A-Type potassium currents active at subthreshold potentials in mouse cerebellar purkinje cells

Voltage-dependent and calcium-independent K⁺ currents were whole-cell recorded from cerebellar Purkinje cells in slices. Tetraethylammonium (TEA, 4 m m ) application isolated an A-type K⁺ current ( I _{k ( a )}) with a peak amplitude, at +20 mV, of about one third of the total voltage-dependent and calcium-independent K⁺ current. The I _{k ( a )} activated at about −60 mV, had a V _0.5 of activation of −24.9 mV and a V _0.5 of inactivation of −69.2 mV. The deactivation time constant at −70 mV was 3.4 ± 0.4 ms, while the activation time constant at +20 mV was 0.9 ± 0.2 ms. The inactivation kinetics was weakly voltage dependent, with two time constants; those at +20 mV were 19.3 ± 3.1 and 97.6 ± 9.8 ms. The recovery from inactivation had two time constants of 60.8 ms (78.4%) and 962.3 ms (21.6%). The I _{k ( a )} was blocked by 4-aminopyridine with an IC₅₀ of 67.6 μM. Agitoxin-2 (2 n m ) blocked 17.4 ± 2.1% of the I _{k ( a )}. Flecainide completely blocked the I _{k ( a )} with a biphasic effect with IC₅₀ values of 4.4 and 183.2 μM. In current-clamp recordings the duration of evoked action potentials was affected neither by agitoxin-2 (2 n m ) nor by flecainide (3 μM), but action potentials that were already broadened by TEA were further prolonged by 4-aminopyridine (100 μM). The amplitude of the hyperpolarisation at the end of depolarising steps was reduced by all these blockers.     

Selectivity and interactions of Ba2+ and Cs+ with wild-type and mutant TASK1 K+ channels expressed in Xenopus oocytes

The acid-sensitive K⁺ channel, TASK1 is a member of the K⁺-selective tandem-pore domain (K2P) channel family. Like many of the K2P channels, TASK1 is relatively insensitive to conventional channel blockers such as Ba²⁺. In this paper we report the impact of mutating the pore-neighbouring histidine residues, which are involved in pH sensing, on the sensitivity to blockade by Ba²⁺ and Cs⁺; additionally we compare the selectivity of these channels to extracellular K⁺, Na⁺ and Rb⁺. H98D and H98N mutants showed reduced selectivity for K⁺ over both Na⁺ and Rb⁺, and significant permeation of Rb⁺. This enhanced permeability must reflect changes in the structure or flexibility of the selectivity filter. Blockade by Ba²⁺ and Cs⁺ was voltage-dependent, indicating that both ions block within the pore. In 100 m m K⁺, the K _D at 0 mV for Ba²⁺ was 36 ± 10 m m  ( n = 6)  , whilst for Cs⁺ it was 20 ± 6.0 m m  ( n = 5)  . H98D was more sensitive to Ba²⁺ than the wild-type (WT); in addition, the site at which Ba²⁺ appears to bind was altered (WT: δ, 0.64 ± 0.16, n = 6; H98D: δ, 0.16 ± 0.03, n = 5, statistically different from WT; H98N: δ, 0.58 ± 0.09, not statistically different from WT). Thus, the pore-neighbouring residue H98 contributes not only to the pH sensitivity of TASK1, but also to the structure of the conduction pathway.     

Descending vasa recta pericytes express voltage operated Na+ conductance in the rat

We studied the properties of a voltage-operated Na⁺ conductance in descending vasa recta (DVR) pericytes isolated from the renal outer medulla. Whole-cell patch-clamp recordings revealed a depolarization-induced, rapidly activating and rapidly inactivating inward current that was abolished by removal of Na⁺ but not Ca⁺ from the extracellular buffer. The Na⁺ current ( I _Na) is highly sensitive to tetrodotoxin  (TTX, K _d= 2.2 n m )  . At high concentrations, mibefradil (10 μ m ) and Ni⁺ (1 m m ) blocked I _Na. I _Na was insensitive to nifedipine (10 μ m ). The L-type Ca⁺ channel activator FPL-64176 induced a slowly activating/inactivating inward current that was abolished by nifedipine. Depolarization to membrane potentials between 0 and 30 mV induced inactivation with a time constant of ∼1 ms. Repolarization to membrane potentials between −90 and −120 mV induced recovery from inactivation with a time constant of ∼11 ms. Half-maximal activation and inactivation occurred at −23.9 and −66.1 mV, respectively, with slope factors of 4.8 and 9.5 mV, respectively. The Na⁺ channel activator, veratridine (100 μ m ), reduced peak inward I _Na and prevented inactivation. We conclude that a TTX-sensitive voltage-operated Na⁺ conductance, with properties similar to that in other smooth muscle cells, is expressed by DVR pericytes.     

Cocaine binds to a common site on open and inactivated human heart (Nav1.5) sodium channels

The inhibition by cocaine of the human heart Na⁺ channel (Na_v1.5) heterologously expressed in Xenopus oocytes was investigated. Cocaine produced little tonic block of the resting channels but induced a characteristic, use-dependent inhibition during rapid, repetitive stimulation, suggesting that the drug preferentially binds to the open or inactivated states of the channel. To investigate further the state dependence, depolarizing pulses were used to inactivate the channels and promote cocaine binding. Cocaine produced a slow, concentration-dependent inhibition of inactivated channels, which had an apparent K _D of 3.4 μM. Mutations of the interdomain III-IV linker that remove fast inactivation selectively abolished this high-affinity component of cocaine inhibition, which appeared to be linked to the fast inactivation of the channels. A rapid component of cocaine inhibition persisted in the inactivation-deficient mutant that was enhanced by depolarization and was sensitive to changes in the concentration of external Na⁺, properties that are consistent with a pore-blocking mechanism. Cocaine induced a use-dependent inhibition of the non-inactivating mutant and delayed the repriming at hyperpolarized voltages, indicating that the drug slowly dissociated when the channels were closed. Mutation of a conserved aromatic residue (Y1767) of the D4S6 segment weakened both the inactivation-dependent and the pore-blocking components of the cocaine inhibition. The data indicate that cocaine binds to a common site located within the internal vestibule and inhibits cardiac Na⁺ channels by blocking the pore and by stabilizing the channels in an inactivated state.     

Tyrosine kinase and phosphatase regulation of slow delayed-rectifier K+ current in guinea-pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the regulation of the cardiac slowly activating delayed-rectifier K⁺ current ( I _Ks) that is important for action potential repolarization. Constitutive I _Ks recorded from guinea-pig ventricular myocytes was suppressed by broad-spectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC₅₀, 4.1 ± 0.6 μ m ), tyrphostin A25 (IC₅₀, 12.1 ± 2.1 μ m ) and genistein (IC₅₀, 64 ± 4 μ m ), but was relatively insensitive to the inactive analogues tyrphostin A1, tyrphostin A63, daidzein and genistin. I _Ks was unaffected by AG1478 (10 μ m ), an inhibitor of epidermal growth factor receptor TK, and was strongly suppressed by the Src TK inhibitor PP2 (10 μ m ) but not by the inactive analogue PP3 (10 μ m ). The results of experiments with forskolin, H89 and bisindolylmaleimide I indicate that the suppression of I _Ks by TK inhibitors was not mediated via inhibition of ( I _Ks-stimulatory) protein kinases A and C. To evaluate whether the suppression was related to lowered tyrosine phosphorylation, myocytes were pretreated with TK inhibitors and then exposed to the phosphotyrosyl phosphatase inhibitor orthovanadate (1 m m ). Orthovanadate almost completely reversed the suppression of I _Ks induced by broad-spectrum TK inhibitors at concentrations around their IC₅₀ values. We conclude that basal I _Ks is strongly dependent on tyrosine phosphorylation of Ks channel (or channel-regulatory) protein.     

Inhibition of HERG K+ Current and Prolongation of the Guinea-Pig Ventricular Action Potential by 4-Aminopyridine

4-Aminopyridine (4-AP) has been used extensively to study transient outward K⁺ current ( I _TO,1) in cardiac cells and tissues. We report here inhibition by 4-AP of HERG (the human ether-à-go-go -related gene) K⁺ channels expressed in a mammalian cell line, at concentrations relevant to those used to study I _TO,1. Under voltage clamp, whole cell HERG current ( I _HERG) tails following commands to +30 mV were blocked with an IC₅₀ of 4.4 ± 0.5 m m . Development of block was contingent upon HERG channel gating, with a preference for activated over inactivated channels. Treatment with 5 m m 4-AP inhibited peak I _HERG during an applied action potential clamp waveform by ∼59 %. It also significantly prolonged action potentials and inhibited resurgent I _K tails from guinea-pig isolated ventricular myocytes, which lack an I _TO,1. We conclude that by blocking the α-subunit of the I _Kr channel, millimolar concentrations of 4-AP can modulate ventricular repolarisation independently of any action on I _TO,1.     

Role of extracellular Ca2+ in gating of CaV1.2 channels

We examined changes in ionic and gating currents in Ca_V1.2 channels when extracellular Ca²⁺ was reduced from 10 m m to 0.1 μ m . Saturating gating currents decreased by two-thirds ( K _D≈ 40 μ m ) and ionic currents increased 5-fold ( K _D≈ 0.5 μ m ) due to increasing Na⁺ conductance. A biphasic time dependence for the activation of ionic currents was observed at low [Ca²⁺], which appeared to reflect the rapid activation of channels that were not blocked by Ca²⁺ and a slower reversal of Ca²⁺ blockade of the remaining channels. Removal of Ca²⁺ following inactivation of Ca²⁺ currents showed that Na⁺ currents were not affected by Ca²⁺-dependent inactivation. Ca²⁺-dependent inactivation also induced a negative shift of the reversal potential for ionic currents suggesting that inactivation alters channel selectivity. Our findings suggest that activation of Ca²⁺ conductance and Ca²⁺-dependent inactivation depend on extracellular Ca²⁺ and are linked to changes in selectivity.     

Proton modulation of ion channels in isolated horizontal cells of the goldfish retina

Transient changes in extracellular pH (pH_o) occur in the retina and may have profound effects on neurotransmission and visual processing due to the pH sensitivity of ion channels. The present study characterized the effects of acidification on the activity of membrane ion channels in isolated horizontal cells (HCs) of the goldfish retina using whole-cell patch-clamp recording. Currents recorded from HCs were characterized by prominent inward rectification at potentials negative to −80 mV, a negative slope conductance between −70 and −40 mV, a sustained inward current, and outward rectification positive to 40 mV. Inward currents were identified as those of inward rectifier K⁺ (Kir) channels and Ca²⁺ channels by their sensitivity to 10 m m Cs⁺ or 20 μ m Cd²⁺, respectively. Both of these currents were reduced when pH_o decreased from 7.8 to 6.8. Glutamate (1 m m )-activated currents were also identified, as were hemichannel currents that were enhanced by removal of extracellular Ca²⁺ and application of 1 m m quinidine. Both glutamate-activated and hemichannel currents were suppressed by a similar reduction of pH_o. When all of these H⁺-inhibited currents were blocked, a small, sustained inward current at −60 mV increased following a decrease in pH_o from 7.8 to 6.8. In addition, slope conductance between −70 and −20 mV increased during this acidification. Suppression of this H⁺-activated current by removal of extracellular Na⁺, and an extrapolated E _rev near E _Na, indicated that this current was carried predominantly by Na⁺ ions.     

Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current ( I _ti). I _ti is the result of three different [Ca]_i-sensitive currents: the Na⁺–Ca²⁺ exchange current, a Ca²⁺-activated chloride current and a Ca²⁺-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca²⁺-activated non-selective cation channel (NSC_Ca) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na⁺ and K⁺) and was slightly permeable to Ca²⁺ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K _d for [Ca²⁺]_i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC_Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca²⁺] overload and thus may be implicated in the genesis of arrhythmias.     

An amiloride-sensitive H+-gated Na+ channel in Caenorhabditis elegans body wall muscle cells

About 30 genes are predicted to encode degenerin/epithelial sodium channels (DEG/ENaCs) in Caenorhabditis elegans but the gating mode of these channels has not been determined. Using the whole-cell configuration of the patch-clamp technique in acutely dissected C. elegans , we investigated the effects of H⁺ as a potential activating factor of DEG/ENaCs on electrical properties of body wall muscle cells. Under current-clamp conditions, decreasing external pH from 7.2 to 6.1 led to a reversible depolarization of muscle cells associated with a decrease in input resistance which was partially inhibited by amiloride. Under voltage-clamp conditions, extracellular acidification activated an inward desensitizing current at −60 mV. In the absence of external Ca²⁺, H⁺-gated channels were found to be slightly more permeable to Na⁺ than to K⁺ and were blocked by amiloride with a K _0.5 of 31 μ m at −60 mV. An inward current could be also activated by protons in a GABA receptor null mutant in the presence of d -tubocurare and in an unc-105 null mutant. These results demonstrate that ion channels sharing common properties with mammalian acid-sensing ion channels (ASICs) are functional in C. elegans muscle which should prove useful for understanding proton sensing in animals.     

Cellular electrophysiology of canine pulmonary vein cardiomyocytes: action potential and ionic current properties

Pulmonary vein (PV) cardiomyocytes play an important role in atrial fibrillation; however, little is known about their specific cellular electrophysiological properties. We applied standard microelectrode recording and whole-cell patch-clamp to evaluate action potentials and ionic currents in canine PVs and left atrium (LA) free wall. Resting membrane potential (RMP) averaged -66 ± 1 mV in PVs and -74 ± 1 mV in LA ( P < 0.0001) and action potential amplitude averaged 76 ± 2 mV in PVs vs. 95 ± 2 mV in LA ( P < 0.0001). PVs had smaller maximum phase 0 upstroke velocity ( V _max: 98 ± 9 vs. 259 ± 16 V s⁻¹, P < 0.0001) and action potential duration (APD): e.g. at 2 Hz, APD to 90 % repolarization in PVs was 84 % of LA ( P < 0.05). Na⁺ current density under voltage-clamp conditions was similar in PV and LA, suggesting that smaller V _max in PVs was due to reduced RMP. Inward rectifier current density in the PV cardiomyocytes was ˜58 % that in the LA, potentially accounting for the less negative RMP in PVs. Slow and rapid delayed rectifier currents were greater in the PV (by ˜60 and ˜50 %, respectively), whereas transient outward K⁺ current and L-type Ca²⁺ current were significantly smaller (by ˜25 and ˜30 %, respectively). Na⁺-Ca²⁺-exchange (NCX) current and T-type Ca²⁺ current were not significantly different. In conclusion, PV cardiomyocytes have a discrete distribution of transmembrane ion currents associated with specific action potential properties, with potential implications for understanding PV electrical activity in cardiac arrhythmias.     

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage-gated Ca²⁺ channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated-patch clamp recordings and hormone-specific radioimmunoassays. Adenosine, but not adenosine 5'-triphosphate (ATP), dose-dependently and reversibly inhibited the transient component of the whole-terminal Ba²⁺ currents, with an IC₅₀ of 0.875 μ m. Adenosine strongly inhibited, in a dose-dependent manner (IC₅₀= 2.67 μ m ), depolarization-triggered AVP and OT release from isolated NH terminals. Adenosine and the N-type Ca²⁺ channel blocker ω-conotoxin GVIA, but not other Ca²⁺ channel-type antagonists, inhibited the same transient component of the Ba²⁺ current. Other components such as the L-, Q- and R-type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω-conotoxin GVIA were able to inhibit the same component of AVP release. A₁ receptor agonists, but not other purinoceptor-type agonists, inhibited the same transient component of the Ba²⁺ current as adenosine. Furthermore, the A₁ receptor antagonist 8-cyclopentyltheophylline (CPT), but not the A₂ receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A₁ receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A₁ receptors, specifically blocks the terminal N-type Ca²⁺ channel thus leading to inhibition of the release of both AVP and OT.     

Molecular correlates of the M-current in cultured rat hippocampal neurons

M-type K⁺ currents ( I _K(M)) play a key role in regulating neuronal excitability. In sympathetic neurons, M-channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K⁺ channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of I _K(M) in hippocampal pyramidal neurons cultured from 5- to 7-day-old rats. RT-PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single-cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M-channel blocker, fully inhibited I _K(M) with an IC₅₀ of 3.6 ± 1.5 μM. In 70 % of these cells, TEA fully suppressed I _K(M) with an IC₅₀ of 0.7 ± 0.1 m m . In the remaining cells, TEA maximally reduced I _K(M) by only 59.7 ± 5.2 % with an IC₅₀ of 1.4 ± 0.3 m m ; residual I _K(M) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to I _K(M) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.     

Passive water and urea permeability of a human Na+–glutamate cotransporter expressed in Xenopus oocytes

The human Na⁺-glutamate transporter (EAAT1) was expressed in Xenopus laevis oocytes. The passive water permeability, L _p, was derived from volume changes of the oocyte induced by changes in the external osmolarity. Oocytes were subjected to two-electrode voltage clamp. In the presence of Na⁺, the EAAT1-specific (defined in Discussion) L _p increased linearly with positive clamp potentials, the L _p being around 23 % larger at +50 mV than at –50 mV. l -Glutamate increased the EAAT1-specific L _p by up to 40 %. The K _0.5 for the glutamate-dependent increase was 20 ± 6 μ m , which is similar to the K _0.5 value for glutamate activation of transport. The specific inhibitor dl - threo -β-benzyloxyaspartate (TBOA) reduced the EAAT1-specific L _p to 72 %. EAAT1 supported passive fluxes of [¹⁴C]urea and [¹⁴C]glycerol. The [¹⁴C]urea flux was increased in the presence of glutamate. The data suggest that the permeability depends on the conformational equilibrium of the EAAT1. At positive potentials and in the presence of Na⁺ and glutamate, the pore is enlarged and water and urea penetrate more readily. The L _p was larger when measured with urea or glycerol as osmolytes as compared with mannitol. Apparently, the properties of the pore are not uniform along its length. The outer section may accommodate urea and glycerol in an osmotically active form, giving rise to larger water fluxes. The physiological role of EAAT1 for water homeostasis in the central nervous system is discussed.     

Protein kinase C mediates up-regulation of tetrodotoxin-resistant, persistent Na+ current in rat and mouse sensory neurones

The tetrodotoxin-resistant (TTX-r) persistent Na⁺ current, attributed to Na_V1.9, was recorded in small (< 25 μm apparent diameter) dorsal root ganglion (DRG) neurones cultured from P21 rats and from adult wild-type and Na_V1.8 null mice. In conventional whole-cell recordings intracellular GTP-γ-S caused current up-regulation, an effect inhibited by the PKC pseudosubstrate inhibitor, PKC19–36. The current amplitude was also up-regulated by 25 μ m intracellular 1-oleoyl-2-acetyl-sn-glycerol (OAG) consistent with PKC involvement. In perforated-patch recordings, phorbol 12-myristate 13-acetate (PMA) up-regulated the current, whereas membrane-permeant activators of protein kinase A (PKA) were without effect. PGE₂ did not acutely up-regulate the current. Conversely, both PGE₂ and PKA activation up-regulated the major TTX-r Na⁺ current, Na_V1.8. Extracellular ATP up-regulated the persistent current with an average apparent K _d near 13 μ m , possibly consistent with P2Y receptor activation. Numerical simulation of the up-regulation qualitatively reproduced changes in sensory neurone firing properties. The activation of PKC appears to be a necessary step in the GTP-dependent up-regulation of persistent Na⁺ current.     

ATP modulates intracellular Ca2+ and firing rate through a P2Y1 purinoceptor in cane toad pacemaker cells

Bipolar sensory neurons within the vomeronasal organ (VNO) are thought to mediate the detection of pheromones in vertebrates. In the mouse, VNO neurons respond to pheromones with a rise in intracellular Ca²⁺ that accompanies a depolarization of the cell. Transduction of the pheromone appears to occur through the activation of a phosphatidylinositol signalling pathway, but the ion channels that respond to this signalling pathway have not been identified. In this report patch-clamp recording from hamster vomeronasal sensory neurons was used to identify second-messenger-gated channels that might play a role in transduction. The results demonstrate that VNO neurons show abundant expression of a Ca^2+-activated non-selective (CaNS) cation channel. The CaNS channel does not discriminate between Na⁺ and K⁺ and has a slope conductance of 22 pS. Half-activation of the channel occurs at a Ca²⁺ concentration of 0.5 m m (at -80 mV). The probability of opening ( P _o) of the channel is further augmented at positive potentials, and shows an e-fold voltage dependence per 37 mV. The channel exhibits rapid rundown following patch excision with P _o decreasing from near 1.0 to near 0. The adenine nucleotides ATP and cAMP block the channel with an apparent affinity of 3 and 42 μ m , respectively (-80 mV). Both the activation of the channel by Ca²⁺ and the block of the channel by adenine nucleotides show a mild voltage dependence, which can be accounted for by the voltage dependence of P _o. The properties of this channel make it a candidate to either directly mediate vomeronasal sensory transduction, or to amplify the primary sensory response.     

Veratridine block of rat skeletal muscle Nav1.4 sodium channels in the inner vestibule

Veratridine (VTD) is an alkaloid toxin found in Liliaceae plants. VTD causes persistent opening of the voltage-gated Na⁺ channel and reduces its single-channel conductance by 75 %. The mechanisms for these different VTD actions are unknown. Recent reports indicate that the VTD receptor aligns closely with the local anaesthetic (LA) receptor, which resides at D1S6, D3S6 and D4S6 of the Na⁺ channel α-subunit. To study this alignment, we created a mutant with cysteine substitutions at three S6 residues (rNav1.4-N434C/L1280C/F1579C). Under voltage-clamp conditions, amitriptyline and bupivacaine remained as potent blockers of this mutant channel when expressed in human embryonic kidney cells, whereas VTD completely failed to cause persistent opening. Unexpectedly, VTD at 100 μ m progressively blocked mutant currents by 90.4 ± 1.6 % ( n = 5), as assayed at 0.1 Hz for 15 min. This VTD block was reversed little during wash-off: ∼70 % of mutant currents did not return in 30 min. An increase in channel opening either by repetitive pulses at 1 Hz or by the inhibition of the fast inactivation hastened the VTD block. Co-application of amitriptyline or bupivacaine, which targeted the LA receptor, prevented this VTD block. Our data suggest that (a) the VTD receptor and the LA receptor overlap extensively, (b) receptor-bound VTD lies in the inner vestibule, and (c) VTD blocks this mutant channel as a bona fide Na⁺ channel blocker. We propose that VTD likewise blocks the wild-type open Na⁺ channel, albeit partially, to decrease the unitary conductance and to stabilize the open conformation for persistent opening.     

Phosphorylation-dependent differences in the activation properties of distal and proximal dendritic Na+ channels in rat CA1 hippocampal neurons

At distal dendritic locations, the threshold for action potential generation is higher and the amplitude of back-propagating spikes is decreased. To study whether these characteristics depend upon Na⁺ channels, their voltage-dependent properties at proximal and distal dendritic locations were compared in CA1 hippocampal neurons. Distal Na⁺ channels activated at more hyperpolarized voltages than proximal (half-activation voltages were −20.4 ± 2.4 mV vs. −12.0 ± 1.7 mV for distal and proximal patches, respectively,   n = 16  ,   P < 0.01  ), while inactivation curves were not significantly different. The resting membrane potential of distal regions also appeared to be slightly but consistently more hyperpolarized than their proximal counterpart. Staurosporine, a non-selective protein kinase inhibitor, shifted the activation curves for both proximal and distal Na⁺ channels to the left so that they overlapped and also caused the resting potentials to be comparable. Staurosporine affected neither the inactivation kinetics of Na⁺ currents nor the reversal potential for Na⁺. These results suggest that the difference in the voltage dependence of activation of distal and proximal Na⁺ channels can be attributed to a different phosphorylation state at the two locations.     

Effects of Perfusion Rate on Permeability of Frog and Rat Mesenteric Microvessels to Sodium Fluorescein

The permeability, P _S, to sodium fluorescein (Stokes-Einstein radius = 0.45 nm) has been measured in single mesenteric capillaries of pithed frogs and anaesthetised rats as perfusion velocity, U , was varied over a range from 400 up to 2000–10 000 μm s⁻¹. P _S increased linearly with U . In 20 frog capillaries, mean (± S.E.M.) P _S (in μm s⁻¹) = 9.35 (± 1.55) U × 10⁻⁵+ 0.244 (± 0.0291). Similarly, in nine rat venules, mean P _S= 1.62 (± 0.385) U × 10⁻⁴+ 0.375 (± 0.025). The flow-dependent component of permeability could be reversibly abolished in frog capillaries by superfusing with 100 μM noradrenaline and by superfusing rat venules with the nitric oxide synthase inhibitor, N ^G-nitro-L-arginine (20 μM). It was shown that changes in microvascular pressure accompanying changes in U during free perfusion could account for only 15 % of the changes in P _S, i.e. 85 % of the changes in P _S were changes in the permeability coefficient itself. A comparison between the changes in P _S with U and the previously described changes in microvascular permeability to K⁺ with U , suggest that if the flow-dependent component of permeability is modelled as a population of pores of constant size, these have radii of 0.8 nm. Such a pathway would limit flow-dependent permeability to small hydrophilic molecules and have minimal effect on net fluid exchange.