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PubMed1987

Acceleration of desensitization by agonist pre-treatment in the snake.

1. The kinetics of carbachol-induced desensitization have been studied in snake twitch-muscle fibres maintained in an isotonic potassium propionate solution and voltage clamped to +50 mV. 2. Microperfusion of carbachol (162-756 microM) induces a transient outward current which peaks within a few seconds and then slowly decays towards the base line. The time course of current decay estimates the time course of desensitization onset. 3. Brief exposure (30 s) to a 'conditioning' concentration of agonist (10.8 microM) accelerates the desensitization onset produced by exposure to higher 'test' concentrations of agonist (162-756 microM). 4. The acceleration of desensitization by pre-treatment with 10.8 microM-carbachol was independent of the duration of exposure between 15 and 60 s. This observation indicated that the mechanism responsible for the alteration in desensitization kinetics by treatment with 10.8 microM-carbachol differed from that responsible for the time-dependent development of desensitization produced in the presence of higher carbachol concentrations. 5. Pre-treatment with the muscarinic agonists, methylcholine and bethanechol, did not accelerate 216 microM-carbachol-induced desensitization, suggesting that the alteration of desensitization kinetics by pre-treatment was specific for nicotinic agonists. 6. The conditioning concentrations of carbachol (5.4-10.8 microM) produced no measurable outward current in muscle fibres voltage clamped to +50 mV. Further, in patch-clamp recordings it was observed that, with these concentrations of carbachol, there was no channel activity in many successful patches voltage clamped to +50 mV and, when present, the frequency of channel activity was very low. These results demonstrated that the alteration in desensitization was not the consequence of significant amounts of either receptor activation or desensitization produced by the conditioning concentration. 7. Exposure to 10.8 microM-carbachol for periods of up to 150 s did not change the amplitude of miniature end-plate currents recorded at end-plates voltage clamped to +50 mV. These results also demonstrated that the acceleration of desensitization by pre-treatment with conditioning concentrations of agonist was not due to partial desensitization occurring during the pre-treatment period. 8. Our results are consistent with the view that there are distinct populations of agonist binding sites on the acetylcholine receptor which separately regulate desensitization and channel opening.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1994

Slowly activating voltage-dependent K+ conductance is apical pathway for K+ secretion in vestibular dark cells.

Dark cell epithelium secretes K+ into the lumen of the vestibular labyrinth by a previously unidentified apical transport mechanism. Previous single-channel patch-clamp studies demonstrated nonselective cation channels and maxi-K+ channels in the apical membrane, but in too low a density to account for transepithelial K+ transport. In this report, we demonstrated with the cell-attached macro-patch-clamp technique an outward apical membrane current at 0-mV pipette voltage, which was stimulated by elevating bath K+ concentration from 3.6 to 25 mM and inhibited by 10 microM bumetanide, similar to their known effects on transepithelial short-circuit current and K+ secretion. Furthermore, the patch current was activated over several seconds by a sustained depolarization and deactivated over several hundred milliseconds by a hyperpolarization. Current-voltage relationships from tail currents were obtained with either NaCl or KCl in the pipette. Depolarization from -40 to +40 mV led to an increased conductance by a factor of 7.3 +/- 1.7 (n = 7) and 19.2 +/- 7.6 (n = 6) for NaCl and KCl, respectively, and to a reversal voltage near the presumed equilibrium potential for K+. The results demonstrate that dark cell K+ secretion occurs via K(+)-selective channels with characteristics similar to those associated with the IsK protein.

Animals↗

PubMed1993

Metabolic regulation of cardiac ATP-sensitive K+ channels.

Activation of ATP-sensitive K+ (KATP) channels has been implicated as a cause of increased cellular K+ efflux and action potential duration (APD) shortening during myocardial ischemia, hypoxia, and selective glycolytic inhibition, since selective KATP channel antagonists partially or completely block increased cellular K+ efflux and APD shortening under these conditions. During substrate-free hypoxia or myocardial ischemia in intact rabbit ventricle, unidirectional K+ efflux rate during systole approximately doubled and APD decreased by approximately 40% after 10 minutes. In patch-clamped guinea pig ventricular myocytes, similar changes could be produced by activation of < 0.5% of the maximal KATP channel conductance. Furthermore, from studying the desensitizing effects of ADPi on the ATP sensitivity of KATP channels in excised inside-out patches, it was estimated that the rapid changes in the cytosolic ATP/ADP ratio during ischemia and hypoxia were of sufficient magnitude to activate KATP channels to this degree. During selective glycolytic inhibition, however, the global cytosolic ATP/ADP ratio in intact heart remained normal despite an increase in cellular K+ efflux comparable to ischemia and hypoxia. In patch-clamped saponin-permeabilized ventricular myocytes, KATP channels were preferentially suppressed by glycolytic ATP production compared to ATP generated by mitochondria or by the creatinine kinase reaction, and functional glycolytic enzymes were found to be associated with KATP channels in excised membrane patches. We hypothesize that sarcolemma-associated glycolytic enzymes may be important in maintaining a high local cytosolic ATP/ADP ratio in the vicinity of KATP channels, where sarcolemmal ATPases are tending to depress the local ATP/ADP ratio.

Action Potentials↗

PubMed1991

Effects of pH on acetylcholine receptor function.

We have examined the effects of changing extracellular pH on the function of nicotinic acetylcholine receptors from Torpedo californica using ion flux and electrophysiological methods. Agonist-induced cation efflux from vesicles containing purified, reconstituted receptors showed a monotonic dependence on external hydrogen ion concentration with maximal fluxes at alkaline pH and no agonist-induced efflux at pH's less than approximately 5. A similar pH dependence was measured for the peak agonist-activated membrane currents measured in microelectrode voltage-clamped Xenopus oocytes induced to express Torpedo receptor through mRNA injection. Half-maximal inhibition occurred at a similar pH in both systems, in the range of pH 6.5-7.0. Single-channel currents from Torpedo ACh receptors measured in patch-clamp recordings were also reduced in amplitude at acid pH with an apparent pKa for block of less than 5. Measurements of channel kinetics had a more complicated dependence on pH. The mean channel open time determined from patch-clamp measurements was maximal at neutral pH and decreased at both acid and alkaline pH's. Thus, both channel permeability properties and channel gating properties are affected by the extracellular pH.

Animals↗

PubMed2004

Electrophysiological approaches to the study of protein translocation in mitochondria.

Electrophysiological techniques have been integral to our understanding of protein translocation across various membranes, and, in particular, the mitochondrial inner and outer membranes. Descriptions of various methodologies (for example, patch clamp, planar bilayers, and tip dip, and their past and potential contributions) are detailed within. The activity of protein import channels of native mitochondrial inner and outer membranes can be studied by directly patch clamping mitochondria and mitoplasts (mitochondria stripped of their outer membrane by French pressing) from various genetically manipulated strains of yeast and mammalian tissue cultured cells. The channel activities of TOM, TIM23, and TIM22 complexes are compared with those reconstituted in proteoliposomes and with those of the recombinant proteins Tom40p, Tim23p, and Tim22p, which play major roles in protein translocation. Studies of the mechanism(s) and the role of channels in protein translocation in mitochondria are prototypes, as the same principles are likely followed in all biological membranes including the endoplasmic reticulum and chloroplasts. The ability to apply electrophysiological techniques to these channels is now allowing investigations into the role of mitochondria in diverse fields such as neurotransmitter release, long-term potentiation, and apoptosis.

Antibodies↗

PubMed1991

Modulation of Ca2+ channels by atrial natriuretic peptide in the bovine adrenal glomerulosa cell.

In the bovine adrenal glomerulosa cell, calcium influx through voltage-dependent calcium channels is critical to maintaining an aldosterone secretory response. In patch clamp, atrial natriuretic peptide (ANP) inhibits T-type calcium channel current yet stimulates L-type calcium channel current. In the present study the channel effects of ANP observed in the patch-clamp configuration were extended and related to populations of cells. We observed the following. (i) The effect of ANP on T-channel current resulted in the reduction in the open state probability. ANP decreased the mean open state duration from 14.2 to 1.8 ms/sweep. (ii) In the weakly depolarized cell stimulated by 8 mM K+, ANP reduced the level of aequorin luminescence (a measure of cytosolic calcium) and completely inhibited the stimulated rate of aldosterone secretion, returning it to prestimulation values. These effects are consistent with a decrease in net calcium channel influx and the reported inhibition of T-channel current. In contrast, the calcium channel blocker, nitrendipine, which at low dose selectively blocks L-type calcium channel flux, only slightly reduced luminescence, and partially inhibited the sustained secretory response. (iii) In the strongly depolarized cell, stimulated by 60 mM K+, ANP increased the level of aequorin luminescence consistent with an increase in net calcium channel influx and the reported stimulation of L-channel current. These results indicate that under physiological conditions the inhibition of T-type calcium channels may be involved in the inhibition of the aldosterone secretion induced by ANP.

Aequorin↗

PubMed1998

Functional and molecular evidence for P2X receptors in LLC-PK1 cells.

Extracellular ATP affects a wide variety of cells via purinergic membrane receptors. One class of purinergic receptors, P2X, consists of ATP-gated, calcium-permeable, cation-selective channels. We performed whole cell patch-clamp studies, intracellular calcium concentration ([Ca2+]i) measurements, and reverse transcription-polymerase chain reaction (RT-PCR) to determine whether P2X receptors are expressed in LLC-PK1 cells. First, in patch-clamp studies, 100 microM ATP depolarized the cell membrane and increased the whole cell conductance of LLC-PK1 cells. This response was dose dependent and inhibited by 100 microM suramin, a P2 receptor antagonist. The ATP-induced conductance was cation selective but did not discriminate between Na+ and K+. ADP, alpha,beta-methylene-ATP, and beta,gamma-methylene-ATP had no effect on the whole cell conductance. Next, 10 microM ATP caused a rapid rise in [Ca2+]i in LLC-PK1 cells. This effect of ATP was inhibited by the absence of extracellular calcium and by suramin but not by pretreatment with pertussis toxin. ADP and beta,gamma-methylene-ATP had little or no effect on [Ca2+]i. Finally, RT-PCR produced a 330-bp fragment from LLC-PK1 cell RNA, whose sequence was 80% identical to the rat P2X1 receptor. We conclude that LLC-PK1 cells express purinergic receptors of the P2X class, which mediate depolarization and calcium entry when activated.

Adenosine Triphosphate↗

PubMedDate not supplied

[Mechanism of signal transduction in photoreceptors of vertebrates: identification of an intracellular transmitter].

A short account is given of the studies which resulted in identification of cGMP as intracellular transmitter which realizes the control of light-depending channels of vertebrate photoreceptors. The following problems are discussed. 1. Proofs of necessary participation of intracellular mediator in the signal transduction in the photoreception act. 2. Competition between cGMP and Ca2+ ions for the role of such mediator in the photoreceptors of vertebrates. Relationship between metabolism of cGMP and Ca2+ which makes impossible identification of the mediator in the in vivo experiments. 3. Patch-clamp-experiments which have shown that conductivity of the rod plasma membrane is controlled by cGMP rather than by Ca2+ ions. Proofs of the identity of cGMP-regulated channels recorded in patch-clamp-experiments and light-dependent channels observed in the in vivo experiments. 4. Analysis of the prospects of further studies of the control mechanism of light-dependent channels.

Animals↗

PubMed2003

Somatostatin directly inhibits substantia gelatinosa neurons in adult rat spinal dorsal horn in vitro.

Effects of somatostatin (SST) on the synaptic transmission to substantia gelatinosa (SG) neurons of adult spinal cord slices were investigated using intracellular recording and blind whole-cell patch-clamp technique. Bath application of SST (1 microM) induced the membrane hyperpolarization that was accompanied by a decrease in input resistance and had the reversal potential of -92 +/- 3 mV (n=5) in the intracellular recording experiment. In patch-clamp experiment, SST (1 microM) induced an outward current with amplitude of 14 +/- 2 pA (n=60) at the holding potential of -60 mV, and was not affected by TTX (n=3). The effect was dose-dependent with EC50 value of 0.82 microM (Hill coefficient: 0.89). The outward current was suppressed when the patch-pipette solution containing potassium channel blockers, Cs+ and tetraethylammonium (TEA), and was inhibited by Ba2+ (200 microM) to 15 +/- 6% of the control (n=3). In addition, the SST current reversed its polarity at potential close to the equilibrium potential of K+ channel calculated by the Nernst equation. No significant changes were found in amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) and dorsal root evoked EPSC (eEPSC) by SST. Also, SST did not affect both of the miniature inhibitory postsynaptic currents (mIPSCs) and evoked inhibitory postsynaptic currents (eIPSCs), mediated by either the GABA or glycine receptor. We conclude that SST activates the K+ channel resulting in postsynaptic hyperpolarization in adult rat SG neurons without affecting presynaptic component of the transmission, which are considered to account, at least a part, for the analgesic effects of SST reported previously.

Action Potentials↗

PubMed2000

Dietary selenium and vitamin E intakes alter beta-adrenergic response of L-type Ca-current and beta-adrenoceptor-adenylate cyclase coupling in rat heart.

Previously we have shown that both insufficient (combined with vitamin E deficiency) and excess intake of selenium (Se) impairs isoproterenol (ISO)-induced contractions of rat papillary muscle. In the present study, we used patch-clamp and biochemical techniques to investigate mechanisms of this effect in rats fed a Se- and vitamin E-deficient, a Se-excess or a normal diet. Whole-cell configuration of patch-clamp technique was used to investigate L-type Ca(2+) currents (I(Ca,L)) and their regulation by beta-adrenergic receptor stimulation in enzymatically isolated single rat ventricular myocytes. Alteration of Se and vitamin E intake did not affect peak I(Ca,L), but the threshold potential of activation was significantly different among groups. Maximal I(Ca,L) responses to ISO were depressed in both experimental groups, but the EC(50) values were not affected. In the Se-deficient group, basal, ISO- or forskolin-induced adenylate cyclase (AC) activity, measured in cardiac membrane preparations, was reduced when compared to the control, whereas 5' guanylyimidodphosphate (GppNHp) stimulated activity was unaffected. Decreased beta-adrenoceptor density and reduced GppNHp-induced affinity shift in ISO binding were also observed in the deficient group. No such differences were present in the excess group. These results suggest that combined Se and vitamin E deficiency interferes with beta-adrenoceptor-AC coupling, whereas excess intake of Se does not affect it. Thus, in the deficient group, the impairment of I(Ca) responses to ISO may be a result of a defect in beta-adrenoceptor-AC pathway. Impairment of I(Ca) response in the excess group, however, appears to have a different underlying mechanism.

Adenylyl Cyclases↗

PubMed2002

Effect of basal lamina of ovarian follicle on T- and L-type Ca(2+) currents in differentiated granulosa cells.

Patch clamp experiments were conducted to study the effects of basal lamina (basement membrane) of chicken ovarian follicle on membrane Ca(2+) currents in differentiated chicken granulosa cells in a homologous system. The whole cell patch clamp technique was used to simultaneously monitor membrane capacitance (an indirect measure of total cell surface area) and currents flowing through voltage-dependent Ca(2+) channels (using Ba(2+) as the charge carrier). Membrane capacitance was smaller in cells incubated on intact basal lamina than in control cells (incubated on tissue culture-treated plastic substratum). Granulosa cells expressed both T- and L-type Ca(2+) currents, and the amplitudes of the currents in cells incubated on intact basal lamina were significantly lower than those of control cells. Also, granulosa cells incubated on intact basal lamina were found to have significantly lower T- or L-type Ca(2+) current densities than control cells. Intact basal lamina that had been stored for 12 mo produced effects on T- and L-type Ca(2+) currents similar to those caused by freshly isolated basal lamina. The basal lamina was solubilized completely in one step and used to coat glass coverslips (uncoated glass coverslips served as controls). Granulosa cells incubated on coverslips precoated with solubilized basal lamina assumed spherical shape similar to those incubated on intact basal lamina. Similar to the observations made for intact basal lamina, the solubilized basal lamina suppressed T- and L-type Ca(2+) currents in the differentiated granulosa cells. Moreover, fibronectin, laminin, and type IV collagen, obtained from commercial sources, attenuated T- and L-type Ca(2+) currents in the differentiated granulosa cells. This interplay between basal lamina and Ca(2+) currents may be one mechanism that subserves the effects of the matrix material on metabolic functions of granulosa cells.

Animals↗

PubMed1993

Satellite glial cells in situ within mammalian prevertebral ganglia express K+ channels active at rest potential.

Patch-clamp experiments were performed on satellite glial cells wrapped around sympathetic neurons in the rabbit coeliac ganglion. With the cleaning method used, the glial cells could be kept in place and were directly accessible to the patch-clamp pipettes. Whole-cell recordings showed that glial cells had almost ohmic properties. Their resting potential (-79.1 +/- 1.2 mV) was found to be very nearly the same as the K+ reversal potential and approximately 20 mV more negative than that of the neurons they encapsulated. Unitary currents from ionic channels present in the glial membrane were recorded in the cell-attached configuration with pipettes filled with various amounts of K+, Na+ and gluconate. Only K(+)-selective channels with slight inwardly rectifying properties (in the presence of 150 mM [K+]o) were detected. These channels were active (Po = 0.7-0.8) at the cell resting potential. The channel conductance, but not its opening probability, was dependent on the [K+] in the pipette. Cl(-)-selective channels (outwardly rectifying and large conductance channels) were detected in excised patches. The properties of the K+ channels (increased inward current with [K+] and detectable outward current at low [K+]) are well suited for siphoning the K+ released by active neurons.

Animals↗

PubMed1994

Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes.

The nicotinic acetylcholine receptor (AChR) is a pentameric complex made up of four types of subunits in the stoichiometry alpha 2 beta gamma delta. These subunits have been shown to be differentially phosphorylated by cAMP-dependent protein kinase (PKA) protein kinase C, and a protein tyrosine kinase. A variety of studies have suggested that phosphorylation of the AChR in vitro and in vivo regulates the rate of desensitization of the receptor. In this study we have used site-specific mutagenesis and patch-clamp techniques to examine the role of phosphorylation in the regulation of desensitization of the AChR expressed in Xenopus oocytes Expression of wild-type AChR in Xenopus oocytes results in the constitutive phosphorylation of the AChR on the gamma and delta subunits. This phosphorylation is apparently due to the high basal level of PKA in oocytes since a specific peptide inhibitor of PKA completely eliminated phosphorylation of the AChR by oocyte extracts in vitro. The phosphorylation of the AChR in oocytes was not significantly enhanced by forskolin or cAMP analogs or by coexpression with the catalytic subunit of PKA, suggesting that the basal activity of PKA in oocytes is sufficient to phosphorylate the receptor to a high stoichiometry. Using site-specific mutagenesis, the sites of phosphorylation were determined to be serines 353 and 354 on the gamma subunit and serines 361 and 362 on the delta subunit. To examine the functional properties of wild-type and mutant receptors lacking phosphorylation sites, we used patch-clamp techniques to measure the responses of out-side-out patches to repetitive pulses of ACh using a rapid perfusion system. Wild-type and mutant receptors showed rapid concentration-dependent activation and desensitization to applied agonist. The time constant of desensitization of ensemble mean currents ranged from several hundred milliseconds at low ACh concentrations to 100-200 msec at saturating concentrations. The desensitization time constants for mutant receptors lacking all phosphorylation sites were significantly slower than wild-type phosphorylated receptors at all concentrations of ACh tested. In addition, mutant receptors that had the serine residues changed to glutamate residues in order to mimic the negative charge of the phosphorylated serine residue produced receptors that had desensitization rates approaching those of the wild-type phosphorylated receptor. These results provide further support that phosphorylation of the nicotinic ACh receptor regulates rate of desensitization.

Animals↗

PubMed1997

Basic FGF enhances calcium permeable channel openings in adult rat cardiac myocytes: implication in the bFGF-induced increase of free Ca2+ content.

Basic fibroblast growth factor (bFGF) has been implicated in the changes in gene expression that, under pathological conditions such as ischemia or volume overload, lead to adult cardiomyocyte hypertrophy. In many tissues, one of the first events following cell activation by growth factors is an enhancement of the intracellular free calcium concentration, generated by fluxes from internal storage compartments and through channels of the plasma membrane. The present study was undertaken to determine whether cardiac myocytes isolated from adult rat ventricles express Ca2+-permeable channels activated by bFGF. Using the cell-attached mode of the patch-clamp technique, we observed that bFGF (from 0.1-10 nM) induced an increase of fast burst openings, mediated by Ca2+-permeable channels with low conductance (15 pS) and voltage-independence. Inside-out patch-clamp experiments revealed that inositol 1,4,5-trisphosphate (5 microM) enhanced the opening of Ca2+-permeable channels with similar properties as the bFGF-induced channels, indicating that IP3 may be a second messenger of this process. Confocal fluorescence imaging of intracellular free calcium provided direct evidence that bFGF induced an increase of cytoplasmic and nucleoplasmic free Ca2+ concentrations which were generated, in part, by Ca2+ influx through the plasma membrane. In conclusion, this study supports the presence, in the plasma membrane of adult cardiac myocytes, of messenger-activated calcium channels which could play key roles in the calcium-dependent pathways that are activated in response to growth factors.

Animals↗

PubMed2005

Short hairpin RNA-mediated selective knockdown of NaV1.8 tetrodotoxin-resistant voltage-gated sodium channel in dorsal root ganglion neurons.

BACKGROUND: Voltage-gated sodium channels comprise a family of closely related proteins, each subserving different physiologic and pathologic functions. NaV1.8 is an isoform of voltage-gated sodium channel implicated in the pathogenesis of inflammatory and neuropathic pain, but currently, there is no isoform-specific inhibitor of any voltage-gated sodium channels. The authors explored the possibility of short hairpin RNA-mediated selective knockdown of NaV1.8 expression. METHODS: DNA constructs designed to transcribe short hairpin RNA targeting NaV1.8 were created and incorporated into recombinant lentiviruses. The virus-induced selective knockdown of NaV1.8 was examined at the protein, messenger RNA, and functional levels using Western blot, immunohistochemistry, reverse-transcription polymerase chain reaction, and patch clamp electrophysiology. RESULTS: Transduction of HEK293 cells stably expressing NaV1.8 or primary dorsal root ganglion neurons with lentivirus expressing short hairpin RNA resulted in the knockdown of NaV1.8 protein and messenger RNA concentrations. Whole cell patch clamp recordings confirmed decrease in the NaV1.8-mediated current density without changes in other biophysical properties. CONCLUSIONS: A selective knockdown of NaV1.8 expression in dorsal root ganglion neurons can be attained by short hairpin RNA delivered with lentivirus. This method may provide a new gene therapy approach to controlling neuronal hyperexcitability and pathologic pain.

Animals↗

PubMed1990

Red light regulates calcium-activated potassium channels in mougeotia plasma membrane.

The alga Mougeotia has a large central chloroplast whose positioning is regulated by photoactivation of phytochrome, possibly via modulation of cytosolic calcium (Serlin B, Roux SJ [1984] Proc Natl Acad Sci USA 81: 6368-6372). We used the patch clamp technique to examine the effects of red and far-red light on ion channel activity in the plasma membrane of Mougeotia protoplasts to determine if ion channels play a role in chloroplast movement. Patch clamping in the cell-attached mode reveals two channels of about 2 and 4 picoamperes amplitude at 0 millivolt (inside pipette) and estimated conductances of 30 and 65 picosiemens. They are activated by red light irradiation after a lag period of about 2 to 5 minutes. Far-red light, when applied immediately after red light irradiation, reverses this activation; otherwise it has no effect. This result implicates phytochrome. The addition of the calcium ionophore, A23187, also activates ion channel activity after a lag of a few minutes. The channels are not specific for calcium since they are present when calcium is removed from the external and pipette media. They are inhibited by quaternary ammonium ions. Thus, we believe they are calcium-activated potassium channels. Their possible role in chloroplast positioning is discussed.

Journal Article↗

PubMed2006

Activation of the cGMP/PKG pathway inhibits electrical activity in rabbit urethral interstitial cells of Cajal by reducing the spatial spread of Ca2+ waves.

In the present study we used a combination of patch clamping and fast confocal Ca2+ imaging to examine the effects of activators of the nitric oxide (NO)/cGMP pathway on pacemaker activity in freshly dispersed ICC from the rabbit urethra, using the amphotericin B perforated patch configuration of the patch-clamp technique. The nitric oxide donor, DEA-NO, the soluble guanylyl cyclase activator YC-1 and the membrane-permeant analogue of cGMP, 8-Br-cGMP inhibited spontaneous transient depolarizations (STDs) and spontaneous transient inward currents (STICs) recorded under current-clamp and voltage-clamp conditions, respectively. Caffeine-evoked Cl- currents were unaltered in the presence of SP-8-Br-PET-cGMPs, suggesting that activation of the cGMP/PKG pathway does not block Cl- channels directly or interfere with Ca2+ release via ryanodine receptors (RyR). However, noradrenaline-evoked Cl- currents were attenuated by SP-8-Br-PET-cGMPs, suggesting that activation of cGMP-dependent protein kinase (PKG) may modulate release of Ca2+ via IP3 receptors (IP3R). When urethral interstitial cells (ICC) were loaded with Fluo4-AM (2 microm), and viewed with a confocal microscope, they fired regular propagating Ca2+ waves, which originated in one or more regions of the cell. Application of DEA-NO or other activators of the cGMP/PKG pathway did not significantly affect the oscillation frequency of these cells, but did significantly reduce their spatial spread. These effects were mimicked by the IP3R blocker, 2-APB (100 microm). These data suggest that NO donors and activators of the cGMP pathway inhibit electrical activity of urethral ICC by reducing the spatial spread of Ca2+ waves, rather than decreasing wave frequency.

Action Potentials↗

PubMed1999

[Antibodies directed to voltage-gated potassium channels in sera from acquired neuromyotonia and related disorders].

Anti-voltage-gated potassium channel (VGKC) antibodies were measured in sera from 21 patients with acquired neuromyotonia (Isaacs syndrome) and related disorders. Seventeen of 21 sera suppressed potassium currents of neuroblastoma cell line (NB-1) using the patch clamp method, and the finding is mostly correlated with myokymic discharge in electromyography. Regarding the pathophysiology of suppression of VGKC, we investigated the effects of sera from 2 ANM patients on channel kinetics using the patch clamp method. The activation and inactivation kinetics of the potassium current were not altered by patients' immunoglobulins. We also investigated the effect of patients' sera on different VGKC subtypes using hKv1.1 and 1.6 transfected CHO cells. The same patients' sera suppressed the expressed VGKCs. Our results suggest that anti-VGKC antibodies may be positive not only in patients with classical ANM (Isaacs syndrome) but also in patients with related disorders who did not fulfill the characteristic symptoms. These disorders may be categorized as potassium channelopathy of the peripheral nervous system. The pathophysiology of the suppression of VGKC by antibodies is not the direct effect on channel kinetic but due to increased degradation or reduced production of VGKC. Epitopes of Anti-VGKC antibodies are still unclear.

Autoantibodies↗