Reduction Of Outward Currents Research Articles

Increased KCNJ18 promoter activity as a mechanism in atypical normokalemic periodic paralysis.

ObjectiveTo identify the genetic basis of a patient with symptoms of normokalemic sporadic periodic paralysis (PP) and to study the effect of KCNJ18 mutations.MethodsA candidate gene approach was used to identify causative gene mutations, using Sanger sequencing. KCNJ18 promoter activity was analyzed in transfected HEK293 cells with a luciferase assay, and functional analysis of Kir2.6 channels was performed with the two-electrode voltage-clamp technique.ResultsAlthough we did not identify harmful mutations in SCN4A, CACNA1S, KCNJ2 and KCNE3, we detected a monoallelic four-fold variant in KCNJ18 (R39Q/R40H/A56E/I249V), together with a variant in the respective promoter of this channel (c.-542T/A). The exonic variants in Kir2.6 did not alter the channel function; however, luciferase assays revealed a 10-fold higher promoter activity of the c.-542A promoter construct, which is likely to cause a gain-of-function by increased expression of Kir2.6. We found that reducing extracellular K+ levels causes a paradoxical reduction in outward currents, similar to that described for other inward rectifying K+ channels. Thus, reducing the extracellular K+ levels might be a therapeutic strategy to antagonize the transcriptionally increased KCNJ18 currents. Consistently, treatment of the patient with K+ reducing drugs dramatically improved the health situation and prevented PP attacks.ConclusionsWe show that a promoter defect in the KCNJ18 gene is likely to cause periodic paralysis, as the observed transcriptional upregulation will be linked to increased Kir2.6 function. This concept is further supported by our observation that most of the PP attacks in our patient disappeared on medical treatment with K+ reducing drugs.

Inhibition of K+ Currents in Type I Vestibular Hair Cells by Gentamicin and Neomycin

Significant ototoxicity limits the use of aminoglycoside (AG) antibiotics. Several mechanisms may contribute to the death of both auditory and vestibular hair cells. In this study the effects of gentamicin and neomycin on K⁺ currents in mature and early postnatal type I vestibular hair cells (HCI) were tested directly. The whole-cell patch clamp technique was used to assess the effects of AG and KCNQ channel modulators on K⁺ currents (I_K) in HCI acutely isolated from gerbil semicircular canals. Extracellular neomycin (1 m<smlcap>M</smlcap>) rapidly reduced peak outward I_K by 16 ± 4% (n = 9) in mature HCI (postnatal days, P, 25-66). Gentamicin (5 m<smlcap>M</smlcap>) reduced outward I_K by 16 ± 3% (n = 8). A similar reduction in outward current was seen in immature HCI (P5-9) that lacked the low-voltage-activated component of I_K observed in mature cells. Intracellular application of gentamicin and neomycin also reduced I_K in mature HCI. Modulators of KCNQ channels were used to probe KCNQ channel involvement. The selective KCNQ antagonist XE991 did not reduce I_K and the neomycin-induced reduction in I_K was not reversed by the KCNQ agonist flupirtine. Application of intracellular poly-<smlcap>D</smlcap>-lysine to sequester PIP₂ did not reduce I_K. Application of the K⁺ channel blocker 4-aminopyridine (4-AP) strongly reduced I_K, and extracellular AG in the presence of 4-AP gave no further inhibition of I_K. In summary, AG significantly reduce the 4-AP-sensitive I_K in early postnatal and mature HCI. K⁺ current inhibition differs from that seen in outer hair cells, since it does not appear to involve PIP₂ sequestration or KCNQ channels.

A Functional Role for the 'Fibroblast-Like Cells' in Gastrointestinal Smooth Muscles (J Physiol 2011;589[Pt 3]:697-710)

The physiologic role of fibroblast-like cells (FLCs) has not been well established. Recently Kurahashi et al1 suggested the involvement of FLCs in neurotransmission by Ca2+-activated K+ (SK3) channels. They demonstrated that a network of more extensively branching cells with platelet-derived growth factor receptor alpha (PDGFRα) existed between the circular and longitudinal muscles in the plane of the myenteric plexus. FLCs (PDGFRα+) were located in the same anatomical spaces and distributed in similar density as interstitial cells of Cajal (ICC), but PDGFRα+ cells were distinct from c-Kit+ ICC, as shown by double labeling. Other double labeling experiments with anti-SK3 antibody revealed that PDGFRα+ cells in both muscle layers and the region of the myenteric plexus expressed SK3. Some other experiments showed that PDGFRα+ cells labeled with enhanced green fluorescent protein (eGFP) produced robust expression of P2ry1 (P2Y1 receptors) and Kcnn3 (SK3 channels). Whole-cell patch clamp results suggested that PDGFRα+ cells express a high density of apamin-sensitive Ca2+-activated K+ channels, which is consistent with the expression of SK3 channels: small amplitude and time-dependent outward currents without voltage-dependent inward currents in step depolarization, and significant reduction of this currents by apamin 300 nM and charybdotoxin (block both BK and IK conductance Ca2+-activated K+ channels) caused a small but significant reduction in outward currents. Single channel patch recording (excised inside-out patch) to change Ca2+ at the intracellular surface produced the results consistent with previous reported data of Ca2+-activated K+ channels. Pharmacological data using purines (ATP, β-NAD and ADP) evoked significant outward currents in PDGFRα+ cells. Apamin and MRS2500 (P2Y1 antagonist) attenuated current amplitudes. The authors concluded that PDGFRα+ cells are a novel class of excitable cells with large current densities attributable to SK channels that are the molecular and ionic apparatus to mediate enteric inhibitory responses to purines in gastrointestinal (GI) muscles.

Contribution of Ca2+-activated K+ channels to central chemosensitivity in cultivated neurons of fetal rat medulla.

Neurons in fetal rat medullary slices that exhibited spontaneous electrical activity after blockade of synaptic transmission were investigated for their response to decreases in extracellular pH. Increases in [H+] (induced either by fixed acid or increases in PCO2) induced a significant increase in the frequency of action potentials, associated with a membrane depolarization, and/or increases in the slope of the interspike depolarization. In addition, CO2/H+ prolonged the repolarizing phase of action potentials and reduced the afterhyperpolarization, suggesting that K+ channels were the primary site of CO2/H+ action. The type of K+ channel that was modulated by CO2/H+ was identified by application of agents that inhibited Ca2+-activated K+ channels either directly (tetraethylammonium chloride, TEA) or indirectly (Cd2+ ions) by inhibiting Ca2+ influx. CO2/H+ effects on neuronal activity were abolished after application of these blockers. The contribution of Ca2+-activated K+ channels to H+ sensitivity of these neurons was confirmed further in voltage-clamp experiments in which outward rectifying I-V curves were recorded that revealed a zero current potential of -70 mV. CO2/H+ induced a prominent reduction in outward currents and shifted the zero current potential to more positive membrane potentials (mean -63 mV). The CO2/H+-sensitive current reversed at -72 mV and was blocked by external application of TEA. It is concluded that CO2/H+ exerts its stimulatory effects on fetal medullary neurons by inhibition of Ca2+-activated K+ channels, either directly or indirectly, by blocking voltage-dependent Ca2+ channels, which in turn results in a reduction of K+ efflux and in cell depolarization.

Effects of growth hormone-releasing peptide-2 (GHRP-2) on membrane Ca2+ permeability in cultured ovine somatotrophs.

The newly synthesized GH-releasing peptide, GHRP-2 (D-Ala-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2), was studied in somatotroph-enriched populations of ovine pituitary cells in primary culture. Nystatin-perforated whole-cell recordings were made on identified somatotrophs after 4-14 days of culture. Using a standard bath solution (containing Na+, Ca2+) and an electrode solution containing K+ in current-clamp recordings, GHRP-2 (10 nM) depolarized the membrane potential of the cells triggering a burst of action potentials. Voltage-clamp recordings indicated that GHRP-2 produced a slowly inactivated inward current with a slight reduction in outward current. The inward current was blocked by the Ca2+ channel blocker, Co2+ (0.5 mM). Ca2+ currents were then isolated using tetraethylammonium bath solution and an electrode solution containing Cs+. Ovine somatotrophs possess transient (T type) and long lasting (L type) Ca2+ currents. The L type current was abolished by addition of nifedipine (3 microM) to the bath solution and T type current was isolated on this basis. Current-voltage relationships indicated an increase in both T and L type Ca2+ currents in response to GHRP-2. The voltage-dependent inactivation curve for T type Ca2+ current was shifted towards a less negative level by the peptide. Intracellular free Ca2+ concentration ([Ca2+]i) in somatotroph-enriched populations was specifically increased by GHRP-2 but this effect was totally abolished by blockade of membrane Ca2+ channels. These data show that GHRP-2 causes an influx of Ca2+ leading to an increase in [Ca2+]i in ovine somatotrophs.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen deprivation inhibits a K+ channel independently of cytosolic factors in rat central neurons.

1. K+ channel modulation has been shown to be an integral and important cellular response to O2 deprivation. Although part of this modulation occurs as a result of changes in concentrations of several cytosolic factors such as ATP and Ca2+, it is unknown whether there are mechanisms other than those originating from the cytosol. To test the hypothesis that membrane-delimited mechanisms participate in the O2-sensing process and are involved in the modulation of K+ channel activity in central neurons, we performed experiments using patch-clamp techniques and dissociated cells from the rat neocortex and substantia nigra. 2. Whole-cell outward currents were studied in voltage-clamp mode using Na(+)-free or low-Na+ (5 mM, with 1 microM tetrodotoxin) extracellular medium plus 0.5 mM Co2+. O2 deprivation produced a biphasic response in current amplitude, i.e. an initial transient increase followed by a pronounced decrease in outward currents. The reduction in outward currents was a reversible process since perfusion with a medium of PO2 > 100 mmHg (1 mmHg = 133 Pa) led to a complete recovery. 3. In cell-free excised membrane patches, we found that a specific K+ current (large conductance, inhibited by micromolar concentrations of ATP and activated by Ca2+) was reversibly inhibited by lack of O2. This was characterized by a marked decrease in channel open-state probability and a slight reduction in unitary conductance. The magnitude of channel inhibition by O2 deprivation was closely dependent on O2 tension. The PO2 level for 50% channel inhibition was about 10 mmHg with little or no inhibition at PO2 > or = 20 mmHg. 4. Single-channel kinetic analysis showed that channel open times consisted of two components and closed times were composed of three. The hypoxia-induced inhibition of K+ channel activity was mediated by selective suppression of the longer time constant channel openings without significantly affecting closed time constants. This led to an increase in frequency of opening and closing and rapid channel flickerings. 5. Our data showed that O2 deprivation had no effect on another K+ current characterized by a much smaller conductance and Ca2+ independence. This provides evidence for the selective nature of the hypoxia-induced inhibition of some species of K+ channels. 6. These results therefore provide the first evidence for regulation of K+ channel activity by O2 deprivation in cell-free excised patches from central neurons.

Characterization of potassium currents in adult rat sensory neurons and modulation by opioids and cyclic AMP

Using the whole-cell patch-clamp technique on acutely dissociated and cultured adult rat sensory neurons, we characterized the K + currents by voltage dependence, kinetics, calcium dependence, and pharmacology. In the presence of Ca channel blockers, the cells heterogeneously expressed transient and sustained outward K + currents. The transient current was a high-threshold A-current which activated at potentials greater than −30 mV and was blocked by 4-aminopyridine. Some of the sustained current was classified as a delayed rectifier. It demonstrated shallow voltage-dependent inactivation and was blocked by tetraethylammonium. Capsaicin produced large reductions in both transient and sustained currents with an ec 50of 8 μM. Likewise, dendrotoxin partially blocked both currents but with an ec 50 of 21 nM. In the absence of Ca channel blockers, a prominent Ca-dependent K + current was observed. The kinetics of whole-cell potassium currents varied widely among cells, perhaps reflecting the different functional properties of sensory neurons. We also investigated the effects of elevating intracellular cyclic AMP and applying opioids on K + currents. Membrane-permanent analogs of cyclic AMP and phosphodiesterase inhibitors caused small reductions in voltage-dependent outward current. In contrast, forskolin produced a large reduction in outward current. This response was not solely mediated by cyclic AMP, since large responses were elicited with an inactive congener, 1,9-dideoxyforskolin, but not with the active, water-soluble congener, 7-deacetyl-6-[N-acetylglycyl]-forskolin. Surprisingly, opioids had no effect on resting or voltage-dependent K + conductances. However, opioid inhibition of Ca 2+ currents and Ca-dependent K 2+ currents was observed. The failure to demonstrate opioid modulation of resting or voltage dependent K 2+ currents suggests that modulation of Ca 2+ currents is the principal mechanism for the inhibitory effect of opioids on sensory neurons.

Modulation by Mg2+ and ADP of ATP-sensitive potassium channels in frog skeletal muscle.

The patch-clamp technique was used to examine the action of intracellular magnesium ions and ADP in the absence of ATP on skeletal muscle ATP-sensitive potassium channels (K-ATP channels). Inside-out patches were excised from the membrane of sarcolemmal blebs which arise spontaneously without enzymatic treatment after a frog muscle fiber is split in half. In the absence of nucleotides, K-ATP channel open probability was not significantly affected by intracellular magnesium even at a concentration (20 mM) which fully blocks cardiac and pancreatic K-ATP channels. On the other hand, Mg2+ ions (10-20 mM) decreased both inward and outward unitary currents. The percent reduction in inward currents (about 8%) was independent of voltage while the reduction in outward currents was larger at higher voltages, suggesting that the former effect resulted from cancellation of surface charges and the latter from rapid channel block. With or without Mg2+, intracellular ADP could either stimulate or inhibit K-ATP channel activity. Low concentrations (1-100 microM) of ADP rapidly and reversibly increased average activity by a factor of 2 to 3. This activation was seen in half of the patches tested and was greater in the presence of mM Mg2+. High concentrations (> 100 microM) of ADP inhibited activity with a half-block concentration of 450 microM in 0 Mg2+, i.e., more than an order of magnitude the value for ATP. ADP inhibition, like ATP inhibition, was partially relieved by mM Mg2+, suggesting that the Mg(2+)-bound ADP forms are less effective than free ADP forms. During exercise, free ADP levels rise and ATP declines while remaining high.(ABSTRACT TRUNCATED AT 250 WORDS)

Slow inward current in single cells isolated from adult human ventricles.

Characteristics of the slow inward current (Isi) in human ventricular myocytes isolated from septal specimens obtained in patients undergoing corrective cardiac surgery were studied using the whole-cell clamp method. A first series of experiments was performed under normal standard superfusion. Clamping from -60 mV evoked an inward current with a threshold at about -35 mV, a maximum around +10 mV and an apparent reversal potential at about +55 mV. No overlapping transient or background outward currents were detected in the -60 to +30 mV potential range, but time-dependent and steady-state outward currents were elicited at potentials above +30 mV. An overlap of steady-state activation and inactivation curves was present between -30 and +10 mV and a slight relief from inactivation was observed for voltages positive to +10 mV. The time course of inactivation consisted of fast and slow phases with time constants differing by a factor of eight. Slow time constants of inactivation were shorter at potentials that elicited larger Isi, and longer at potentials inducing smaller Isi. Recovery from inactivation evolved slowly with 100% reactivation occurring in about 4000 ms. Switching the holding potential from -60 to -40 mV led to a reversible decline of Isi without any change of the decay time constants. Isi was significantly increased by 0.1 microM isoproterenol. Total or partial inhibition by inorganic (2 mM Mn2+, 3 mM Co2+, 1 mM Cd2+) and organic (1 microM methoxyverapamil, 5 microM diltiazem) calcium antagonists did not unmask any transient outward current. However, a consistent increase of Isi was reversibly observed with 3 mM 4-aminopyridine while using standard solutions. A second series of experiments carried out with K(+)- and Na(+)-free solutions did not demonstrate any significant change from data observed with standard solutions except a reduction of outward currents at steps above +30 mV and alteration of inactivation kinetics. In this experimental setting, 4-aminopyridine also increased Isi but to a lesser degree. We conclude that Isi, as compared to the outward currents, is dominant in the diseased human ventricular cells we have studied.

Cholinergically-induced changes: outward currents in hair cells isolated from the semicircular canal of the frog

Two cholinergically-induced modulations of membrane conductances have been identified in hair cells isolated from the crista ampullaris of the leopard frog (Rana pipiens), using the whole cell recording configuration of the patch clamp technique. Of 56 crista hair cells tested, 28 showed drug-induced changes in membrane current or membrane potential which were repeatable and could be reversed with washout of drug.The predominant effect (observed in 20 hair cells) of acetylcholine (Arch, 100μM to 1mM) or carbachol (1μM to 50μM) applied to these hair cells was the reduction of an outward current corresponding to a change in conductance of approximately −0.22 nS. This action by Ach on hair cells has been inferred from previous studies of afferent fiber discharge which reported an increase in firing rate with stimulation of efferent fibers or exogenous application of cholinomimetics (Bernard et al., 1985; Valli et al., 1986; Guth et al., 1986; Norris et al., 1988). The Ach-induced reduction in outward current was associated with a depolarization of the zero-current membrane potential by approximately +2.5 mV.In a total of 8 hair cells, an Ach-induced reversible increase in outward current was recorded. Changes in conductance were approximately +0.13 nS and were associated with a hyperpolarization of the zero-current membrane potential by approximately −2.2 mV. This current increase is likely to be responsible for the inhibitory post-synaptic potentials (IPSPs) which have previously been recorded intracellularly from acoustico-lateralis hair cells during stimulation of the efferent innervation (Flock and Russell, 1976; Ashmore and Russell, 1982; Art et al., 1984, 1985).Of the remaining 28 hair cells, six cells failed to exhibit any change in membrane conductance or membrane potential in the presence of cholinomimetics while an additional 15 cells exhibited decreases, and 7 cells exhibited increases in outward conductance, during application of Ach or carbachol, which were neither reversible with washout nor repeatable.The Ach-induced decrease in outward current could be reversible blocked by removal of Ca2+ from the external solution.The antagonism of the Ach-induced decrease on outward current by atropine (10−5 M) suggests that this current may correspond to a facilitatory become-preferring Ach receptor mediated response previously identified in the isolated semicircular canal (Norris et al., 1988).The enhanced conductance observed in the presence of Ach in some cells may be related to the suppressive actions of Ach in the isolated semicircular canal also reported by us (Norris et al. 1988) and to the inhibitory actions of efferent stimulation and Ach application previously investigated in turtle cochlear hair cells (Art et al., 1984).The possible identities of these conductances are discussed in relation to ionic currents previously identified in hair cells.

Isolation of calcium current and its sensitivity to monovalent cations in dialysed ventricular cells of guinea-pig.

The ion selectivity of the Ca2+ channels in single ventricular cells of guinea-pig was studied using a 'giga-ohm seal' patch electrode for voltage clamp and internal dialysis. To isolate the Ca2+ channel current, currents through the Na+ channel and K+ channels were minimized by replacing external Na+ with Tris+ and removing K+ from both sides of the membrane. With 5 mM-ATP and 5 mM-EGTA in the pipette solution, the Ca2+ current was well maintained for more than 30 min in K+- and/or Na+-free external solution. Substitution of Cs+ for intracellular K+ eliminated the region of negative slope conductance in the steady-state current-voltage curve and shifted the zero-current potential or resting potential from -80 to -31 mV. After Cs+ substitution, a large inward current still flowed via inwardly rectifying K+ channels, but was abolished by removing external K+, which resulted in reduction of the resting membrane slope conductance to 1% of the control value. A decaying outward current attributable to the inwardly rectifying K+ channel was observed on depolarization in 5.4 mM-external K+ solution with Cs+-rich internal solution after blocking Ca2+ current. The induction of that current caused an apparent decrease of Ca2+ channel current when the K+-rich internal solution was switched to the Cs+-rich one at an external K+ concentration of 5.4 mM. When inwardly rectifying K+ current was suppressed by exposure to K+-free external solution, replacement of intracellular K+ with Cs+ caused no significant change in the Ca2+ current. With Cs+-rich solution in the electrode, the decaying outward current was responsible for an apparent depression of the Ca2+ current observed when extracellular K+ was increased. When the K+ current was abolished by 0.2 mM-extracellular Ba2+, changes in external K+ concentration did not affect the Ca2+ current, excluding the possibility of a direct inhibitory action of K+ on the Ca2+ channel. A time- and voltage-dependent outward current attributed to Cs+ was observed at potentials above +30 mV in Na+-, K+-free external solution with Cs+-rich internal solution. This current persisted in the presence of 20 mM-intracellular TEA Cl and 5 mM-extracellular 4-aminopyridine. Inorganic Ca2+ channel blockers, such as Co2+ or Cd2+, not only suppressed the inward Ca2+ current but also caused some reduction in outward current. Thus the blocker-sensitive peak current reversed at around +75 mV.(ABSTRACT TRUNCATED AT 400 WORDS)