Effect of specific ion channel blockers on cultured schwann cell proliferation

Mitogenesis in a variety of tissues is known to be inhibited by K⁺ channel blockers such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Using radiolabeled thymidine as a proliferation index we have examined what role, if any, specific K⁺ channels have in cultured Schwann cells that have been induced to proliferate by pre-exposure to mitogens. TEA and 4-AP are “broad-spectrum” in that they block a variety of different types of K⁺ channel. In contrast, we found that α-dendrotoxin (α-DTX), a specific blocker of the type 1 fast delayed rectifier current (the largest component of Schwann cell K⁺ current) does not affect proliferation, suggesting that type 1 current may not be involved in mitogenesis. This suggestion is supported by our finding that the values of the K_D for the mitogenic effect (722 nM, 4-AP; 13 mM, TEA) are much larger than the corresponding electrophysiological values for type 1 channels (0.1 mM, 4-AP; 0.2 mM, TEA). Charybdotoxin (200 nM) and iberiotoxin (100 nM), inhibitors of Ca²⁺-activated K⁺ channels, cesium (5 mM), an inhibitor of inward rectifier channels, and furosemide (100 μM), which blocks Na⁺/K⁺/Cl⁻ cotransport, all had no effect on proliferation. Interestingly, 4,4′-diisothiocyanatostilbene 2,2′-disulphonate (DIDS), which blocks voltage-gated Cl⁻ channels, reduced proliferation. In summary, broad-spectrum K⁺ channel blockers inhibit Schwann cell proliferation, but inhibitors specific for type 1, Ca²⁺-activated, and inward rectifier K⁺ channels do not. Whether the inhibition is mediated by type 2 K⁺ channels, by an as yet unidentified Schwann cell K⁺ channel, or by another mechanism remains unclear. GLIA 22:113–120, 1998. © 1998 Wiley-Liss, Inc.     

Adult rat optic nerve oligodendrocyte progenitor cells express a distinct repertoire of voltage- and ligand-gated ion channels

Cultured oligodendrocyte progenitor cells derived from the developing central nervous system (CNS) express a pattern of ion channels that is distinct from mature oligodendrocytes and other cell types of the CNS. In the present study, we used the whole-cell patch-clamp technique and the fura-2-based Ca⁺⁺ imaging system to study the ion channel expression of oligodendrocyte progenitor cells derived from the optic nerves of adult rats. We found that the adult oligodendrocyte progenitor cell membrane is dominated by K⁺ currents, both delayed outward and inward rectifying. The inwardly rectifying K⁺ currents were often as large as the outward delayed rectifying K⁺ currents. The delayed rectifying outward currents were partially blocked by 50 mM tetraethylammonium or 1 mM 4-aminopyridine, but not by 2 or 5 mM BaCl₂. This suggests that the delayed rectifier channels expressed by adult progenitor cells are different from those expressed by perinatal cells. Most adult oligodendrocyte progenitor cells showed no or only small A-type K⁺ currents. Both Ca⁺⁺ and Na⁺ channels were also detected in these cells. Furthermore, adult progenitor cells responded to the neurotransmitters GABA and kainate and the pharmacology of these responses indicated that these cells express GABA_A receptors and kainate receptors that are Ca⁺⁺ -permeable. Our study suggests that adult oligodendrocyte progenitor cells are electrophysiologically distinct and that these cells share electrophysiological characteristics with both perinatal progenitor cells and immature oligodendrocytes. © 1995 Wiley-Liss, Inc.     

Microglial K+ channel expression in young adult and aged mice

The K⁺ channel expression pattern of microglia strongly depends on the cells' microenvironment and has been recognized as a sensitive marker of the cells' functional state. While numerous studies have been performed on microglia in vitro, our knowledge about microglial K⁺ channels and their regulation in vivo is limited. Here, we have investigated K⁺ currents of microglia in striatum, neocortex and entorhinal cortex of young adult and aged mice. Although almost all microglial cells exhibited inward rectifier K⁺ currents upon membrane hyperpolarization, their mean current density was significantly enhanced in aged mice compared with that determined in young adult mice. Some microglial cells additionally exhibited outward rectifier K⁺ currents in response to depolarizing voltage pulses. In aged mice, microglial outward rectifier K⁺ current density was significantly larger than in young adult mice due to the increased number of aged microglial cells expressing these channels. Aged dystrophic microglia exhibited outward rectifier K⁺ currents more frequently than aged ramified microglia. The majority of microglial cells expressed functional BK‐type, but not IK‐ or SK‐type, Ca²⁺‐activated K⁺ channels, while no differences were found in their expression levels between microglia of young adult and aged mice. Neither microglial K⁺ channel pattern nor K⁺ channel expression levels differed markedly between the three brain regions investigated. It is concluded that age‐related changes in microglial phenotype are accompanied by changes in the expression of microglial voltage‐activated, but not Ca²⁺‐activated, K⁺ channels. GLIA 2015;63:664–672     

Activation of outward current by pituitary adenylate cyclase activating polypeptide in mouse microglial cells

In order to investigate the interaction between the nervous and immune systems, we have analyzed the effect of one of the neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP), on microglia cells by the patch-clamp method. Puff application of PACAP38 onto mouse microglial cells induced an outward current in a dose-dependent manner. Reversal potentials of the outward current were dependent on external K⁺ concentrations ([K⁺]_o) and independent of [Cl⁻]_o. Ion channel blockers of potassium currents, quinine (1 mM), tetraethylammonium (TEA, 20 mM) and 4-aminopyridine (4-AP, 5 mM), suppressed the outward current with a potency order of quinine>TEA>4-AP. PACAP27 also induced outward current less effectively than PACAP38. A fragment of PACAP38 [PACAP(6-38)], known as an inhibitor for PACAP38, suppressed the outward current. These data suggest that PACAP38 activates a quinine-sensitive K⁺ outward current and modulates activities in microglia. They indicate that the immune system in the brain can be modulated by neurotransmitters, the mediators of neurons. J. Neurosci. Res. 51:382–390, 1998. © 1998 Wiley-Liss, Inc.     

Ionic channel currents in cultured neurons from human cortex

Ionic channels in human cortical neurons have not been studied extensively. HCN-1 and HCN-1A cells, which recently were established as continuous cultures from human cortical tissue, have been shown by histochemical and immunochemical methods to exhibit a neuronal phenotype, but expression of functional ionic channels was not demonstrated. For the present study, HCN-1 and HCN-1A cells were cultured in Dulbecco's modified Eagle's medium with 15% fetal calf serum, in some cases supplemented with 10 ng/ml nerve growth factor, 10 μM forskolin, and 1 mM dibutyryl cyclic adenosine monophosphate to promote differentiation. Cells or membrane patches were voltage clamped using conventional patch clamp techniques. In HCN-1A cells, we identified a tetrodotoxin-sensitive Na⁺ current, two types of Ca²⁺ channel current, including L-type current and a second type that in some respects resembled N-type current, and four types of K⁺ current, including a delayed outward rectifier that showed voltage-dependent inactivation, two types of noninactivating Ca²⁺-activated K⁺ channels with slope conductances of 146 and 23 pS (K⁺ _iK⁺ _o 145 mM/5 mM), and less frequently, a noninactivating, intermediate conductance channel that was not sensitive to internal Ca²⁺. When HCN-1A cells were examined after 3 days of exposure to differentiating agents, pronounced morphological changes were evident but no differences in ionic currents were apparent. HCN-1 cells also exhibited K⁺ and Ca²⁺ channel currents, but Na⁺ currents were not detected in these cells. Our data provide additional evidence indicating a functional neuronal phenotype for HCN-1A cells, and represent the most comprehensive survey to date of the variety of ionic channels expressed by human cortical neurons. © 1993 Wiley-Liss, Inc.     

Endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells

Endogenous voltage-gated potassium currents were investigated in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells using whole-cell voltage clamp recording. Depolarizing voltage steps from −70 mV triggered an outwardly rectified current in nontransfected HEK293 cells. This current had an amplitude of 296 pA at +40 mV and a current density of 19.2 pA/pF. The outward current was eliminated by replacing internal K⁺ with Cs⁺ and suppressed by the K⁺ channel blockers tetraethylammonium and 4-aminopyridine. Raising external K⁺ attenuated the outward current and shifted the reversal potential towards positive potentials as predicted by the Nernst equation. The current had a fast activation phase but inactivated slowly. These features implicate delayed rectifier (I_K)-like channels as mediators of the observed current, which was comparable in size to I_K currents in many other cells. A small native inward rectifier current but no transient outward current I_A, the M current I_M, or Ca²⁺-dependent K⁺ currents were detected in HEK293 cells. In contrast to these findings in HEK293 cells, little or no I_K-like current was detected in CHO cells. The difference in endogenous voltage-activated currents in HEK293 and CHO cells suggest that CHO cell lines are a preferred system for exogenous K⁺ channel expression. J. Neurosci. Res. 52:612–617, 1998. © 1998 Wiley-Liss, Inc.     

Single ion channel currents in neuropile glial cells of the leech central nervous system

The patch-clamp technique was used to investigate the activity of single ion channels in neuropile glial (NG) cells in the central nervous system (CNS) of the medicinal leech, Hirudo medicinalis. We found evidence for two distinct Cl^? channels that could be distinguished by their basic electrical properties and their responses to different inhibitors on single ion channels currents. In the Inside-out configuration in symmetrical Cl solutions, these channels showed current-voltage relationships with slight outward rectification, mean conductances of 70 and 80 pS, and reversal potentials near 0 mV. Significant permeability to Na⁺, K⁺, or SO₄^2? could not be detected. The open-state probability of the 70 pS Cl^? channel increased with membrane depolarization, whereas the open-state probability of the 80 pS Cl^? channel was voltage-independent. The application of the stilbene derivative DIDS (100 μM) to the cytoplasmic side of the glial cell membrane blocked both Cl^? channels. The activity of the 70 pS channel was blocked irreversibly by DIDS, whereas the activity of the 80 pS channel reappeared after wash-out of DIDS. Both channels were blocked reversibly by 1 mM Zn²⁺. K⁺ channels could only be observed occasionally in the soma membrane of the NG cells. We have characterized a 60 pS K⁺ channel with a high selectivity for K⁺ over Na⁺. The low density of K⁺ channels in the soma membrane may indicate a non-uniform distribution of this channel type in NG cells. © 1993 Wiley-Liss, Inc.     

Single potassium channels in neuropile glial cells of the leech central nervous system

We performed patch-clamp experiments to identify distinct K⁺ channels underlying the high K⁺ conductance and K⁺ uptake mechanism of the neuropile glial cell membrane on the single-channel level. In the soma membrane four different types of K⁺ channels were characterized, which were found to be distributed in clusters. Since no other types of K⁺ channels were observed, these appear to be the complete repertoire of K⁺ channels expressed in the soma region of this cell type. The outward rectifying 42 pS K⁺ channel could markedly contribute to the high K⁺ conductance and the maintenance of the membrane potential, since it shows the highest open probability of all channels. The channel gating occurred in bursts and patch excision decreased the open probability. The outward rectifying 74 pS K⁺ channel was rarely active in the cell-attached configuration; however, patch excision enhanced its open probability considerably. This type of channel may be involved in neuron-glial crosstalk, since it is activated by both depolarizations and increases in the intracellular Ca²⁺ concentration, which are known to be induced by neurotransmitter release following the activation of neurons. The 40 pS and 83 pS K⁺ channels showed inward rectifying properties, suggesting their involvement in the regulation of the extracellular K⁺ content. The 40 pS K⁺ channel could only be observed in the inside-out configuration. The 83 pS channel was activated following patch excision. At membrane potentials more negative than −60 mV, flickering events indicated voltage-dependent gating.     

Functional identification of an outwardly rectifying pH‐ and anesthetic‐sensitive leak K+ conductance in hippocampal astrocytes

Astrocytes function as spatial K⁺ buffers by expressing a rich repertoire of K⁺ channels. Earlier studies suggest that acid‐sensitive tandem‐pore K⁺ channels, mainly TWIK‐related acid‐sensitive K⁺ (TASK) channels, mediate part of the passive astroglial membrane conductance. Here, using a combination of electrophysiology and pharmacology, we investigated the presence of TASK‐like conductance in hippocampal astrocytes of rat brain slices. Extracellular pH shifts to below 7.4 (or above 7.4) induced a prominent inward (or outward) current in astrocytes in the presence of tetrodotoxin, a Na⁺ channel blocker, and 4,4′‐diisothiocyanatostilbene‐2,2’‐disulfonate, a co‐transporter blocker. The pH‐sensitive current was insensitive to quinine, a potent blocker of tandem‐pore K⁺ channels including TWIK‐1 and TREK‐1 channels. Voltage‐clamp analysis revealed that the pH‐sensitive current exhibited weak outward rectification with a reversal potential of ?112 mV, close to the Nernst equilibrium potential for K⁺. Furthermore, the current–voltage relationship was well fitted with the Goldman–Hodgkin–Katz current equation for the classical open‐rectifier ‘leak’ K⁺ channel. The pH‐sensitive K⁺ current was potentiated by TASK channel modulators such as the volatile anesthetic isoflurane but depressed by the local anesthetic bupivacaine. However, unlike TASK channels, the pH‐sensitive current was insensitive to Ba²⁺ and quinine. Thus, the molecular identity of the pH‐sensitive leak K⁺ channel is unlikely to be attributable to TASK channels. Taken together, our results suggest a novel yet unknown leak K⁺ channel underlying the pH‐ and anesthetic‐sensitive background conductance in hippocampal astrocytes.     

Light-increased cGMP and K conductance in the hyperpolarizing receptor potential of Onchidium extra-ocular photoreceptors

The phototransduction mechanism of the extra-ocular photoreceptor cells Ip-2 and Ip-1 in the mollusc Onchidium ganglion was examined. Previous work showed that the depolarizing receptor potential of another extra-ocular photoreceptor cell, A-P-1 is produced by a decrease of the light-sensitive K⁺ conductance activated by a second messenger, cGMP and is inactivated by the hydrolysis of cGMP. Here, a hyperpolarizing receptor potential of Ip-2 or Ip-1 was associated with an increase in membrane conductance. When Ip-2 or Ip-1 was voltage-clamped near the resting membrane potential, light induced an outward photocurrent corresponding to the above hyperpolarization. The spectral sensitivity had a peak at 510 nm. The shift of reversal potentials of the photocurrent depended on the Nernst equation of K⁺-selective conductance. The photocurrent was blocked by 4-AP and l-DIL, which are effective blockers of the A-P-1 light-sensitive K⁺ conductance. These results suggested that the hyperpolarization is mediated by increasing a similar light-sensitive K⁺ conductance to that of A-P-1. The injection of cGMP or Ca²⁺ into a cell produced a K⁺ current that mimicked the photocurrent. 4-AP and l-DIL both abolished the cGMP-activated K⁺ current, while TEA suppressed only the Ca²⁺-activated K⁺ current. These results indicated that cGMP is also a second messenger that regulates the light-sensitive K⁺ conductance. The photocurrent was blocked by LY-83583, a guanylate cyclase (GC) inhibitor, but was unaltered by zaprinast, a phosphodiesterase (PDE) inhibitor. Together, the present results suggest that increasing the internal cGMP in Ip-2 or Ip-1 cells light-activates GC rather than inhibits PDE, thereby leading to an increase of the light-sensitive K⁺ conductance and the hyperpolarization.     

Plasma gelsolin protects HIV-1 gp120-induced neuronal injury via voltage-gated K+ channel Kv2.1

Plasma gelsolin (pGSN), a secreted form of gelsolin, is constitutively expressed throughout the central nervous system (CNS). The neurons, astrocytes and oligodendrocytes are the major sources of pGSN in the CNS. It has been shown that levels of pGSN in the cerebrospinal fluid (CSF) are decreased in several neurological conditions including HIV-1-associated neurocognitive disorders (HAND). Although there is no direct evidence that a decreased level of pGSN in CSF is causally related to the pathogenesis of neurological disorders, neural cells, if lacking pGSN, are more vulnerable to cell death. To understand how GSN levels relate to neuronal injury in HAND, we studied the effects of pGSN on HIV-1 gp120-activated outward K⁺ currents in primary rat cortical neuronal cultures. Incubation of rat cortical neurons with gp120 enhanced the outward K⁺ currents induced by voltage steps and resulted in neuronal apoptosis. Treatment with pGSN suppressed the gp120-induced increase of delayed rectifier current (I_K) and reduced vulnerability to gp120-induced neuronal apoptosis. Application of Guangxitoxin-1E (GxTx), a Kv2.1 specific channel inhibitor, inhibited gp120 enhancement of I_K and associated neuronal apoptosis, similar effects to pGSN. Western blot and PCR analysis revealed gp120 exposure to up-regulate Kv2.1 channel expression, which was also inhibited by treatment with pGSN. Taken together, these results indicate pGSN protects neurons by suppressing gp120 enhancement of I_K through Kv2.1 channels and reduction of pGSN in HIV-1-infected brain may contribute to HIV-1-associated neuropathy.     

Ionic mechanisms of action of prion protein fragment PrP(106–126) in rat basal forebrain neurons

Prion diseases are neurodegenerative disorders that are characterized by the presence of the misfolded prion protein (PrP). Neurotoxicity in these diseases may result from prion‐induced modulation of ion channel function, changes in neuronal excitability, and consequent disruption of cellular homeostasis. We therefore examined PrP effects on a suite of potassium (K⁺) conductances that govern excitability of basal forebrain neurons. Our study examined the effects of a PrP fragment [PrP(106–126), 50 nM] on rat neurons using the patch clamp technique. In this paradigm, PrP(106–126) peptide, but not the “scrambled” sequence of PrP(106–126), evoked a reduction of whole‐cell outward currents in a voltage range between –30 and +30 mV. Reduction of whole‐cell outward currents was significantly attenuated in Ca²⁺‐free external media and also in the presence of iberiotoxin, a blocker of calcium‐activated potassium conductance. PrP(106–126) application also evoked a depression of the delayed rectifier (I_K) and transient outward (I_A) potassium currents. By using single cell RT‐PCR, we identified the presence of two neuronal chemical phenotypes, GABAergic and cholinergic, in cells from which we recorded. Furthermore, cholinergic and GABAergic neurons were shown to express K_v4.2 channels. Our data establish that the central region of PrP, defined by the PrP(106–126) peptide used at nanomolar concentrations, induces a reduction of specific K⁺ channel conductances in basal forebrain neurons. These findings suggest novel links between PrP signalling partners inferred from genetic experiments, K⁺ channels, and PrP‐mediated neurotoxicity. © 2010 Wiley‐Liss, Inc.     

Effects of 4-aminopyridine on calcium action potentials and calcium current under voltage clamp in spinal neurons

We examined the effects of 4-aminopyridine (4-AP) on calcium conductance mechanisms in cultured mouse spinal cord neurons. At low concentrations ( 1mM), 4-AP enhanced Ca²⁺-dependent transmitter release and prolonged the duration of Ca²⁺-dependent action potentials. Voltage clamp studies indicated that 4-AP directly facilitates Ca²⁺ entry through voltage sensitive channels apart from an effect on K⁺ currents. These results may help to explain why the drug promotes Ca²⁺-dependent transmitter release at peripheral and central synapses.     

Intracellular Action of Spermine on Neuronal Ca2+ and K+ Currents

Intra-and extracellular effects of the polyamine spermine on electrical activity and membrane currents of identified neurons in the abdominal ganglion of Aplysia californica were studied under current-and voltage-clamp conditions. Lonophoretic injection of spermine reduced the amplitude of action potentials and altered their time course as well as spontaneous discharge activity. Investigation of membrane currents showed that intracellular spermine suppressed the total outward current but increased the inward rectifier current. After separation of ion currents it was found that the voltage-activated, delayed K⁺ outward current and the Ca²⁺ inward current were reduced by intracellular spermine in a dose- and voltage-dependent manner. The block of the K⁺ current can be described by a voltage-dependent reaction, where one spermine molecule binds to one channel. The binding constant K_b, at zero voltage, and the effective valency, zδ, had values of 176/M and 0.41 for cell R-15, 223/M and 0.64 for cell L-11, and 137/M and 0.42 for cell L-3. Apparently, more than one spermine cation is needed to block one Ca²⁺ channel, since the coefficient n, which absorbs the molecularity and cooperativity of the reaction, had non-integral values between 1.34 and 2.22. The binding constant K_b and the effective valency zδ had values of 265/M and 0.64 for cell R-15, 821M and 0.56 for cell L-4, and 263/M and 0.51 for cell L-6. Intracellular spermine also blocked the Ca²⁺-activated K⁺ current induced by ionophoretic Ca²⁺-injections, but increased the current at prolonged times after spermine injection. Extracellular spermine had no effect on electrical activity or on membrane currents. The results indicate that intracellular spermine affects the electrical discharge activity of neurons by acting as a blocker and/or modulator at voltage-dependent membrane conductances.     

The potassium channels Kv1.5 and Kv1.3 modulate distinct functions of microglia

Activation of microglia by LPS leads to an induction of cytokine and NO release, reduced proliferation and increased outward K⁺ conductance, the latter involving the activation of Kv1.5 and Kv1.3 channels. We studied the role of these channels for microglial function using two strategies to interfere with channel expression, a Kv1.5 knockout (Kv1.5^−/−) mouse and an antisense oligonucleotide (AO) approach. The LPS-induced NO release was reduced by AO Kv1.5 and completely absent in the Kv1.5^−/− animal; the AO Kv1.3 had no effect. In contrast, proliferation was augmented with both, loss of Kv1.3 or Kv1.5 channel expression. After facial nerve lesion, proliferation rate was higher in Kv1.5^−/− animals as compared to wild type. Patch clamp experiments confirmed the reduction of the LPS-induced outward current amplitude in Kv1.5^−/− microglia as well as in Kv1.5- or Kv1.3 AO-treated cells. Our study indicates that induction of K⁺ channel expression is a prerequisite for the full functional spectrum of microglial activation.     

Activation and modulation of calcium-activated non-selective cation channels from embryonic chick sensory neurons

We have shown that calcium-activated non-selective (CAN) channels from embryonic chick sensory neurons are permeable to both Na⁺ and K⁺ and are not blocked by TTX, TEA, or 4-AP. These neuronal CAN channels are activated by sub-micromolar cytoplasmic Ca²⁺ with negative cooperativity. The effect of Ca²⁺ is to decrease the closed times of the channel with little effect on the time the channel remains open. Isolated neuronal CAN channels can be phosphorylated by cAMP-dependent protein kinase (PKA). The effect of phosphorylation is to shorten channel open time and to minimize the effect of Ca²⁺ on channel closed time.     

Voltage-dependent membrane currents of cultured human neurofibromatosis type 2 Schwann cells

Previous experimental observations indicate that inhibition of voltage-dependent K⁺ currents suppresses proliferation of normal Schwann cells. In the present study we tested the opposite relationship, i.e., whether Schwann cells from tumors with abnormally high rates of proliferation would have an increase in membrane K⁺ currents. Whole-cell membrane currents were studied in cultured cells from schwannomas of two neurofibromatosis type 2 (NF2) patients (n = 53), one patient with a sporadic schwannoma (n = 22), and two control subjects (n = 41). Five different types of voltage-dependent membrane currents were found in all of the Schwann cells tested. Membrane depolarization activated outward K⁺ and Cl⁻ currents; quinidine was found to block the K⁺ current (IC₅₀ ≈ 1 μM), and NPPB reduced the Cl⁻ current. Ba²⁺-sensitive inward rectifier K⁺ currents, fast Na⁺ currents, and a transient, inactivating K⁺ current were less frequently observed. On average, NF2 cells were found to have statistically significant higher membrane potential and larger non-inactivating K⁺ outward current as compared to controls. Electrophysiological parameters of Schwann cells from a sporadic schwannoma showed a tendency for larger outward currents; however, the difference did not reach statistical significance. Together the data support the suggestion of a possible link between K⁺ outward current and proliferation of Schwann cells. GLIA 24:313–322, 1998. © 1998 Wiley-Liss, Inc.     

4-Aminopyridine stimulates B-50 (GAP-43) phosphorylation in rat synaptosomes

Recently, we have shown that stimulation of [³H]-noradrenaline release from hippocampal slices by 4-aminopyridine (4-AP) is accompanied by an enhancement of the phosphorylation of B-50, a major presynaptic substrate of protein kinase C (PKC). PKC has been implicated in the regulation of transmitter release. In this study, we investigated the effects of 4-AP on B-50 phosphorylation in synaptosomes from rat brain and compared the effects of 4-AP with those of depolarization with K⁺, in order to gain more insight into the mechanism of action of 4-AP. B-50 phosphorylation was stimulated by incubation with 4-AP for 2 minutes at concentrations ranging from 10 μM to 5 mM. 4-AP (100 μM) stimulated B-50 phosphorylation already within 15 seconds; longer incubations revealed a sustained increase in the presence of 4-AP. B-50 phosphorylation was also stimulated by depolarization with 30 mM K⁺ for 15 seconds. The effects of both 4-AP or K⁺ depolarization on B-50 phosphorylation were abolished at low extracellular Ca²⁺ concentrations. The increase in B-50 phosphorylation induced by 4-AP seemed to be dependent on the state of depolarization, since the effect of 4-AP was largest under nondepolarizing conditions. Comparing the effects of 4-AP and K⁺ depolarization on B-50 phosphorylation suggests that a different mechanism of action is involved. These results indicate that the stimulation of B-50 phosphorylation by 4-AP in hippocampal slices can be attributed to a direct action of 4-AP on presynaptic terminals. In addition, our results support the hypothesis that B-50 phosphorylation by PKC is involved in Ca²⁺-dependent transmitter release evoked by 4-AP. This research was supported by CLEO-TNO grant A66 of the Dutch Epilepsy Foundation.     

Impaired capillary-to-arteriolar electrical signaling after traumatic brain injury

Traumatic brain injury (TBI) acutely impairs dynamic regulation of local cerebral blood flow, but long-term (>72 h) effects on functional hyperemia are unknown. Functional hyperemia depends on capillary endothelial cell inward rectifier potassium channels (Kir2.1) responding to potassium (K⁺) released during neuronal activity to produce a regenerative, hyperpolarizing electrical signal that propagates from capillaries to dilate upstream penetrating arterioles. We hypothesized that TBI causes widespread disruption of electrical signaling from capillaries-to-arterioles through impairment of Kir2.1 channel function. We randomized mice to TBI or control groups and allowed them to recover for 4 to 7 days post-injury. We measured in vivo cerebral hemodynamics and arteriolar responses to local stimulation of capillaries with 10 mM K⁺ using multiphoton laser scanning microscopy through a cranial window under urethane and α-chloralose anesthesia. Capillary angio-architecture was not significantly affected following injury. However, K⁺-induced hyperemia was significantly impaired. Electrophysiology recordings in freshly isolated capillary endothelial cells revealed diminished Ba²⁺-sensitive Kir2.1 currents, consistent with a reduction in channel function. In pressurized cerebral arteries isolated from TBI mice, K⁺ failed to elicit the vasodilation seen in controls. We conclude that disruption of endothelial Kir2.1 channel function impairs capillary-to-arteriole electrical signaling, contributing to altered cerebral hemodynamics after TBI.