Pipette Voltage Research Articles

The Role of the C-Terminal Domain on the Gating Properties of Corynebacterium Glutamicum Mechanosensitive Channel MscCG

The gram-positive soil bacterium Corynebacterium glutamicum exports a large amount of glutamate through the mechanosensitive channel MscCG. This process is utilized for the world industrial glutamate production. MscCG belongs to a subfamily of bacterial MscS-like channels, which function in osmoregulation, however this channel shows structural and functional differences compared to MscS. To understand the role of the carboxyl terminal domain of MscCG, the chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in E. coli spheroplasts characterized by a lower activation threshold and slow channel closing compared to MscS. The chimeric channel MscS-(C-MscCG) was also successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for the Corynebacterium glutamicum cells. This contribution to the gating hysteresis of the C-terminal domain of MscCG provides the channel with gating properties highly suitable for release of intracellular solutes.

The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum

The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes.

The Right-Side-Out Orientation of MscS in Liposomal Membranes

The bacterial mechanosensitive channel MscS plays a crucial role in the protection of bacterial cells against hypo-osmotic shock. MscS functional characteristics have extensively been studied in both giant spheroplasts and liposomes. Despite many studies of MscS reconstituted into liposomes the channel orientation in liposomal membranes is still unknown. We examined the orientation of MscS in liposomes by patch-clamp and confocal microscopy. using its previously determined electrophysiological and pharmacological properties we were able to determine that in liposomes MscS retains the right-side-out orientation as in giant spheroplasts based on the following evidence: (i) I-V curves recorded in both spheroplast and liposome preparations exhibited strong outward rectification at both negative and positive pipette pressures. (ii) MscS activation ratio in liposome patches at positive relative to negative pipette voltages and vice versa showed positive correlation at both positive and negative pipette pressures similar to MscS in inside-out excised spheroplast patches. (iii) MscS exhibited a voltage-dependent hysteresis upon application of saw-tooth pressure ramps in both spheroplasts and liposomes. In both spheroplasts and liposomes the hysteresis was more pronounced upon positive pipette voltages compared to negative voltages. (iv) 2.5% of 2,2,2-trifluoroethanol (TFE) caused MscS inactivation in liposome patches when added to the cytoplasmic side of MscS, whereas addition of TFE to the periplasmic side did not inactivate the channel, although it caused a shift of the channel activation towards lower pipette pressures. We obtained a similar result when applying TFE to MscS in spheroplast patches. In conclusion, our findings strongly indicate that the cytoplasmic domain of MscS in liposome membrane patches faces the bath solution as in spheroplast patches. Consequently, upon liposome reconstitution MscS channels preserve their right-side-out orientation comparable to what was previously reported for the MscL channels.Supported by the NH & MRC.

The protective effect of osmoprotectant TMAO on bacterial mechanosensitive channels of small conductance MscS/MscK under high hydrostatic pressure

Activity of the bacterial mechanosensitive channels of small conductance MscS/MscK of E. coli was investigated under high hydrostatic pressure (HHP) using the “flying-patch” patch-clamp technique. The channels were gated by negative pipette voltage and their open probability was measured at HHP of 0.1 to 80 MPa. The channel open probability decreased with increasing HHP. When the osmolyte methylamine N-oxide (TMAO) was applied to the cytoplasmic side of the inside-out excised membrane patches of E. coli giant spheroplasts the inhibitory effect of HHP on the channel activity was suppressed at pressures of up to 40 MPa. At 40 MPa and above the channel open probability decreased in a similar fashion with or without TMAO. Our study suggests that TMAO helps to counteract the effect of HHP up to 40 MPa on the MscS/MscK open state by “shielding” the cytoplasmic domain of the channels.

Cardiolipin Effects on the Gating Behaviour and Reconstitution of MscL and MscS

The mechanosensitive channels of large (MscL) and small (MscS) conductance act as osmosensors in bacterial cells against hypo-osmotic shock. MscL has been extensively studied by reconstitution into liposomes2,3, however MscS has proved more difficult to reconstitute, requiring high protein-lipid ratios4,5. We recently published an improved reconstitution method for both MscL and MscS in soy azolectin6, a mixture that contains lipids, sugars and sterols. We have expanded these results and show here the effect of both individual and mixtures of lipids on the reconstitution and channel gating behaviour of co-reconstituted MscL and MscS. Introduction of the highly charged lipid cardiolipin causes rapid gating of MscS (Fig 1A) in comparison to soy azolectin (Fig 1B), indicating that lipid charge may play a significant role on channel gating dynamics.View Large Image | View Hi-Res Image | Download PowerPoint SlideFig 1 (A) MscS/MscL co- reconstitution in soy azolectin. (B) MscS/MscL co- reconstitution in mixture of phosphatidyl ethanolamine/ phosphatidyl choline/cardiolipin at a wt/wt ratio of 7:2:1, recordings at a pipette voltage of +30 mV.Supported by the Australian Research Council and the National Health and Medical Research Council of Australia.

Sulfonylureas Correct Trafficking Defects of Disease-causing ATP-sensitive Potassium Channels by Binding to the Channel Complex

ATP-sensitive potassium (K(ATP)) channels mediate glucose-induced insulin secretion by coupling metabolic signals to beta-cell membrane potential and the secretory machinery. Reduced K(ATP) channel expression caused by mutations in the channel proteins: sulfonylurea receptor 1 (SUR1) and Kir6.2, results in loss of channel function as seen in congenital hyperinsulinism. Previously, we reported that sulfonylureas, oral hypoglycemic drugs widely used to treat type II diabetes, correct the endoplasmic reticulum to the plasma membrane trafficking defect caused by two SUR1 mutations, A116P and V187D. In this study, we investigated the mechanism by which sulfonylureas rescue these mutants. We found that glinides, another class of SUR-binding hypoglycemic drugs, also markedly increased surface expression of the trafficking mutants. Attenuating or abolishing the ability of mutant SUR1 to bind sulfonylureas or glinides by the following mutations: Y230A, S1238Y, or both, accordingly diminished the rescuing effects of the drugs. Interestingly, rescue of the trafficking defects requires mutant SUR1 to be co-expressed with Kir6.2, suggesting that the channel complex, rather than SUR1 alone, is the drug target. Observations that sulfonylureas also reverse trafficking defects caused by neonatal diabetes-associated Kir6.2 mutations in a way that is dependent on intact sulfonylurea binding sites in SUR1 further support this notion. Our results provide insight into the mechanistic and structural basis on which sulfonylureas rescue K(ATP) channel surface expression defects caused by channel mutations.

Effect of high hydrostatic pressure on the bacterial mechanosensitive channel MscS

We have investigated the effect of high hydrostatic pressure on MscS, the bacterial mechanosensitive channel of small conductance. Pressure affected channel kinetics but not conductance. At negative pipette voltages (corresponding to membrane depolarization in the inside-out patch configuration used in our experiments) the channel exhibited a reversible reduction in activity with increasing hydrostatic pressure between 0 and 900 atm (90 MPa) at 23 degrees C. The reduced activity was characterized by a significant reduction in the channel opening probability resulting from a shortening of the channel openings with increasing pressure. Thus high hydrostatic pressure generally favoured channel closing. Cooling the patch by approximately 10 degrees C, intended to order the bilayer component of the patch by an amount similar to that caused by 50 MPa at 23 degrees C, had relatively little effect. This implies that pressure does not affect channel kinetics via bilayer order. Accordingly we postulate that lateral compression of the bilayer, under high hydrostatic pressure, is responsible. These observations also have implications for our understanding of the adaptation of mechanosensitive channels in deep-sea bacteria.

The “Dashpot” Mechanism of Stretch-dependent Gating in MscS

The crystal structure of the small conductance mechanosensitive channel (MscS) has been an invaluable tool in the search for the gating mechanism, however many functional aspects of the channel remain unsettled. Here we characterized the gating of MscS in Escherichia coli spheroplasts in a triple mutant (mscL − , mscS − , mscK −) background. We used a pressure clamp apparatus along with software developed in-lab to generate dose–response curves directly from two-channel recordings of current and pressure. In contrast to previous publications, we found that MscS exhibits essentially voltage-independent activation by tension, but at the same time strong voltage-dependent inactivation under depolarizing conditions. The MscS activation curves obtained under saturating ramps of pressure, at different voltages, gave estimates for the energy, area, and gating charge for the closed-to-open transition as 24 kT, 18 nm2, and +0.8, respectively. The character of activation and inactivation was similar in both K+ and Na+ buffers. Perhaps the most salient and intriguing property of MscS gating was a strong dependence on the rate of pressure application. Patches subjected to various pressure ramps from 2.7 to 240 mmHg/s revealed a midpoint of activation almost independent of rate. However, the resultant channel activity was dramatically lower when pressure was applied slowly, especially at depolarizing pipette voltages. It appears that MscS prefers to respond in full to abrupt stimuli but manages to ignore those applied slowly, as if the gate were connected to the tension-transmitting element via a velocity-sensitive “dashpot.” With slower ramps, channels inactivate during the passage through a narrow region of pressures below the activation midpoint. This property of “dumping” a slowly applied force may be important in environmental situations where rehydration of cells occurs gradually and release of osmolytes is not desirable. MscS often enters the inactivated state through subconducting states favored by depolarizing voltage. The inactivation rate increases exponentially with depolarization. Based on these results we propose a kinetic scheme and gating mechanism to account for the observed phenomenology in the framework of available structural information.

Single-cell electroporation

Using modified patch-clamp methodology, we demonstrated that it is possible to insert genes or other compounds routinely into single cells by electroporation. When the cell is indented by a small-tipped microelectrode, a voltage of 10 V or less in the pipette is divided by the pipette resistance and the series resistance of the cleft between the pipette tip and the cell surface. The voltage at the cell membrane can be high enough to cause localized dielectric breakdown of the membrane and create pores that allow compounds in the pipette to enter the cell. Rectangular pulses from 20 micros to more than 300 ms are effective, as are frequencies from DC to 5 kHz. The most significant parameter was the total time for which the voltage was applied. Pipette voltages of 2-10 V were required, with larger genes requiring larger voltages. With optimal parameters, transfection rates in excess of 80% were also possible routinely. This approach offers an effective alternative to intracellular pressure injection and iontophoresis for placing genes, drugs, and other compounds in cells. Because of the small size of the electrode tips, substances can be inserted in cells from almost any location on their surfaces. In addition, the small tips electroporated only a limited area and so did little cell damage.

Apical nonspecific cation channels in everted collecting tubules of potassium-adapted ambystoma.

We observed intermediate conductance channels in approximately 20% of successful patch-clamp seals made on collecting tubules dissected from Ambystoma adapted to 50 mm potassium. These channels were rarely observed in collecting tubules taken from animals which were maintained in tap water. Potassium-adaptation either leads to an increase in the number of channels present or activates quiescent channels. In cell-attached patches the conductance averaged 30.3 +/- 2.4 (9) pS. Since replacement of the chloride in the patch pipette with gluconate did not change the conductance, the channel carries cations, not anions. Notably, channel activity was observed at both positive and negative pipette voltages. When the pipette was voltage clamped at 0 mV or positive voltages, the current was directed inward, consistent with the movement of sodium into the cell. The pipette voltage at which the polarity of the current reversed (movement of potassium into the pipette) was -29.6 +/- 6.5(9) mV. Open probability at 0 mV pipette voltage was 0.08 +/- 0.03 and was unaffected when the apical membrane was exposed to either 2 x 10(-6) or 2 x 10(-5) m of amiloride. Exposure of the basolateral surface of the tubule to a saline containing 15 mm potassium caused a significant increase (P less than 0.001) in the open probability of these channels to 0.139 +/- 0.002 without affecting the conductance of the apical channel. These data illustrate the presence of an intermediate conductance, poorly selective, amiloride-insensitive cation channel in native vertebrate collecting tubule. We postulate that, at least in amphibia, this channel may be used to secrete potassium.

A Novel Voltage Clamp Technique for Mapping Ionic Currents from Cultured Skeletal Myotubes

The biophysical properties and cellular distribution of ion channels largely determine the input/output relationships of electrically excitable cells. A variety of patch pipette voltage clamp techniques are available to characterize ionic currents. However, when used by themselves, such techniques are not well suited to the task of mapping low-density channel distributions. We describe here a new voltage clamp method (the whole cell loose patch (WCLP) method) that combines whole-cell recording through a tight-seal pipette with focal extracellular stimulation through a loose-seal pipette. By moving the stimulation pipette across the cell surface and using a stationary whole-cell pipette to record the evoked patch currents, this method should be suitable for mapping channel distributions, even on large cells possessing low channel densities. When we applied this method to the study of currents in cultured chick myotubes, we found that the cell cable properties and the series resistance of the recording pipette caused significant filtering of the membrane currents, and that the filter characteristics depended in part upon the distance between the stimulating and recording pipettes. We describe here how we determined the filter impulse response for each loose-seal pipette placement and subsequently recovered accurate estimates of patch membrane current through deconvolution.

E.coli PhoE porin has an opposite voltage-dependence to the homologous OmpF.

We used patch clamp analysis to compare the electrophysiological behavior of two related porins from Escherichia coli, the anion-specific PhoE and the cation-selective OmpF. Outer membrane fractions were obtained from strains expressing just one of these porin types, and the channels were reconstituted into liposomes without prior purification. We show that the orientation of the reconstituted channels is not random and is the same for both PhoE and OmpF. Like cation-selective porins, PhoE shows fast and slow gating to closed levels of various amplitudes, testifying that the channels visit multiple functional states and behave as cooperative entities. The voltage-dependence of PhoE closure is asymmetric, but strikingly, occurs at voltages of inverse polarity from those promoting closures of OmpC and OmpF. Both slow kinetics and inverse voltage-dependence are removed when 70 amino acids from the N-terminal of OmpF are introduced into the homologous region of PhoE. This novel observation regarding the voltage-dependence of the two channel types, along with published results on PhoE and OmpF mutants, allows us to propose a molecular mechanism for voltage sensing and sensor charge movements in bacterial porins. It also offers new cues on the possible physiological relevance in bacteria of this common form of channel modulation.

Apical Na+ conductance in maturing rabbit principal cell.

Net Na+ absorption in microperfused rabbit cortical collecting ducts (CCDs) is low during the 1st wk of postnatal life, increasing substantially thereafter [L. M. Satlin. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F57-F65, 1994]. To establish whether the low rate of Na+ absorption observed immediately after birth is due to a low apical Na+ permeability of the neonatal principal cell, we used the patch-clamp technique in split-open CCDs isolated from maturing rabbits to estimate conductance, number (N), and open probability (Po) of apical Na+ channels in principal cells. With LiCl in the pipette and a NaCl or potassium gluconate solution, warmed to 37 degrees C, in the bath, inward currents with a conductance of approximately 11 pS (n = 23) were observed in 17% of cell-attached patches at 1 wk, 41% of patches at 2 wk, and 43% of patches at 5 wk. The mean N per patch in the 1st wk (0.22 +/- 0.09; n = 36) was significantly less than that observed in the 2nd (1.38 +/- 0.39; n = 34) and 5th (1.24 +/- 0.37; n = 21) wk of life. Po, studied at positive pipette voltages, was significantly lower in the 1st wk (0.085 +/- 0.035; n = 5) than in the 2nd wk (0.345 +/- 0.063; n = 9) and 5th wk (0.291 +/- 0.058; n = 4). To confirm that the 11-pS channel represented the amiloride-sensitive apical Na+ channel, cell-attached patches in CCDs isolated from 2-wk-old rabbits were studied with 0.5 microM amiloride added to the LiCl pipette solution. Amiloride led to > 90% reductions in mean open and closed times of the 11-pS conductance, consistent with blockade of the channel. These data indicate that N and Po of apical amiloride-sensitive Na+ channels in principal cells increase significantly after birth.

Estimation of the junctional resistance between electrically coupled receptor cells in Necturus taste buds.

Junctional resistance between coupled receptor cells in Necturus taste buds was estimated by modeling the results from single patch pipette voltage clamp studies on lingual slices. The membrane capacitance and input resistance of coupled taste receptor cells were measured to monitor electrical coupling and the results compared with those calculated by a simple model of electrically coupled taste cells. Coupled receptor cells were modeled by two identical receptor cells connected via a junctional resistance. On average, the junctional resistance was approximately 200-300 M omega. This was consistent with the electrophysiological recordings. A junctional resistance of 200-300 M omega is close to the threshold for Lucifer yellow dye-coupling detection (approximately 500 M omega). Therefore, the true extent of coupling in taste buds might be somewhat greater than that predicted from Lucifer yellow dye coupling. Due to the high input resistance of single taste receptor cells (> 1 G omega), a junctional resistance of 200-300 M omega assures a substantial electrical communication between coupled taste cells, suggesting that the electrical activity of coupled cells might be synchronized.

Effect of basolateral or apical hyposmolarity on apical maxi K channels of everted rat collecting tubule

We are able to evert and perfuse rat cortical collecting tubules (CCT) at 37 degrees C. Patch-clamp techniques were used to study high-conductance potassium channels (maxi K) on the apical membrane. Under control conditions (150 mM Na+ and 5 mM K+ in pipette and bathing solutions), the slope conductance averaged 109.8 +/- 6.6 pS (12 channels), and reversal potential (expressed as pipette voltage) was +26.3 +/- 2.4 mV. The percent of time the channel spends in the open state and unitary current when voltage was clamped to 0 mV were 1.4 +/- 0.7% and 3.12 +/- 0.42 pA, respectively. In six patches voltage clamped to 0 mV, the isosmotic solution perfused through the everted tubule (basolateral surface) was exchanged for one made 70 mosmol/kgH2O hyposmotic to the control saline. Open probability increased from 0.019 to 0.258, an increase of 0.239 +/- 0.065 (P < 0.005). In four patches where a maxi K channel was evident, no increase in open probability was observed when a hyposmotic saline was placed on the apical surface. However, when vasopressin was present on the basolateral surface, apical application of hyposmotic saline resulted in a series of bursts of channel activity. The average increase in open probability during bursts was (0.055 +/- 0.017, P < 0.005). We conclude that one function of the maxi K channel located in the apical membrane of the rat CCT may be to release intracellular solute (potassium) during a volume regulatory decrease induced by placing a dilute solution on the basolateral surface or when the apical osmolarity is reduced in the presence of vasopressin. These data are consistent with the hypothesis that the physiological role of the channel is to regulate cell volume during water reabsorption.

Amiloride-sensitive apical membrane sodium channels of everted Ambystoma collecting tubule

Patch clamp methods were used to characterize sodium channels on the apical membrane of Ambystoma distal nephron. The apical membranes were exposed by everting and perfusing initial collecting tubules in vitro. In cell-attached patches, we observed channels whose mean inward unitary current averaged 0.39 +/- 0.05 pA (9 patches). The conductance of these channels was 4.3 +/- 0.2 pS. The unitary current approached zero at a pipette voltage of -92 mV. When clamped at the membrane potential the channel expressed a relatively high open probability (0.46). These characteristics, together with observation that doses of 0.5 to 2 microM amiloride reversibly inhibited the channel activity, are consistent with the presence of the high amiloride affinity, high sodium selectivity channel reported for rat cortical collecting tubule and cultured epithelial cell lines. We used antisodium channel antibodies to identify biochemically the epithelial sodium channels in the distal nephron of Ambystoma. Polyclonal antisodium channel antibodies generated against purified bovine renal, high amiloride affinity epithelial sodium channel specifically recognized 110, 57, and 55 kDa polypeptides in Ambystoma and localized the channels to the apical membrane of the distal nephron. A polyclonal antibody generated against a synthetic peptide corresponding to the C-terminus of Apx, a protein associated with the high amiloride affinity epithelial sodium channel expressed in A6 cells, specifically recognized a 170 kDa polypeptide. These data corroborate that the apically restricted sodium channels in Ambystoma are similar to the high amiloride affinity, sodium selective channels expressed in both A6 cells and the mammalian kidney.

Modulation of large conductance calcium-activated K+ channel by membrane-delimited protein kinase and phosphatase activities.

Large conductance Ca(2+)-activated K+ channel was identified and studied in excised inside-out membrane patches of freshly dispersed smooth muscle cells from rabbit gastric antrum. The current-voltage relationship of the single channel was linear from -80 to +80 mV of pipette voltage in which single channel conductance was 249 +/- 17.8 pS (n = 19) in symmetrical concentration of K+ (145 mM) across the patch. Activity of the channel (NPo) depended not only on cytoplasmic calcium concentration but also on membrane potential. MgATP increased NPo in a dose-dependent manner and Mg2+ was prerequisite for the effect. Okadaic acid (100 nM), inhibitor of protein phosphatases, increased NPo further in the presence of MgATP. Therefore, it would be concluded that activity of the calcium-activated K+ channel in gastric smooth muscle cells was controlled by phosphorylation state of the channel protein and the state is further modulated by membrane-delimited protein kinase and protein phosphatase activities.

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

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

Differential block of cardiac delayed rectifier current by class Ic antiarrhythmic drugs: evidence for open channel block and unblock.

The aim was to compare the effects of the class Ic antiarrhythmic drugs flecainide, encainide, and recainam on the delayed rectifier current, IK. Membrane currents were studied using the single suction pipette voltage clamp technique in freshly dissociated cat ventricular myocytes bathed in HEPES buffered physiological saline at 32 degrees C. Flecainide and encainide decreased IK with IC50 values of 2.1 microM and 6 microM, respectively. Recainam (100 microM) reduced IK by only 7 (SEM 3)% after 20-30 min exposure and by 19% after an 80 min exposure (IC50 > 400 microM). None of the compounds blocked the inward rectifier, IK1. Block of IK by flecainide and encainide increased with depolarisation following a voltage dependence similar to that describing channel activation. Flecainide and encainide also slowed the time course of the IK tail currents, consistent with drug dissociating from open channels. The observed voltage dependence for IK block by flecainide and encainide resembles the interaction reported between these agents and the excitatory sodium channel, ie, depolarisation enhances block while repolarisation leads to removal of block. The results further suggest that the electrophysiological profile of class Ic agents can have a markedly different ionic basis, ie, K+ channel block by flecainide and encainide is balanced by a potent block of sodium channels, while recainam appears to be a weak but relatively specific blocker of sodium channels only. These differences are not readily accommodated by the current Harrison-Vaughan-Williams classification scheme, and suggest the possibility that potentially important drug specific differences can exist within the same antiarrhythmic drug class.

A voltage‐dependent potassium current in rabbit coronary artery smooth muscle cells.

1. Voltage- and time-dependent outward currents were recorded from relaxed enzymatically isolated smooth muscle cells from the rabbit left descending coronary artery using a single pipette voltage clamp technique. The calcium-activated potassium current was blocked by inclusion of EGTA in the pipette solution and CdCl2 in the extracellular bath. 2. Outward currents were elicited with depolarizing voltage steps to potentials positive to -20 mV. Long (5 s) voltage steps revealed slow inactivation of the current with a time constant of nearly 3 s at +60 mV. Potassium was identified as the predominant charge carrier by reversal potential measurements in potassium substitution experiments. 3. The results of kinetic analyses compared favourably with the Hodgkin-Huxley model for a delayed rectifier with some deviations. The sigmoid current onset was best fitted by raising the activation variable (n) to the second power. Deactivation tail currents were consistently found to be comprised of two exponential components. The kinetics of activation and deactivation were strongly voltage-dependent from -80 to +60 mV. 4. Envelope of tails experiments showed that the scaled tail current amplitudes followed the kinetic behaviour of current activation. The contribution of each of the two exponential tail components was also measured in these experiments. They did not reveal kinetically separable currents, nor were they differentially altered by 4-aminopyridine (4-AP), tetraethylammonium (TEA), or elevated [K+]o. 5. The steady-state voltage-dependence curves for both activation and inactivation were well fitted by a Boltzmann distribution with V1/2 = -5.60 mV and k = -8.66 mV for n infinity act and V1/2 = -24.20 mV and k = 5.16 mV for n infinity act. Super-imposition of the two curves revealed a 'window' of voltage where channels are available for activation without completely inactivating. 6. Neither of the commonly used potassium channel blockers, TEA or 4-AP, were particularly effective blockers of IK, reducing current by only 50-70% at an extracellular concentration of 10 mM. TEA block was mildly voltage-dependent and was more effective in reducing current towards the end of a 500 ms depolarization. 4-AP, on the other hand, demonstrated considerable voltage-dependence and preferentially reduced early currents. 7. Outward currents recorded from guinea-pig and human coronary artery myocytes under the same conditions as in the rabbit cell experiments displayed similar characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)