Double whole-cell patch-clamp characterization of gap junctional channels in isolated insect epidermal cell pairs

Double whole-cell patch-clamp methods were used to characterize Junctional membrane conductances in epidermal cell pairs isolated from the prepupal integument of the flour beetle, Tenebrio molitor. The mean initial Junctional conductance in 267 cell pairs was 9.5 ± 1.0 nS (range 0–95 nS). Well-coupled cell pairs uncoupled spontaneously with a half-time of 7.6 min. Adding 5 mM ATP to the pipette solution stabilized coupling with less than a 50% drop occurring after 30 min. Nonjunctional membrane potential was the major determinant of Junctional conductance with transjunctional potential playing a minor role. Junctional conductance approached 0 pA at nonjunctional membrane potentials greater than 0 mV and increased with hyperpolarization. The voltage at half-maximal conductance was –26 mV. The time course of the reversible changes in Junctional conductance were slow (30 sec) with time-dependent decay occurring faster and recovery occurring slower with increasing depolarization. Single gap Junctional channel activity was recorded in uncoupling cell pairs and in poorly coupled ATP-stabilized cell pairs. One main single channel conductance was observed in each cell pair. The mean single channel conductances from all cell pairs in this study ranged from 197–347 pS (mean 248 pS). Single channel conductance was linear over the ±60 mV transjunctional voltage range tested. A broad range of subconductance states of the main state representing 5% of the total open time of measurable main state events was observed. Single channel activity was strongly dependent on the nonjunctional membrane potential, increasing with hyperpolarization.We gratefully acknowledge the helpful advice of Dr. Stephen Sims. This work was supported by NSERC of Canada grant No. A6797 to S.C. D.C. was supported by an NSERC scholarship for part of this work.     

Potassium-39 NMR of K+ interaction with the gramicidin channel and NMR-derived conductance ratios for Na+, K+ and Rb+

Summary A potassium-39 NMR study of potassium ion interaction with the gramicidin transmembrane channel in phospholipid bilayers at high ion activity is reported which allows determination of a weak binding constant, K _b ^w 8.3/m, and an off-rate constant for the weak site,k _off ^w 2.6×10⁷/sec. These values are interpreted with the aid of additional NMR data as the binding constant for formation of the doubly occupied channel state and the rate constant for an ion leaving the doubly occupied state. Considering the singly occupied channel state for the potassium ion to be electrically silent at 1 molar ion activity, as with the sodium ion, the single-channel conductance for 100 mV and 30°C calculated to be 29 pS, and using the same approximation with previous NMR results on the sodium and rubidium ions, reasonable conductance ratios were calculated. Further experimental estimates of the other three constants with the experimental location of binding sites and Eyring rate theory to introduce voltage dependence allowed a more complete calculation of the two-site channel. The single-channel conductance for potassium ion is calculated to be 24 pS at 1m activity and 26 pS at 0.6m activity, which compares for diphytanoyl phosphatidylcholine membranes to an experimental most probable single-channel conductance of 25 pS and a mean channel conductance of 20 pS. The calculated conductance ratios using NMR-derived constants were (K)/(Na)=2.0 and (Rb)/(Na)=4.3. These results are close to the experimental values and provide further basis for the use of NMR of quadrupolar ions to provide information on the ionic mechanism of channel transport.     

Protein interactions with lipid bilayers: The channels of kidney plasma membrane proteolipids

Summary Proteolipids extracted from bovine kidney plasma membrane induce irreversible changes in the electrical properties of lipid bilayers formed from diphytanoyl phosphatidylcholine. The interaction with the proteolipid produces channels which are cation selective. At low protein concentrations (i.e., <0.6 g/ml), the single-channel conductance is approximately 10 pS in 100mm KCl and 3 pS in 100mm NaCl. In the presence of protein concentrations above 1 g/ml, another population of channels appears. These channels have a conductance of about 100 pS in 100mm KCl and 30 pS in 100mm NaCl. Further, these channels are voltage dependent in KCl, closing when the voltage is clamped at values 30 mV. The steady-state membrane conductance, measured at low voltages, was found to increase proportional to a high power (2–3) of the proteolipid concentration present in one of the aqueous phases. In 100mm NaCl, the conductance increases at protein concentrations above 5 g/ml, whereas in 100mm KCl in increases at protein concentrations above 0.6 g/ml. These measurements indicate that the higher steady-state conductance observed in KCl at a given proteolipid concentration in a multi-channel membrane presumably results because more channels incorporate in the presence of KCl than in the presence of NaCl.The two major fractions which comprise the proteolipid complex were also tested on bilayers. It was found that both fractions are required to produce the effects described.     

Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating conditions

Short term (15 min) effects of activators of protein kinase A (PKA), PKC and PKG on cardiac macroscopic (g_j) and single channel (_j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell voltage-clamp, PKC activation by 100 nM TPA increased g_j by 16 ± 2% (mean ± S.E.M, n=9), 1.5 mM of the PKG activator 8-bromo-cGMP (8Br-cGMP) decreased g_j by 26 ± 2% (n=4), whereas 1.5 mM of the PKA activator 8Br-cAMP did not affect g_j (1 ± 5%, n=11). Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two _j sizes of 20 pS and 40–45 pS. Under control conditions, the larger events were most frequently observed. Whereas 8Br-cAMP did not change this distribution, TPA or 8Br-cGMP shifted the _j distribution to the lower sizes. Diffusion of 6-carboxyfluorescein (6-CF), a gap junction permeant tracer, from the injected cell to neighboring cells was studied on small clusters of neonatal rat cardiomyocytes. Under control conditions, 6-CF labeled 8.4 ± 0.4 cells (mean ± S.E.M, n=31). Whereas 8Br-cAMP did not change the extent of dye transfer (8.1 ± 0.5 cells, n=10), TPA restricted the diffusion of 6-CF to 2.2 ± 0.2 cells (n=30) and 8Br-cGMP to 3.5 ± 0.3 cells (n=10). This suggests that permeability and single channel conductance of Cx43 gap junction channels are parallel related. Altogether, these results point to the differential modulation of electrical and metabolic coupling of cardiac cells by various phosphorylating conditions.     

Angiotensin II,vasopressin and GTP[γ-S] inhibit inward-rectifying K+ channels in porcine cerebral capillary endothelial cells

Summary Cerebral capillaries from porcine brain were isolated. and endothelial cells were grown in primary culture. The whole-cell tight seal patch-clamp method was applied to freshly isolated single endothelial cells, and cells which were held in culture up to one week. With high K⁺ solution in the patch pipette and in the bath we observed inward-rectifying K⁺ currents, showing a time-dependent decay in part of the experiments. Ba²⁺ (1–10mm) in the bath blocked this current, whereas outside tetraethylammonium (10mm) decreased the peak current but increased the steady-state current. Addition of 1 m of angiotensin II or of arginine-vasopressin to the extracellular side caused a time-dependent inhibition of the inward-rectifying K⁺ current in part of the experiments. Addition of 100 m GTP[-S] to the patch pipette blocked the K⁺ inward rectifier. In cell-attached membrane patches two types of single inward-rectifying K⁺ channels were observed, with single channel conductances of 7 and 35 pS. Cell-attached patches were also obtained at the antiluminal membrane of intact isolated cerebral capillaries. Only one type of K⁺ channel withg=30 pS was recorded. In conclusion, inwardly rectifying K⁺ channels, which can be inhibited by extracellular angiotensin II and arginine-vasopressin, are present in cerebral capillary endothelial cells. The inhibition of this K⁺ conductance by GTP[-S] indicates that G-proteins are involved in channel regulation. It is suggested that angiotensin II and vasopressin regulate K⁺ transport across the blood-brain barrier, mediating their effects via G-proteins.     

Modulation of inner mitochondrial membrane channel activity

Three classes of inner mitochondrial membrane (IMM) channel activities have been defined by direct measurement of conductance levels in membranes with patch clamp techniques in 150 mM K Cl. The 107 pS activity is slightly anion selective and voltage dependent (open with matrix positive potentials). Multiple conductance channel (MCC) activity includes several levels from about 40 to over 1000 pS and can be activated by voltage or Ca²⁺. MCC may be responsible for the Ca²⁺-induced permeability transition observed with mitochondrial suspensions. A low conductance channel (LCC) is activated by alkaline pH and inhibited by Mg²⁺. LCC has a unit conductance of about 15 pS and may correspond to the inner membrane anion channel, IMAC, which was proposed from results obtained from suspension studies. All of the IMM channels defined thus far appear to be highly regulated and have a low open probability under physiological conditions. A summary of what is known about IMM channel regulation and pharmacology is presented and possible physiological roles of these channels are discussed.     

Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells

Summary Using the patch clamp technique we have identified a small conductance ion channel that typically occurs in clusters on the apical plasma membrane of pancreatic duct cells. The cell-attached current/voltage (I/V) relationship was linear and gave a single channel conductance of about 4 pS. Since the reversal potential was close to the resting membrane potential of the cell, and unaffected by changing from Na⁺-rich to K⁺-rich pipette solutions, the channel selects for anions over cations in cell-attached patches. The open state probability was not voltagedependent. Adding 25mm-bicarbonate to the bath solution caused a slight outward rectification of theI/V relationship, but otherwise, the characteristics of the channel were unaffected. In excised, inside-out, patches theI/V relationship was linear and gave a single channel conductance of about 4 pS. A threefold chloride concentration gradient across the patch (sulphate replacement) shifted the single channel current reversal potential by –26 mV, indicating that the channel is chloride selective. Stimulation of duct cells with secretin (10nm), dibutyryl cyclic AMP (1mm) and forskolin (1 m) increased channel open state probability and also increased the number of channels, and/or caused disaggregation of channel clusters, in the apical plasma membrane. Coupling of this channel to a chloride/bicarbonate exchanger would provide a mechanism for electrogenic bicarbonate secretion by pancreatic duct cells.     

Single-channel K+ currents inDrosophila muscle and their pharmacological block

Summary Four types of nonvoltage-activated potassium channels in the body-wall muscles ofDrosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA). Ba²⁺, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The K_ST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130mm KCl solution), was blocked by TEA (K _d 35mm), and was the most sensitive to Ba²⁺ (complete block at 10^–4 m). A Ca²⁺-activated potassium channel. K_CF 72pS (130/130mm KCl), was gated open at>10^–8 m Ca²⁺, was the least sensitive to Ba²⁺ (K _d of 3mm) and TEA (K _d of 100mm), and was not affected by quinidine. K₂ was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1mm. It also exhibited a half block at 0.3mm Ba²⁺ and 25mm TEA. A fourth channel type, K₃, was the most sensitive to TEA (half block<1mm). It displayed a partial block to Ba²⁺ at 10mm, but no block by 0.1mm quinidine. The blocking effects of TEA, Ba²⁺ and quinidine were reversible in all channels studied. The actions of TEA and Ba²⁺ appeared qualitatively different: in all four channels. TEA reduced the apparent unitary conductance, whereas Ba²⁺ decreased channel open probability.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.     

K-current fluctuations in inward-rectifying channels of frog skeletal muscle

Summary K currents and K-current fluctuations were recorded in inwardly rectifying K channels of frog skeletal muscle under voltage-clamp conditions. External application of 0.2 to 10mm Cs reduces the inward mean K current but produces a distinct increase of the spectral density of K-current fluctuations. The additional fluctuations arise from the random blocking by Cs ions. From the variance of current fluctuations, the steady-state current and the probability of the open unblocked channel an effective single-channel conductance ^* was calculated. ^* strongly depends on the external Cs concentration (7.8 pS at 0.2mm Cs, 2.1 pS at 10mm Cs). This dependence is interpreted in terms of a two-step blocking process: (1) a fast exchange of Cs ions between the external solution and a first binding site inside the channel which leads to the Cs-modulated effective single-channel conductance, and (2) a slow Cs binding to a second site deeper in the channel which produces the observed current fluctuations. With this hypothesis we obtained a real single-channel conductance of 10 pS and a real density ofn4 inwardly rectifying channels per m² of muscle surface area.     

Comparative study of K channel behavior inβ cell lines with different secretory responses to glucose

Summary The patch-clamp technique was used to identify and investigate two K channels in the cell membrane of the HIT cell, an insulin secreting cell line with glucose-sensitive secretion. Channel characteristics were compared with those of glucose-modulated K channels in the RINm5F cell, an insulin secreting cell line in which secretion is largely glucose insensitive. A 65.7 pS channel, identified with the ATP-sensitive K channel was seen in HIT cell-attached patches. Channel activity was dose-dependently inhibited by glucose, by 50 and 100% at 450 m and 8mm glucose, respectively, similar to the values previously reported for RIN cells. In inside-out patches channel activity was 50% inhibited by 56 m ATP and completely blocked between 500 m and 1mm, again, similar to the values reported for RIN cells.As in RIN cells a second, considerably larger (184 pS), K channel was glucose sensitive; the glucose sensitivity was similar to that in RIN cells with 50 and 100% channel inhibition at 7.5 and 25mm, respectively. After patch excision the mean channel conductance increased from 184 to 215 pS. Under these conditions activity was strongly calcium dependent in the rangepCa 5–7, identifying this as a calcium- and voltage-dependent K (K(Ca,V)) channel; the calcium sensitivity was similar to that of the adult rat cell K(Ca,V) channel. In inside-out RIN cell patches, the large K channel was less abundant but displayed a similar conductance (223 pS). However, its calcium sensitivity was more than 10 times lower than in HIT cells, similar to that of the K(Ca,V) channel in the neonatal rat cell, which also displays a reduced secretory response to glucose. Based on these observations, it is proposed that the low calcium sensitivity of the K(Ca,V) channel may be causally associated with secretory deficiency in RIN cells and the immature secretory response of the neonatal cell.     

Regulation of maxi-K+ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation

Summary Using the patch-clamp technique we have identified a Ca²⁺-sensitive, voltage-dependent, maxi-K⁺ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of 200 pS in excised patches bathed in symmetrical 150mm K⁺, and was blocked by 1mm Ba²⁺. Channel openstate probability (P _o ) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted theP _o /voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration,P _o remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca²⁺ concentration from 2mm to 0.1 m. However, activated channels were still blocked by 1mm Ba²⁺. ChannelP _o was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase., We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K⁺ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca²⁺. Physiologically, these K⁺ channels may contribute to the basolateral K⁺ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.     

Membrane stretch activates a high-conductance K+ channel in G292 osteoblastic-like cells

Summary A high-conductance K⁺-selective ion channel was studied in excised membrane patches from human G292 osteoblast-like osteosarcoma cells. Channel conductance averaged 170 pS in symmetric solutions of 153mm KCl, and 135 pS when the pipette was filled with standard saline (150mm NaCl). The probability of the channel being in an open state (P _open) increased with membrane potential, internal calcium, and applied negative pressure. At pCa7, channel activity was observed at membrane potentials greater than 60 mV, while at pCa3, channel activity was seen at 10 mV. Likewise, in the absence of applied pressure, channel openings were rare (P _open = 0.02), whereas with –3 cm Hg applied pressure,P _open increased to 0.40. In each case, i.e., voltage, calcium concentration, and pressure, the increase inP _open resulted from a decrease in the duration of long-closed (interburst) intervals and an increase in the duration of long-open (burst) intervals. Whole-cell responses were consistent with these findings. Hypotonic shock produced an increase in the amplitude and conductance of the outward macroscopic current and a decrease in its rise time, and both single-channel and whole-cell currents were blocked by barium. It is suggested that the voltage-gated, calcium dependent maxi-K⁺ channel in G292 osteoblastic cells is sensitive to membrane stretch and may be directly involved in osmoregulation of these cells. Further, stretch sensitivity o£ the maxi-K⁺ channel in osteotrophic cells may represent an adaptation to stresses associated with mechanical loading of mineralized tissues.     

Membrane and junctional properties of dissociated frog lens epithelial cells

Summary Individual cells and cell pairs were isolated from frog lens epithelium. Individual cells were whole cell voltage clamped and the current-voltage relationship was determined. The cells had a mean resting voltage of –54.3 mV and a mean input resistance of 1.4 G. The current-voltage relationship was linear near the cell resting voltage, but showed decreased resistance with large depolarization or hyperpolarization. Junctional currents between pairs of cells were recorded using the dual whole cell voltage-clamp technique. The corrected junctional resistance was 15.5 M (64.5 nS). The junctional current-voltage relationship was linear. A combination of ATP and cAMP, in the electodes, stabilized junctional resistance. Currents recorded when uncoupling was nearly complete, showed evidence of single connexon gating events. A single-channel conductance of about 100 pS was prominent. Dye spread between isolated cell pairs was demonstrated using Lucifer Yellow CH in a whole cell configuration. Photodamage to the cells due to the dye was apparent. Dye loaded cells, in the presence of exciting light, showed decreased resting voltages, decreased input resistances and morphological changes. Glutathione (20mm) delayed this damage.     

Specificity of tetraethylammonium and quinine for three K channels in insulin-secreting cells

Summary The effects of tetraethylammonium (TEA) and quinine on Ca-activated [K(Ca)]. ATP-sensitive [K(ATP)]K channels and delayed-rectifier K current [K(dr)] have been studied in cultured insulin-secreting HIT cells using the patch-clamp technique. K(Ca) and K(ATP) channels were identified in excised, outside/ out patches using physiological solutions and had unitary conductances of 60.8±1.3 pS (n=31) and 15.4±0.3 pS (n=40). respectively. Macroscopic K(dr) current (peak current=607±100 pA at +50 mV,n=14) were recorded in the presence of 100 m cadmium and 0.5 m tetrodotoxin. Tetraethylammonium (TEA) blocked all three channel types but was more effective on K(Ca) channels (EC₅₀=0.15mm) than on K(ATP) channels (EC₅₀=15mm) or K(dr) currents (EC₅₀=3mm). Quinine also blocked all three currents but was less effective on K(Ca) channels (EC₅₀=0.3mm) while equally effective against K(ATP) channels and K(dr) currents (EC₅₀=0.025mm). TEA blocked K(Ca) and K(ATP)_channels by reducing their single-channel conductances and decreasing the probability of K(ATP) channel opening. Quinine blocked K(Ca) channels by reducing the single-channel conductance, but blocked K(ATP) channels by reducing the probability of channel opening. Reinterpretation of previous microelectrode studies in light of these findings suggest that, (i) only K(ATP) channels are active in low glucose, (ii) both K(Ca) and K(dr) channels may assist Ca-spike repolarization, and (iii) K(Ca) channels play no role in forming the burst pattern of Ca spiking in the B cell.     

ATP-sensitive potassium channels in adult mouse skeletal muscle: Characterization of the ATP-binding site

Summary Single K⁺-selective channels were studied in excised inside-out membrane patches from dissociated mouse toe muscle fibers. Channels of 74 pS conductance in symmetrical 160mm KCl solutions were blocked reversibly by 10 m internal ATP and thus identified as ATP-sensitive K⁺ channels. The channels were also blocked reversibly bymm concentrations of internal adenosine, adenine and thymine, but not by cytosine and uracil. The efficacy of the reversible channel blockers was higher when they were present in internal NaCl instead of KCl solutions. An irreversible inhibition of ATP-sensitive K⁺ channels was observed after application of several sulphydryl-modifying substances in the internal solution: 0.5mm chloramine-T, 50mm hydrogen peroxide or 2mm n-ethylmaleimide (NEM). Largeconductance Ca-activated K⁺ channels were not affected by these reagents. The presence of 1mm internal ATP prevents the irreversible inhibition of ATP-sensitive K⁺ channels by NEM. The results suggest that internal Na⁺ ions increase the affinity of the ATP-sensitive K⁺ channel to ATP and to other reversible channel blockers and that a functionally important SH-group is located at or near the ATP-binding site.     

Electrical coupling between cells in islets of langerhans from mouse

Summary Two microelectrodes have been used to measure membrane potentials simultaneously in pairs of mouse pancreatic islet cells. In the presence of glucose at concentrations between 5.6 and 22.2mm, injection of currenti into cell 1 caused a membrane potential change in this cell,V ₁, and, provided the second microelectrode was less than 35 m away, in a second impaled cell 2,V ₂. This result establishes that there is electrical coupling between islet cells and suggests that the space constant of the coupling ratio within the islet tissue is of the order of a few -cell diameters. The current-membrane potential curvesi-V ₁ andi-V ₂ are very similar. By exchange of the roles of the microelectrodes, no evidence of rectification of the current through the intercellular pathways was found. Removal of glucose caused a rapid decrease in the coupling ratioV ₂ /V ₁ . In steady-state conditions, the coupling ratio increases with the concentration of glucose in the range from 0 up to 22mm. Values of the equivalent resistance of the junctional and nonjunctional membranes have been estimated and found to change with the concentration of glucose. Externally applied mitochondrial blockers induced a moderate increase in the junctional resistance possibly mediated by an increase in intracellular Ca²⁺.     

Effects of irreversible and reversible cholinesterase inhibitors on single acetylcholine-activated channels

Summary The use of cholinesterase (CHE) inhibitors provided valuable information about the mechanism(s) of neuromuscular transmission, but questions on side effects at the level of AChactivated channels were raised. Patch-clamp recording was used to study the effects of prostigmine (PST) and methanesulfonyl fluoride (MSF), a reversible and an irreversible cholinesterase inhibitor, respectively, on ACh-activated channels. We found that these drugs diminish the average dwell time of elementary currents from around 5 msec (control) to less than 1 msec in the presence of PST (20 m) or MSF (5 mm) (at room temperature). With MSF the ACh-activated channel conductance of the most frequently observed amplitude class decreased from 45 pS (control) to 30 pS, but not in the presence of PST. In control conditions there were also amplitude classes of 60 and 24 pS, with probabilities of occurrence <10%. In the presence of 1.5 mm MSF, where current dwell time was not affected, additional subconductance states of 19 and 36 pS were observed and may be due to partial blockade of the open channel. We conclude that the drug of choice to be used in studies on the role of CHE in the neuromuscular transmission is MSF, because at 20 m PST, where blockade of ACh-activated channels is significant, cholinesterase was reported to be partially inhibited, whereas at 1 mm MSF it is fully inhibited and the dwell time of ACh-activated channels is not affected.This research was supported by the Ministry of Science and Technology of Republic of Slovenia. M.S. would like to acknowledge the Italian Board of Education (M.P.I.) for support. The analog-to-digital converter (CED 1401) was kindly donated by The Wellcome Trust.     

Regulation of K+ channels in the basolateral membrane ofNecturus oxyntic cells

Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K⁺ over Na⁺ and Cl^–, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca²⁺ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca²⁺. The presence of K⁺ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.     

Anion channels from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl₂ buffers (cis, 100mm/trans, 25mm), single channel conductances at –40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (P _Na:P _Ba:P _Cl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.230.041 and 0.050.031, respectively. Both channels exhibited characteristic voltage-dependent bursting activities. Open probability for type I channels had a maximum of 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from thecis side, while it blocked type II channels from either side.