and activation/inactivation gating overlap could be measured even after 1.5-s-duration pre-pulses.We suggest that TTX-s late sodium current results from channel openings different from those generating transient current. As in large-diameter sensory neurons, TTX-s channels generating late openings may play a key role in controlling membrane excitability. In contrast, a single population of high-threshold TTX-r channels may account for both transient and late TTX-r currents.     

Regulation of Nav1.6 and Nav1.8 peripheral nerve Na+ channels by auxiliary β-subunits

Voltage-gated Na(+) (Na(v)) channels are composed of a pore-forming α-subunit and one or more auxiliary β-subunits. The present study investigated the regulation by the β-subunit of two Na(+) channels (Na(v)1.6 and Na(v)1.8) expressed in dorsal root ganglion (DRG) neurons. Single cell RT-PCR was used to show that Na(v)1.8, Na(v)1.6, and β(1)-β(3) subunits were widely expressed in individually harvested small-diameter DRG neurons. Coexpression experiments were used to assess the regulation of Na(v)1.6 and Na(v)1.8 by β-subunits. The β(1)-subunit induced a 2.3-fold increase in Na(+) current density and hyperpolarizing shifts in the activation (-4 mV) and steady-state inactivation (-4.7 mV) of heterologously expressed Na(v)1.8 channels. The β(4)-subunit caused more pronounced shifts in activation (-16.7 mV) and inactivation (-9.3 mV) but did not alter the current density of cells expressing Na(v)1.8 channels. The β(3)-subunit did not alter Na(v)1.8 gating but significantly reduced the current density by 31%. This contrasted with Na(v)1.6, where the β-subunits were relatively weak regulators of channel function. One notable exception was the β(4)-subunit, which induced a hyperpolarizing shift in activation (-7.6 mV) but no change in the inactivation or current density of Na(v)1.6. The β-subunits differentially regulated the expression and gating of Na(v)1.8 and Na(v)1.6. To further investigate the underlying regulatory mechanism, β-subunit chimeras containing portions of the strongly regulating β(1)-subunit and the weakly regulating β(2)-subunit were generated. Chimeras retaining the COOH-terminal domain of the β(1)-subunit produced hyperpolarizing shifts in gating and increased the current density of Na(v)1.8, similar to that observed for wild-type β(1)-subunits. The intracellular COOH-terminal domain of the β(1)-subunit appeared to play an essential role in the regulation of Na(v)1.8 expression and gating.     

Electrophysiological characteristics of rat gustatory cyclic nucleotide--gated channel expressed in Xenopus oocytes

The complementary DNA encoding gustatory cyclic nucleotide--gated ion channel (or gustCNG channel) cloned from rat tongue epithelial tissue was expressed in Xenopus oocytes, and its electrophysiological characteristics were investigated using tight-seal patch-clamp recordings of single and macroscopic channel currents. Both cGMP and cAMP directly activated gustCNG channels but with markedly different affinities. No desensitization or inactivation of gustCNG channel currents was observed even in the prolonged application of the cyclic nucleotides. Single-channel conductance of gustCNG channel was estimated as 28 pS in 130 mM of symmetric Na(+). Single-channel current recordings revealed fast open-close transitions and longer lasting closure states. The distribution of both open and closed events could be well fitted with two exponential components and intracellular cGMP increased the open probability (P(o)) of gustCNG channels mainly by increasing the slower opening rate. Under bi-ionic conditions, the selectivity order of gustCNG channel among divalent cations was determined as Na(+) approximately K(+) > Rb(+) > Li(+) > Cs(+) with the permeability ratio of 1:0.95:0.74:0.63:0.49. Magnesium ion blocked Na(+) currents through gustCNG channels from both intracellular and extracellular sides in voltage-dependent manners. The inhibition constants (K(i)s) of intracellular Mg(2+) were determined as 360 +/- 40 microM at 70 mV and 8.2 +/- 1.5 mM at -70 mV with z delta value of 1.04, while K(i)s of extracellular Mg(2+) were as 1.1 +/- 0.3 mM at 70 mV and 20.0 +/- 0.1 microM at -70 mV with z delta of 0.94. Although 100 microM l-cis-diltiazem blocked significant portions of outward Na(+) currents through both bovine rod and rat olfactory CNG channels, the gustCNG channel currents were minimally affected by the same concentration of the drug.     

Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons

C-type dorsal root ganglion (DRG) neurons can generate tetrodotoxin-resistant (TTX-R) sodium-dependent action potentials. However, multiple sodium channels are expressed in these neurons, and the molecular identity of the TTX-R sodium channels that contribute to action potential production in these neurons has not been established. In this study, we used current-clamp recordings to compare action potential electrogenesis in Na(v)1.8 (+/+) and (-/-) small DRG neurons maintained for 2-8 h in vitro to examine the role of sodium channel Na(v)1.8 (alpha-SNS) in action potential electrogenesis. Although there was no significant difference in resting membrane potential, input resistance, current threshold, or voltage threshold in Na(v)1.8 (+/+) and (-/-) DRG neurons, there were significant differences in action potential electrogenesis. Most Na(v)1.8 (+/+) neurons generate all-or-none action potentials, whereas most of Na(v)1.8 (-/-) neurons produce smaller graded responses. The peak of the response was significantly reduced in Na(v)1.8 (-/-) neurons [31.5 +/- 2.2 (SE) mV] compared with Na(v)1.8 (+/+) neurons (55.0 +/- 4.3 mV). The maximum rise slope was 84.7 +/- 11.2 mV/ms in Na(v)1.8 (+/+) neurons, significantly faster than in Na(v)1.8 (-/-) neurons where it was 47.2 +/- 1.3 mV/ms. Calculations based on the action potential overshoot in Na(v)1.8 (+/+) and (-/-) neurons, following blockade of Ca(2+) currents, indicate that Na(v)1.8 contributes a substantial fraction (80-90%) of the inward membrane current that flows during the rising phase of the action potential. We found that fast TTX-sensitive Na(+) channels can produce all-or-none action potentials in some Na(v)1.8 (-/-) neurons but, presumably as a result of steady-state inactivation of these channels, electrogenesis in Na(v)1.8 (-/-) neurons is more sensitive to membrane depolarization than in Na(v)1.8 (+/+) neurons, and, in the absence of Na(v)1.8, is attenuated with even modest depolarization. These observations indicate that Na(v)1.8 contributes substantially to action potential electrogenesis in C-type DRG neurons.     

Isolation and kinetic analysis of inward currents in neuroblastoma cells

The suction pipette method for combined voltage clamp and intracellular dialysis was applied to isolate the two components of voltage-gated inward current across membranes of NIE-115 neuroblastoma cells. In order to analyze the kinetic behavior of the Na+ and Ca2+ channels responsible for generating these components, current through K+ channels was effectively blocked by substituting impermeant Cs+ for internal and external K+. Block was confirmed independently by examining the effects of the application of external tetraethylammonium or Cd2+; and comparing the time course of Ca2+ tail currents with the decay of current during a maintained depolarization. Na+ currents studied at 8-10 degrees C, developed as a fourth order process giving a maximum e-fold conductance change for a 3 mV depolarization, with half activation occurring at -10 mV. The instantaneous current-voltage relationship was linear. Time constants of the activation parameter (m) varied from 0.5 ms (-50 mV) to 3-4 ms (-10 to -40 mV) at 10 degrees C. Inactivation (h) was a first order process having a time constant between 4 ms (+10 to +60 mV) and 225 ms (-60 mV). Steady-state inactivation for Na+ channels attained a value of 0.5 at -50 mV. A slow inactivation process, however, also is involved in gating of Na+ channels, and has a time course at least two orders of magnitude slower than that for h. The temperature sensitivity of Na+ currents was found to be similar to that found for other preparations. Ca2+ currents were studied at 24-29 degrees C in the presence of internal ethyleneglycolbis-(aminoethylether)-tetra-acetate (EGTA) and an external Ca2+ concentration of 20 mM. Ca2+ channel activation could also be described by a fourth order process giving an e-fold conductance change for a 5-6 mV change in potential and the half activation potential of -13 mV. Internal EGTA (20 mM) did not abolish inactivation of Ca2+ currents and no recovery from inactivation caused by a prepulse could be measured as the prepulse potential approached the null potential for Ca2+ influx. Time constants of both activation and inactivation of Ca2+ channels were measured between -20 and +50 mV. Currents through K+ channels could be completely eliminated by substitution of K+ with Cs+, although a residual non-linear leakage current remained, in addition to currents through the Na+ and Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Voltage-gated potassium channels activated during action potentials in layer V neocortical pyramidal neurons

To investigate voltage-gated potassium channels underlying action potentials (APs), we simultaneously recorded neuronal APs and single K(+) channel activities, using dual patch-clamp recordings (1 whole cell and 1 cell-attached patch) in single-layer V neocortical pyramidal neurons of rat brain slices. A fast voltage-gated K(+) channel with a conductance of 37 pS (K(f)) opened briefly during AP repolarization. Activation of K(f) channels also was triggered by patch depolarization and did not require Ca(2+) influx. Activation threshold was about -20 mV and inactivation was voltage dependent. Mean duration of channel activities after single APs was 6.1 +/- 0.6 ms (mean +/- SD) at resting membrane potential (-64 mV), 6.7 +/- 0.7 ms at -54 mV, and 62 +/- 15 ms at -24 mV. The activation and inactivation properties suggest that K(f) channels function mainly in AP repolarization but not in regulation of firing. K(f) channels were sensitive to a low concentration of tetraethylammonium (TEA, 1 mM) but not to charybdotoxin (ChTX, 100 nM). Activities of A-type channels (K(A)) also were observed during AP repolarization. K(A) channels were activated by depolarization with a threshold near -45 mV, suggesting that K(A) channels function in both repolarization and timing of APs. Inactivation was voltage dependent with decay time constants of 32 +/- 6 ms at -64 mV (rest), 112 +/- 28 ms at -54 mV, and 367 +/- 34 ms at -24 mV. K(A) channels were localized in clusters and were characterized by steady-state inactivation, multiple subconductance states (36 and 19 pS), and inhibition by 5 mM 4-aminopyridine (4-AP) but not by 1 mM TEA. A delayed rectifier K(+) channel (K(dr)) with a unique conductance of 17 pS was recorded from cell-attached patches with TEA/4-AP-filled pipettes. K(dr) channels were activated by depolarization with a threshold near -25 mV and showed delayed long-lasting activation. K(dr) channels were not activated by single action potentials. Large conductance Ca(2+)-activated K(+) (BK) channels were not triggered by neuronal action potentials in normal slices and only opened as neuronal responses deteriorated (e.g., smaller or absent spikes) and in a spike-independent manner. This study provides direct evidence for different roles of various K(+) channels during action potentials in layer V neocortical pyramidal neurons. K(f) and K(A) channels contribute to AP repolarization, while K(A) channels also regulate repetitive firing. K(dr) channels also may function in regulating repetitive firing, whereas BK channels appear to be activated only in pathological conditions.     

Identification of a novel voltage-gated Na+ channel rNav1.5a in the rat hippocampal progenitor stem cell line HiB5

A conditionally immortalised cell line, HiB5, derived from embryonic hippocampal precursor cells expressed a voltage-gated Na+ channel with electrophysiological characteristics shifted to more negative voltages and a lower sensitivity to tetrodotoxin [concentration required for half-maximal inhibition (IC50) 0.9 microM] compared with endogenous neuronal Na+ channels. The channel activation and steady-state inactivation occurred at very negative potentials with the threshold for activation at -55 mV and half-maximal inactivation at -78.7 mV. The channel was blocked by lamotrigine and sipatrigine voltage and state dependently, with potencies 5-20 times higher (IC50 12 and 1.8 microM at -80 mV respectively) than the corresponding block of endogenous Na+ channels from neurones and cloned rNa(v)1.2a (rBIIA) alpha-subunits. Both compounds slowed the channel's recovery from inactivation. Whereas lamotrigine and sipatrigine had similar effects on the fast inactivated state, the effect of sipatrigine on the slow inactivation state was more pronounced, rendering this compound overall the more effective. The molecular subtype mainly expressed by HiB5 cells was identified using RT-PCR and was a novel splice variant of rNa(v)1.5 (rNa(v)1.5a). It differs from the known rNa(v)1.5 version in that it lacks 53 amino acids in the intracellular loop between domains II and III. rNa(v)1.5a was also detected in mRNA derived from rat whole brain.     

Membrane properties and electrogenesis in the distal axons of small dorsal root ganglion neurons in vitro

Although it is generally thought that sensory transduction occurs at or close to peripheral nerve endings, with action potentials subsequently propagating along the axons of dorsal root ganglia (DRG) neurons toward the central nervous system, the small diameter of nociceptive axons and their endings have made it difficult to estimate their membrane properties and electrogenic characteristics. Even the resting potentials of nociceptive axons are unknown. In this study, we developed the capability to record directly with patch-clamp electrodes from the small-diameter distal axons of DRG neurons in vitro. We showed using current-clamp recordings that 1) these sensory axons have a resting potential of -60.2 ± 1 mV; 2) both tetrodotoxin (TTX)-sensitive (TTX-S) and TTX-resistant (TTX-R) Na(+) channels are present and available for activation at resting potential, at densities that can support action potential electrogenesis in these axons; 3) TTX-sensitive channels contribute to the amplification of small depolarizations that are subthreshold with respect to the action potential in these axons; 4) TTX-R channels can support the production of action potentials in these axons; and 5) these TTX-R channels can produce repetitive firing, even at depolarized membrane potentials where TTX-S channels are inactivated. Finally, using voltage-clamp recordings with an action potential as the command, we confirmed the presence of both TTX-S and TTX-R channels, which are activated sequentially during action potential in these axons. These results provide direct evidence for the presence of TTX-S and TTX-R Na(+) channels that are functionally available at resting potential and contribute to electrogenesis in small-diameter afferent axons.     

Blocking and modifying actions of octanol on Na channels in frog myelinated nerve

The actions of externally applied n-octanol on Na channels in myelinated frog nerve fibres were studied under voltage clamp conditions. Upon octanol application peak Na inward currents declined in two phases: 90% of the reduction occurred in less than 2 min but a steady-state was reached only after 15 min. During washout the currents came to a stable level within 10 min. The reduction of Na inward currents by octanol was dependent on the amplitude and duration of prepotentials. At the resting potential (VH = 0 mV) 0.4 mM octanol reduced peak Na inward currents at V = 60 mV by 50%. After a prepulse of -60 mV and 50 ms duration Na currents decreased only by 20%. At a hyperpolarizing holding potential of VH = -28 mV 0.7 mM octanol reduced peak inward Na currents to one half. Octanol depressed Na currents at all potentials by approximately the same factor. The Na reversal potential VNa remained unchanged. 0.7 mM external octanol shifted the Na activation curve m infinity (V) by 5 mV to more positive and the inactivation curve h infinity (V) by 14 mV to more negative potentials. The midpoint slopes of both curves were reduced. The time constants of Na activation and inactivation at small depolarizations were decreased. The conductance gamma of a single Na channel and the number No of conducting Na channels per node were determined from nonstationary Na current fluctuations. 0.7 mM octanol increased gamma by a factor of 1.6 and reduced No by a factor of 0.34. It is concluded that octanol blocks some Na channels and modifies the remaining unblocked channels.     

Inactivation of cardiac Na+ channel simply through open states as revealed by single-channel analysis in guinea pig ventricular myocytes

Inactivation of the cardiac Na(+) channel was analyzed by recording channel currents from a cell-attached patch containing only one functional Na(+) channel in guinea-pig ventricular myocytes. A two-step test pulse, first to variable levels (Pulse 1) and then to -30 mV (Pulse 2) was applied from a holding potential of -140 mV. When a cumulative histogram was determined for the latency of first opening, the histogram was well fitted with a single exponential function at -70 to -30 mV of Pulse 1. The activation time course of ensemble average was virtually single exponential. Although the ensemble average of 500 sweeps showed various extents of inactivation during Pulse 1, the saturation level of the cumulative first-latency histogram at the end of the two-step pulse was almost constant (0.7-0.8), irrespective of Pulse 1. Even when the interval between successive test pulses was prolonged from 70 to 970 ms, the saturation level of the histogram was not modified. These findings are consistent with inactivation only through the open state. Thus, the apparent "blank sweep inactivation" does not necessarily indicate direct inactivation from closed states. These findings support the hypothesis that the inactivation of cardiac Na(+) channel occurs exclusively through the open state.     

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.     

Kinetics and Gβγ modulation of Cav2.2 channels with different auxiliary β subunits

Modulation of calcium channels by both auxiliary subunits and G proteins was studied in cell-attached patches from COS-7 cells transfected with Ca(v)2.2 channel subunits (N-type, alpha(1)B and either beta(1b) or beta(2a)). These were co-expressed with either Gbeta(1)gamma(2) or the Gbetagamma-binding domain of beta-adrenergic-receptor kinase-1 to sequester endogenous Gbetagamma. Since G protein modulation of Ca(v) channels may affect both inactivation and activation, we examined Gbetagamma modulation of Ca(v)2.2 channels in the presence of two different beta-subunits that affect inactivation differently and compared in detail the single-channel characteristics of N-type channels expressed with either of these beta-subunit isoforms. The single-channel mean amplitude and mean open time were not influenced by the transfection combination. However, the mean closed time at +40 mV was increased for both beta(1b) and beta(2a)-subunits by co-transfection with Gbeta(1)gamma(2). This effect was absent at lower voltages as examined for channels with the beta(1b)-subunit. The distribution of latency-to-first-opening of Ca(v)2.2 channels was similar for both beta-subunit isoforms. However, the inclusion of the beta(2a) subunit resulted in channels with an additional, prominent, slow activation phase. Co-transfection of Gbeta(1)gamma(2) with Ca(v)2.2 channels markedly reduced the ensemble current amplitude and slowed the first latency. The inhibition imposed by Gbeta(1)gamma(2) was largely independent of the beta-subunit species. Facilitation of Gbetagamma-modulated currents (the channel response following a large and brief depolarising prepulse) was observed for channels with both beta-subunits and involved mainly enhancement of the activation, as assessed by the faster first latency. The inactivation process was strongly dependent on the beta-subunit species, with beta(1b) supporting inactivation and beta(2a) reducing this process. This difference was assessed by estimation of both steady-state inactivation (prepulse influence on test pulse responses) and the inactivation time course during depolarisation. At +40 mV, channels with the beta(1b)-subunit had a fast component of inactivation (time constant ~180 ms, 50%) and a slow phase with time constant of approximately 1 s, while the beta(2a)-subunit supported only a very slow inactivation process with time constant of approximately 5 s. Co-transfection of Gbeta(1)gamma(2) with the Ca(v)2.2 channel had no effect on the inactivation properties with either beta-subunit. In summary, we show that the inactivation properties of expressed Ca(v)2.2 channels depend largely on the beta-subunit species and to a minor extent only on the presence or absence of the Gbetagamma modulator. Furthermore, the activation, amplitude, mean open and closed times and G protein modulation of N-type channels were similar for both beta(1b)- and beta(2a)-subunits.     

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.     

Properties and distribution of single voltage-gated calcium channels in adult hippocampal neurons.

1. The properties of single voltage-gated calcium channels were investigated in acutely exposed CA3 and CA1 pyramidal neurons and granule cells of area dentata in the adult guinea pig hippocampal formation. 2. Guinea pig hippocampal slices were prepared in a conventional manner, then treated with proteolytic enzymes and gently shaken to expose the somata of the three cell types studied. Standard patch-clamp techniques were used to record current flow through calcium channels in cell-attached membrane patches with isotonic barium as the charge carrier. 3. Single-channel current amplitudes were measured at different membrane potentials. Single-channel current-voltage plots were constructed and single-channel slope conductances were found to fall into three classes. These were (approximately) 8, 14, and 25 pS, and were observed in all three cell types. 4. The three groups of channels differed from each other in voltage dependence of activation: from a holding potential of -80, the small-conductance channel began to activate at about -40 to -30 mV, the medium-conductance channel at about -20 mV, and the large-conductance channel at approximately 0 mV. 5. Ensemble averages of single-channel currents during voltage steps revealed differences in voltage-dependent inactivation. The small-conductance channel inactivated completely within approximately 50 ms during steps from -80 to -10 mV or more positive. Steps to less positive potentials resulted in less inactivation. The medium-conductance channel displayed variable inactivation during steps from -80 to 0 mV. Inactivation of this channel during a 160-ms step ranged from virtually zero to approximately 100%. The large-conductance channel displayed no significant inactivation during steps as long as 400 ms. 6. The large-conductance channel was strikingly affected by the dihydropyridine agonist Bay K8644 (0.5-2.0 microM), resulting in a high probability of channel opening, prolonged openings, and an apparent increase in the number of channels available for activation. The medium and small-conductance channels were not noticeably affected by the drug. 7. The large-conductance channel could be induced to open at very negative membrane potentials by holding the patch for several seconds at 20 or 30 mV and stepping to -30 or -40 mV. This process was enhanced by Bay K8644, resulting in prolonged openings at potentials as negative as -100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Toxin γ of the scorpionTityus serrulatus modifies both activation and inactivation of sodium permeability of nerve membrane

The effects of the major neurotoxic fraction isolated from scorpion venom of Tityus serrulatus, TiTx gamma, on peripheral nerve membrane of Xenopus laevis were studied under current- and voltage-clamp conditions. 700 nmol/l TiTx gamma depolarized the membrane and induced spontaneous activity (150 s-1, maximum value), which ceased within a few minutes. It reduced the amplitude of the action potentials from 109 mV to 52 mV and increased their duration from 1.25 ms to 4.5 ms. 440 nmol/l TiTx gamma induced inward Na current flow at resting potential. The descending branch of the Na current-voltage curve was flattened and shifted approximately 10 mV to more negative potentials. Maximum Na permeability was reduced to about 20%. Both development of and recovery from inactivation of Na permeability were slowed. The steepness of the steady-state inactivation curve was decreased, but the mid-potential changed only insignificantly. No prepulse was necessary to elicit either a shift of activation or an inward current at resting potential. Expressing the toxin effect either in terms of the decrease of Na peak current or of the slowing of inactivation, half-maximum effects were found with 0.3 +/- 0.1 and 3.7 +/- 0.7 mumol/l TiTx gamma, respectively.