To Tetraethylammonium Research Articles

Functional and Molecular Differences between Voltage-Gated K+Channels of Fast-Spiking Interneurons and Pyramidal Neurons of Rat Hippocampus

We have examined gating and pharmacological characteristics of somatic K+ channels in fast-spiking interneurons and regularly spiking principal neurons of hippocampal slices. In nucleated patches isolated from basket cells of the dentate gyrus, a fast delayed rectifier K+ current component that was highly sensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP) (half-maximal inhibitory concentrations <0.1 mM) predominated, contributing an average of 58% to the total K+ current in these cells. By contrast, in pyramidal neurons of the CA1 region a rapidly inactivating A-type K+ current component that was TEA-resistant prevailed, contributing 61% to the total K+ current. Both types of neurons also showed small amounts of the K+ current component mainly found in the other type of neuron and, in addition, a slow delayed rectifier K+ current component with intermediate properties (slow inactivation, intermediate sensitivity to TEA). Single-cell RT-PCR analysis of mRNA revealed that Kv3 (Kv3.1, Kv3.2) subunit transcripts were expressed in almost all (89%) of the interneurons but only in 17% of the pyramidal neurons. In contrast, Kv4 (Kv4.2, Kv4.3) subunit mRNAs were present in 87% of pyramidal neurons but only in 55% of interneurons. Selective block of fast delayed rectifier K+ channels, presumably assembled from Kv3 subunits, by 4-AP reduced substantially the action potential frequency in interneurons. These results indicate that the differential expression of Kv3 and Kv4 subunits shapes the action potential phenotypes of principal neurons and interneurons in the cortex.

Potassium conductance in the androgen-sensitive prostate cancer cell line, LNCaP: involvement in cell proliferation.

Very little is known about the expression of ion channels in prostate cells (both normal and malignant), and their possible role in physiological and pathological functions. We therefore studied ion conductances and their role in the proliferation of LNCaP cells, an androgen-sensitive human prostate cancer cell line. We applied patch-clamp recording techniques for electrophysiological studies, and 3H-thymidine incorporation and protein content assays for cell growth studies. Only one type of voltage-dependent ion conductance, a potassium K+ conductance, was identified. This current, which was depressed by a rise in intracellular Ca2+, had a high sensitivity to tetraethylammonium (TEA) (with half-block at 2 mM) and was also inhibited by 2 nM alpha-dendrotoxin (DTX) and 20 nM mast-cell degranulating peptide (MCDP). K+ channel inhibitors inhibited [3H]thymidine incorporation and protein content, in a dose-dependent fashion, indicating that K+ channels are involved in cell growth. We conclude from our findings that the human cancer prostate cell line LNCaP has a new type of K+ channel, likely to play an essential role in the physiology of these cells and, more specifically, in cell proliferation.

Potassium currents and membrane excitability of neurons in the rat's dorsal nucleus of the lateral lemniscus.

1. The contribution of voltage-activated outward potassium currents to membrane excitability of neurons in the rat's dorsal nucleus of the lateral lemniscus (DNLL) was studied in a brain slice preparation using whole cell patch-clamp and intracellular recordings. Voltage-clamp methods and pharmacological manipulations were used to examine the currents regulating membrane dynamics in DNLL. 2. A delayed sustained outward current was evoked by applying depolarizing voltage steps across the cell membrane from a holding potential of -50 mV. An additional transient outward current was evoked when the depolarizing steps were preceded by a hyperpolarizing prepulse of -110 or -120 mV. 3. The transient outward current peaked within 6.8 ms of the onset of a depolarizing pulse. It decayed with a time constant of 12.3 ms for a 60-mV depolarizing voltage shift. Half-inactivation of this current occurred at -81.3 mV. The time constant for removal of the inactivation was 17.4 ms. The transient current had a high sensitivity to 4-aminopyridine (4-AP). 4. The sustained current was activated more slowly and was more sensitive to tetraethylammonium (TEA) than the transient current. The sustained current had both Ca2+-dependent and Ca2+-independent components. The Ca2+-dependent portion emerged at potentials of about -35 mV and was activated fully at +10 mV. The Ca2+-independent component was activated at potentials more positive than -40 mV and increased in magnitude with further depolarization. Inactivation of the Ca2+-independent component was voltage dependent. Also, TEA suppressed the Ca2+-independent compound. 5. The transient current in DNLL neurons closely resembled the A current (IA) described for hippocampal and other neurons in both kinetics and pharmacology. The Ca2+-independent component of the sustained current resembled the K current (IK) described for other neurons in both its properties of activation and inactivation and its pharmacology. 6. The outward current of some DNLL neurons was found to contain a dendrotoxin-sensitive component. This component reached its peak at 6.8 ms and had voltage-sensitive time constants of decay of 25.5 and 8.5 ms with voltage steps of 40 and 60 mV, respectively. 7. Application of 4-AP and TEA markedly prolonged the spike width, abolished the fast component of the after hyperpolarization and depolarized the cell membrane. Also, the number of action potentials produced by positive current injection increased under the influence of 4-AP and TEA. Membrane excitability and spike repolarization were dependent on both 4-AP-sensitive transient and TEA-sensitive sustained currents. 8. Neurons in DNLL typically exhibit a steady discharge of action potentials in response to sustained membrane depolarization. The rate and temporal pattern of production of action potentials in these cells are determined by the combination of transient and sustained potassium channels.

Intracellular calcium stores and oscillatory contractions in arteries from genetically hypertensive rats.

Strips of tail artery from stroke-prone spontaneously hypertensive rats (SHRSP), but not from normotensive Wistar Kyoto (WKY) rats, exhibit oscillatory activity after stimulation with norepinephrine. In addition, oscillatory activity is observed in response to tetraethylammonium (TEA) in vessels from both SHRSP and WKY rats. Mechanistically, the oscillatory contractions are associated with calcium (Ca2+)-driven action potentials. We have tested the hypothesis that intracellular Ca2+ stores participate in the generation of norepinephrine-induced oscillatory contractions in tail arteries from SHRSP. Additionally, the role of intracellular Ca2+ stores on TEA-induced contractions were evaluated. Contractile force in strips of tail artery from SHRSP and WKY rats was measured, using standard muscle bath procedures, and the effect of interventions that affect the storage of intracellular Ca2+ on the oscillatory contractions was evaluated. Depletion of intracellular Ca2+ stores, with ryanodine, or inhibition of Ca2+ uptake into the sarcoplasmic reticulum (SR), with thapsigargin and cyclopiazonic acid (CPA), did not inhibit oscillatory contractions induced by norepinephrine in SHRSP vessels. However, these agents inhibited the amplitude of TEA-induced contractions in WKY strips. Bay K 8644 and A23187 inhibited TEA-induced oscillatory contractions in WKY vessels. In SHRSP tail artery Bay K 8644 inhibited both norepinephrine and TEA-induced contractions, while A23187 did not have any effect. The phospholipase C inhibitor, NCDC (3X 10(-5) M), blocked oscillatory activity induced by norepinephrine in SHRSP tail artery and TEA-induced oscillations both in SHRSP and WKY vessels. These observations suggest that Ca2+ release and Ca2+ uptake into intracellular Ca2+ stores are not involved in the contraction-relaxation cycles that characterize norepinephrine-induced oscillatory activity in SHRSP tail artery. Similarly, SR Ca2+ stores may modulate but are not essential for TEA-induced oscillatory contractions.

The pharmacological properties of K+ currents from rabbit isolated aortic smooth muscle cells

1. Using the whole-cell patch-clamp technique, the effects of several K+ channel blocking drugs on K+ current recorded from rabbit isolated aortic smooth muscle cells were investigated. 2. Upon depolarization from -80 mV, outward K+ current composed of several distinct components were observed: a transient, 4-aminopyridine (4-AP)-sensitive component (I1) and a sustained component (Isus), comprising a 4-AP-sensitive delayed rectifier current (IK(V)), and a noisy current which was sensitive to tetraethylammonium (TEA), and probably due to Ca(2+)-activated K+ current (IK(Ca)). 3. Several drugs in clinical or experimental use have as part of their action an inhibitory effect on specific K+ channels. Because of their differential K+ channel blocking effects, these drugs were used in an attempt to characterize further the K+ channels in rabbit aortic smooth muscle cells. Imipramine, phencyclidine, sotalol and amitriptyline failed to block selectively any of the components of K+ current, and were thus of little value in isolating individual channel contributions. Clofilium showed selective block of IK(V) in the presence of TEA, but only at low stimulation frequencies (0.07 Hz). At higher frequencies (1 Hz) of depolarization, both I1 and IK(V) were suppressed to a similar extent. Thus, the blocking action of clofilium was use-dependent. 4. The voltage-dependent inactivation of I1 and the delayed rectifier were very similar although a brief (100 ms) pre-pulse to -30 mV could preferentially inactivate I1. Together with the non-selective blocking effects of the K+ channel blockers, similarities in the activation and inactivation of these two components suggest that they may not exist as separate ionic channels, but as distinct kinetic states within the same K+ channel population. 5. The effects of all of these drugs on tension were examined in strips of rabbit aorta. The non-specific K+ channel blockers caused only minor increases in basal tension. TEA and 4-AP by themselves caused significant increases in tension and were even more effective when applied together. There appeared to be no correlation between the effects of the drugs tested on tension and their actions on currents recorded from isolated myocytes. Thus tension studies are an inappropriate means of investigating the mechanism of action of these drugs, and studies on ionic currents in isolated myocytes cannot easily predict drug actions on intact tissues.

Comparison of voltage-dependent potassium currents in rat pyramidal neurons acutely isolated from hippocampal regions CA1 and CA3

1. The properties of voltage-gated potassium currents were studied in acutely isolated rat hippocampal pyramidal cells from area CA1 and CA3 at postnatal ages of day 6-8, 9-14, and 26-29 (P6-8, P9-14, and P26-29) with the use of the whole cell version of the patch-clamp technique. 2. The outward current pattern of all cells under investigation could be separated in a fast transient A current (IA) and a delayed rectifier-like current (IK). 3. In both preparations, IA activated and inactivated rapidly. Vh describing steady-state inactivation was -84.5 mV in CA3 cells and -85.5 mV in CA1 cells. The activation behavior was characterized by Vh = -23.8 mV in CA3 cells and -27.2 mV in CA1 cells. The removal of inactivation was monoexponential both in CA1 and CA3 neurons with time constants of 32.1 and 28.5 ms, respectively. IA was insensitive to tetraethylammonium (TEA), dendrotoxin (300 nM), and mast cell degranulating peptide (200 nM), but could be blocked with 5 mM 4-aminopyridine (4-AP) by approximately 80%. In both preparations, A currents did not depend on Ca2+ influx. 4. Delayed rectifier currents (IK) in CA1 and CA3 pyramidal neurons decayed along a double exponential time course. Steady-state inactivation was described by Vh = -79.5 mV in CA3 cells and -76.0 mV in CA1 cells. The activation curves were characterized by midpoints of -3.8 mV in CA3 cells and of -1.4 mV in CA1 cells. The removal of inactivation was monoexponential in CA1 and CA3 neurons with time constants of 210.3 and 202.4 ms, respectively. All kinetic properties were identical for the differentially decaying components of IK. In CA1 cells IK was blocked by TEA at +30 mV with an IC50 of 0.98 mM. In CA3 cells the corresponding IC50 value was 1.05 mM. About 20% of IK were insensitive to TEA. IK was partially blocked by approximately 30% with 100 microM 4-AP. Mast cell degranulating peptide (100-200 nM) and dendrotoxin (50-300 nM) had no effect on IK. 6. Perfusion of charybdotoxin (30 nM), Cd2+ (300 microM), La3+ (10 microM), or Ca(2+)-free solutions resulted in the isolation of a small noninactivating outward current component. Around 10% of IK appeared to be Ca2+ dependent in CA1 neurons. In CA3 pyramidal cells Ca(2+)-dependent outward currents seemed to be somewhat larger with approximately 20%. 7. In CA1 as well as in CA3 cells, the kinetic and pharmacological properties of IA and IK remained stable during postnatal development. However, the contribution of IA and IK to the whole cell current varied with age. IA was more prominent in CA1 cells of age group P6-8 than in age-matched CA3 cells. CA3 cells had smaller A currents and larger delayed rectifier currents than CA1 pyramidal cells. Current densities of IA and IK were analyzed during development to assess changes in the expression of these currents. With increasing postnatal age, the expression of IA was downregulated in both preparations. This effect was more pronounced in CA3 than in CA1 cells. In contrast, IK was upregulated during the same developmental period. This increase in the expression of IK was with approximately 300% much larger in CA1 cells than in CA3 cells with only approximately 50%.

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: evidence for tetrameric channel formation.

Unitary currents were recorded from inside-out membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by beta functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent Kd of 80 microM at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent Kd at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%.(ABSTRACT TRUNCATED AT 250 WORDS)

Theophylline affects three different potassium currents in dissociated rat cortical neurones.

1. The effects of theophylline in pyramidal neurones acutely dissociated from the rat frontal cortex were investigated in the whole-cell configuration, using the nystatin-perforated patch-clamp technique. 2. Ten millimolar theophylline evoked triphasic responses: a small slow outward current (Iso), then a large transient outward current (Ito) and finally a slow sustained inward current (Isi). The reversal potentials of the three current components shifted 56-58 mV for a 10-fold change in extracellular K+ concentration, thereby indicating that all these current components were predominantly carried by K+. 3. Iso had no voltage dependence, whereas Ito showed a steep outward rectification. Iso was relatively resistant to tetraethylammonium (TEA) with an IC50 of 10 mM. Ito was susceptible to submillimolar TEA with an IC50 of 0.8 mM. 4. Isi was a net inward current mainly resulting from suppression of the M-current (IM). 5. These three current components had a distinct concentration dependence; in particular, Isi was evoked at a relatively lower concentration range. 6. Ito was not observed when the intracellular Ca2+ was chelated by 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) of 10 mM, using the conventional whole-cell recording configuration, whereas both Iso and Isi were retained but gradually diminished. 7. In Ca(2+)-free external solution, these responses were fully elicited by the first application of theophylline. However, Ito disappeared during successive applications and Iso, but not Isi, also decreased. Similar results were obtained in the presence of ryanodine. 8. Theophylline apparently affects three different kinds of K+ currents in rat cortical neurones. Both Iso and Ito depend on internal calcium mobilized from an intracellular Ca2+ store by theophylline, while Isi was not primarily mediated by a change in [Ca2+]i.

Forskolin inhibition of K + current in pregnant rat uterine smooth muscle cells

Two kinds of outward K + currents were examined in single smooth muscle cells from pregnant rat uterus, using whole-cell voltage clamp. The first and faster component was more sensitive to 4-aminopyridine (4-AP), whereas the second and slower (delayed) component was more sensitive to tetraethylammonium (TEA). A possible third K + component (Ca activated K + current) was not recorded as the pipette solution included EGTA. Forskolin inhibited the outward current in a concentration-dependent manner (50% inhibition occurred at about 30 μM); it affected the delayed component rather than the fast component. 8-Bromo-cAMP did not alter the outward current. In addition, inhibitors of protein kinase A and GDP-βS and GTP-γS did not affect the forskolin-induced inhibition. These results indicate that forskolin inhibition of the delayed component of the outward current is independent of cAMP generation in the pregnant rat myometrial cells. Therefore, forskolin seems to directly inhibit specific K + channels, as was reported for several other cell types.

Voltage-gated potassium currents in acutely dissociated rat cortical neurons.

1. We describe three outward K+ current components in acutely dissociated neurons from rat sensorimotor cortex on the basis of inactivation kinetics and voltage dependence. 2. The fast A current (IAf) was completely inactivated at -40 mV and half-inactivated at -52 mV. It activated [time to peak (TTP) 8 ms at -10 mV] and was inactivated (tau inact = 12 ms at -10 mV) rapidly. Recovery from inactivation had a time constant of approximately 80 ms at -100 mV. It was insensitive to tetraethyl ammonium (TEA) and dendrotoxin but was blocked by 4-aminopyridine (4-AP, IC50 = 1 mM). 3. The slowly inactivating current (IKS) was the largest current seen in acutely dissociated adult neurons. It was completely inactivated at -40 mV, half-inactivated at -98 mV, and was kinetically slower (TTP = 130 ms at -10 mV; tau inact = 293 ms at -10 mV) than the fast A current. Deactivation tails were fit with the sum of two exponentials with time constants of 2-10 and 15-40 ms. IKS recovered from inactivation with a time constant of approximately 1,200 ms at -100 mV. 4. There were two components that inactivated with even slower kinetics. The very slowly inactivating current (IKSS) was operationally defined as the current remaining after a 5-s hold at -40 mV. One component inactivated with a time constant of 1,927 ms at -10 mV. The other component showed no inactivation over a 5-s test command, but in 40- to 50-s steps to -10 mV, inactivated with a tau of approximately 20 s. The very slowly inactivating current activated with similar kinetics to IKS (TTP = 121 ms at -10 mV), and two deactivation tails, with kinetics similar to those after the -100 mV prepulse, were observed after holding at -40 mV. 5. Both IKS and IKSS were sensitive to TEA. Seventy-six percent (76%) of IKSS was blocked by 30 mM TEA. Two components to the TEA block were present for IKSS, with IC50s of 88 microM (67% of blockable current) and 7 mM (33%). Seventy percent (70%) of IKS was blocked by 30 mM TEA. For the IKS current, there were also two effective concentrations, with IC50s of 8 microM (21% of blockade current) and 3 mM (79%). 6. IKS and IKSS were also sensitive to 4-AP. Seventy-six percent (76%) of IKSS was blocked by 3-5 mM 4-AP. IKSS exhibited two components of 4-AP block.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in the electrical properties of chick ciliary ganglion neurones during embryonic development.

1. Whole-cell recording techniques were used to examine the expression of ionic currents in chick ciliary ganglion neurones dissociated acutely at various stages of embryonic development. Currents were also examined in dissociated cells that had been maintained in vitro for several days. 2. Voltage-activated, tetrodotoxin (TTX)-sensitive Na+ currents (INa) could be detected in all cells tested between stage 25 and stage 40 (embryonic days 4.5-14). INa increased in both amplitude and density throughout development, but no obvious changes in kinetics or sensitivity to TTX were observed. 3. High-threshold Ca2+ currents (ICa) were also detectable between stage 25 and stage 40. ICa increased in both amplitude and density throughout this time. No obvious changes in kinetics or voltage dependence were observed. 4. Delayed rectifier K+ currents (IDR) and A-currents (IA) could be detected in Ca(2+)-free salines, and distinguished on the basis of differences in kinetics, voltage dependence, and sensitivity to tetraethylammonium (TEA). IA was either absent, or present at very low densities at stages 26-30, but showed a sharp increase in density thereafter. In contrast, IDR was detectable as early as stage 25, and did not display a significant increase in density during development. 5. Ca(2+)-activated K+ currents (IK(Ca)) were either undetectable or present at very low density between stage 26 and stage 30 (embryonic days 5-9) but showed a large increase in amplitude and density thereafter. 6. Ionic currents were examined in age-matched cells dissociated acutely on embryonic day 13, or isolated on embryonic day 9 and maintained in vitro for an additional 4 days. Most of the cells maintained in culture for 4 days did not express detectable IK(Ca), and had significantly reduced IA compared to acutely isolated controls. The cultured cells expressed normal densities of IDR, ICa and INa. 7. All ionic currents increased in amplitude during normal embryonic development, and all but IDR increased in density. The largest change in density generally occurred between stages 30 and 40, during which time ciliary ganglion neurones form synapses with target tissues. 8. Isolation of ciliary neurones from the in ovo environment prevented the normal development of IA and IK(Ca), suggesting that the expression of these channels is controlled by one or more extrinsic environmental factors. In contrast, the normal expression of INa, ICa and IDR is not dependent upon extrinsic factors.

Multiple subunits of a voltage-dependent potassium channel contribute to the binding site for tetraethylammonium

RNAs encoding a wild-type (RBK1) and a mutant (RBK1(Y379V,V381 T); RBK1 ∗) subunit of voltage-dependent potassium channels were injected into Xenopus oocytes. When expressed separately, they made homotetrameric channels that differed about 100-fold in sensitivity to tetraethylammonium (TEA). Mixtures of channels having one, two, or three low affinity subunits were expressed by injecting various proportions of RBK1 and RBK1 ∗ RNAs. The affinity for TEA of these three channel species was deduced by fitting concentration-response curves for the inhibition of potassium currents. DNAs were also concatenated to construct a sequence that encoded two connected subunits, and channels that contained four, two, or no TEA-sensitive subunits were expressed. The results suggest that bound TEA interacts simultaneously with all four subunits.

Modulatory effects of FMRF-NH2 on outward currents and oscillatory activity in heart interneurons of the medicinal leech

Using single-electrode voltage clamp, heart interneurons of the medicinal leech were shown to possess both a rapidly inactivating outward current, IA, and a more slowly inactivating outward current, IK. IA and IK could be separated by their voltage sensitivity and kinetic properties. FMRF-NH2 (Phe-Met-Arg-Phe-NH2) modulates IK by shifting both steady state activation and inactivation to more hyperpolarized potentials, but it does not affect the time constants. IA and IK appear to use K+ as a charge carrier; a change in the external [K+] produced a shift in the apparent reversal potential in the direction predicted with potassium as the charge carrier. Both IA and IK are sensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP), and TEA and 4-AP both interfere with the effects of FMRF-NH2 on IK. The biophysical properties of IA and of IK in the presence and absence of FMRF-NH2 were incorporated into a Hodgkin-Huxley model of these currents that could reproduce voltage-clamp data. FMRF-NH2 produces two apparently dissimilar effects on the heartbeat rhythm--acceleration and disruption. We suggest that both effects could result from the hyperpolarizing shifts in steady state activation and inactivation of IK.

Tea‐sensitive potassium channels and inward rectification in regenerated rat sciatic nerve

Sucrose gap and intra-axonal recording techniques were used to identify the types of ion channels and inward rectification that are present in regenerated axons of adult (greater than 8 weeks) rat sciatic nerve after crush injury. In sucrose gap recordings, 4-aminopyridine (4-AP) led to slight broadening of the compound action potential (CAP) in normal nerve, and a greater broadening in regenerated nerves. By 12 days after sciatic nerve crush, regenerated nerves manifested an afterhyperpolarization (AHP) lasting up to 250 ms that was sensitive to tetraethylammonium (TEA). A similar TEA-sensitive AHP could be elicited with repetitive stimulation. Hyperpolarizing constant current steps (0.1 to 0.5 mA; 600-900 ms duration) applied across the sucrose gap through regenerated axons evoked membrane hyperpolarizations with a depolarizing, Cs(+)-sensitive relaxation in the response to hyperpolarization, which is characteristic of inward rectification, occurring after about 70 ms. The relaxation was present as early as 21 days after nerve crush. Intra-axonal recordings showed burst firing in 4-AP that was terminated by an AHP that temporally correlated with the TEA-sensitive AHP, and a relaxation in the response to hyperpolarizing current, similar to that of whole nerve recordings. The results demonstrate that in addition to voltage-sensitive sodium channels and 4-AP-sensitive potassium channels, there are TEA-sensitive and inwardly rectifying channels on mammalian regenerated peripheral nerve axons.

Single K + currents during differentiation of embryonic muscle cells in vitro

After 3–7 days in culture, chicken myotubes possess five types of K + channel: two high-conductance channels of 195 and 105 pS which are sensitive to tetraethylammonium (TEA), an ATP-sensitive channel of 64 pS and two low-conductance channels of 40 and 15 pS which are insensitive to TEA and ATP. The same population of channels is to be found in EGTA-treated muscle cells with blocked fusion and, with the exception of the ATP-sensitive channel, also in 1-day-old myoblasts. There are differences between myoblasts and myotubes in the percentage of incidence of individual channel types. High-conductance K + channels are most frequently to be observed in myotubes, but they are rare in myoblasts and EGTA-treated cells where low-conductance K + channels predominate.

Serotonergic modulation of two potassium currents in the pleural sensory neurons of Aplysia

1. The properties of membrane currents that were modulated by serotonin (5-HT) were investigated with two-electrode voltage-clamp techniques in sensory neuron somata isolated from the pleural ganglion of Aplysia californica. The modulatory effects of 5-HT were revealed by computer subtraction of current responses elicited in the presence of 5-HT from current responses elicited prior to the application of 5-HT. The complexities of the resulting 5-HT difference currents (I5-HT) suggested that 5-HT modulated more than one component of membrane current. 2. The 5-HT difference currents appeared to have at least two distinct components. One component was clearly evident at membrane potentials more negative than -10 mV was relatively voltage independent and did not inactivate. A second component was activated at membrane potentials more positive than -10 mV, had complex kinetics, and was highly voltage dependent. In an attempt to identify the membrane currents that were modulated by 5-HT, we compared the pharmacologic sensitivity of I5-HT to that of previously described K+ currents. 3. The two components of I5-HT had different sensitivities to agents that block K+ currents. The relatively voltage-independent component of I5-HT was not blocked by 2 mM 4-aminopyridine (4-AP) and was relatively insensitive to tetraethylammonium (TEA) (estimated Kd of 92 mM). In contrast, the voltage-dependent component of I5-HT was blocked by 4-AP (2 mM) and moderate concentrations of TEA (estimated Kd of 5 mM). 4. The K+ current blockers that were used to examine I5-HT were also used to examine voltage-activated membrane currents. Externally applied TEA blocked the delayed or voltage-dependent K+ current (IK.V) with an estimated dissociation constant (Kd) of 8 mM and a membrane current similar to the Ca2+-activated K+ current (IK.Ca) with an estimated Kd of 0.4 mM. In addition, externally applied 4-AP (2 mM) blocked IK.V. Thus TEA and 4-AP were equipotent in blocking both IK.V and the voltage-dependent component of I5-HT. 5. The suggestion that I5-HT contained multiple components was supported further by examining the modulatory effects of adenosine 3',5'-cyclic monophosphate (cAMP) that mediates some actions of 5-HT on membrane currents in these cells. cAMP difference currents (IcAMP) were similar to the relatively voltage-independent component of I5-HT. The subsequent addition of 5-HT to solutions already containing cAMP resulted in 5-HT difference currents similar to the voltage-dependent component of I5-HT.(ABSTRACT TRUNCATED AT 400 WORDS)

Single potassium channels recorded from vascular smooth muscle cells.

Single-channel activities were recorded from smooth muscle cells of vascular fragments isolated from rat pancreas. Three K-selective channel types were identified and characterized from cell-attached and inside-out membrane patches. Mean single-channel slope conductances of the three channel types, under conditions of symmetrical K (145 mM), were 43, 91, and 276 pS. The channel types exhibited differential sensitivity to tetraethylammonium (TEA) and intracellular Ca concentrations ([Ca2+]i). Intracellular TEA (10 mM) was effective in blocking the small conductance channel whereas the large conductance channel was found to be sensitive to [Ca2+]i. The properties of the large-conductance K channel are consistent with its identification as a calcium-activated maxi K channel described in a variety of tissues. Each of the channel types showed sensitivity to membrane potential, with increasing opening transitions at depolarizing membrane potentials. The small conductance channel type was observed to be active at resting membrane potentials. The generality of each of these channel types to other smooth muscle cell preparations and possible physiological implications are discussed.

Three potassium currents in mouse motor nerve terminals.

A study of the K conductance of the presynaptic membrane has been performed in the triangularis sterni muscle of the mouse. External currents generated in the presynaptic terminals upon invasion by action potentials have been recorded using microelectrodes inserted into the perineurium of preterminal nerve bundles. The voltage-dependent K current could be pharmacologically dissected into fast (IKf) and slow (IKs) components. While both are sensitive to 3,4-diaminopyridine (3,4-DAP), only IKf is sensitive to tetraethylammonium (TEA). Uranyl (100-200 microM) and guanidine (5-10 mM) produced a near complete block of IKf and IKs, which can explain their facilitatory effect upon transmitter release. The third K current of presynaptic terminals is Ca2+-dependent, but was activated also by Sr2+. This current could be suppressed by nanomolar doses of charybdotoxin; it is also sensitive to TEA but not to 3,4-DAP, uranyl or guanidine.

Membrane current responses of NG108‐15 mouse neuroblastoma x rat glioma hybrid cells to bradykinin.

1. Membrane current responses to focal application of bradykinin (BK) were recorded in voltage-clamped NG108-15 neuroblastoma x glioma hybrid cells. 2. BK produced sequential outward and inward currents at clamp potentials between -60 and -30 mV, designated IBK(out) and IBK(in), respectively. 3. The outward current IBK(out) was accompanied by an increased membrane conductance. Ramp current-voltage (I-V) curves yielded a reversal potential (VBK) of -80 +/- 5.6 mV (mean +/- S.D., n = 9) in 5.4 mM [K+]o. VBK showed a positive shift on raising [K+]o, compatible with a primary increase in K+ conductance. Subtracted I-V curves indicated that the underlying conductance was not strongly voltage dependent between -120 and -40 mV. 4. IBK(out) was inhibited by d-tubocurarine (dTC, 0.1-0.5 mM) but was insensitive to tetraethylammonium (TEA) below 5 mM. 5. The inward current IBK(in) was accompanied by a fall in membrane conductance. This was associated with the inhibition of a time- and voltage-dependent K+ current, IM. In consequence, IBK(in) was strongly voltage dependent and dissipated, usually without reversal, on hyperpolarizing the cell beyond -70 mV in 5.4 mM [K+]o. Reversal to an outward current negative to -40 mV could be obtained on raising [K+]o to 54 mM. 5. Both IBK(in) and IBK(out) persisted when ICa was blocked with Co2+ or Cd2+. IBK(out) slowly diminished in Ca2+-free, Mg2+-substituted solution. 6. The Ca2+ spike current ICa and the Ca2+-activated K+ current IAHP were inhibited during IBK(out) or after Ca2+ injections. BK did not affect the voltage-activated K+ current IK(V) recorded in Co2+ solution. 7. It is concluded that the dual response to BK results from opposing effects on two different species of K+ current. IBK(out) results from activation of a Ca2+-dependent, voltage-insensitive K+ conductance, probably mediated by a transient rise in intracellular Ca2+. It is suggested that the Ca2+ is released from an intracellular store. IBK(in) results primarily from inhibition of the Ca2+-independent, voltage-gated K+ current, IM. This effect is not replicated by a rise of intracellular Ca2+ and must therefore be generated by another mechanism.

A reappraisal of the evidence for a direct action of tetraethylammonium on the chick biventer cervicis muscle equilibrated with lidocaine

1. 1. Contractures of the chick biventer cervicis muscle were recorded in vitro in response to exogenous acetylcholine (ACh) and to tetraethylammonium (TEA) and concentration response curves (CRC) produced for these drugs. 2. 2. In BVC muscles equilibrated with lidocaine (1.07 mM), physostigmine (3.63 μM) blocked TEA induced contractures in a reversible non-competitive manner. It had a similar action on ACh induced contractures. 3. 3. Lidocaine (1.07 mM) had no action on the ACh CRC except that it reduced the response to the highest concentration of ACh tested. 4. 4. The data of this and of previous experiments was analyzed and a theory proposed to account for TEA contractures. 5. 5. It suggested that background extravesicular ACh release results in the passage of K + into the transverse tubular system (TT). 6. 6. In the presence of TEA blocking the K + channels accumulation of K + in the TT no longer occurs. 7. 7. Na +/K + exchange is depressed in favour of Na +/Ca 2+ exchange and the resulting rise in intracellular Ca 2+ acting on the sarcoplasmic reticulum leads to contraction. 8. 8. Lidocaine potentiates the contraction by blocking Ca 2+ uptake into the sarcoplasmic reticulum.