Single-channel Slope Conductance Research Articles

Abstract 485: Enhanced Large Conductance Ca+2 -sensitive K+ Channels (bk) Activity Impairs the Myogenic Response in the Cerebral Vasculature of Fawn Hooded Hypertensive (fhh) Rat

Our recent studies have revealed that the myogenic response in cerebral arteries and autoregulation of cerebral blood flow is impaired in FHH and that transfer of a 2.4 Mb region of chromosome 1 from BN into FHH.1 BN congenic strain restores these responses. The present study examined the role of large conductance calcium activated potassium (BK) channel in altering the myogenic response in FHH rats. Whole-cell patch-clamp of cerebral vascular smooth muscle cells (VSMC) revealed a 4.6 fold increase in outward potassium (K) channel current densities (pA/pF) in FHH rats compared to FHH.1 BN congenic strain. Iberiotoxin (IBTX -a selective BK channel inhibitor) sensitive current densities are significantly greater in FHH rats compared with the FHH.1 BN congenic strain (FHH rats: +40mV; pre IBTX 43.1 ± 7.2, after IBTX 11.8 ± 2.2 pA/pF versus pre IBTX 5.6 ± 1 pA/pF and after IBTX 4.1 ± 0.6 pA/pF in the FHH.1 BN congenic strain). In excised patches, the BK channel exhibited similar single-channel slope conductance for FHH and the FHH.1 BN rats (208.9 pS versus 208.7 pS). However, the open channel probability (NP o ) was ~10 fold higher in FHH rats than in FHH.1 BN rats (1μM free (Ca +2 ) i : +40mV: FHH; 0.8 ± 0.04 versus 0.08 ± 0.004 in FHH.1 BN rats). Voltage and Ca 2+ sensitivity of the BK channel is similar in cerebral VSMC isolated from FHH and FHH.1 BN rats. Middle cerebral arterioles (MCA) isolated from FHH rats increased in diameter from 142 ± 16 to 157 ± 19 μm when pressure was increased from 40 to 140 mmHg. In contrast, the diameter of the MCA decreased by 49% in the FHH.1 BN congenic strain from 127 ± 16 to 65 ± 13 μm. Pharmacological block of BK channel by IBTX (100nM) restored myogenic response in FHH rats but had no effect in FHH.1 BN rats. These results indicate that the impaired myogenic response of the cerebral vessels in FHH rats is mediated via a gene and mechanism that enhances BK channel activity.

Palmitoylation of the S0-S1 Linker Regulates Cell Surface Expression of Voltage- and Calcium-activated Potassium (BK) Channels

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium- and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by ∼55% in the C53:54:56A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.

Gap junction channel activity in short-term cultured human detrusor myocyte cell pairs: gating and unitary conductances

Several independent lines of investigation indicate that intercellular communication through gap junctions modulates bladder physiology and, moreover, that altered junctional communication may contribute to detrusor overactivity. However, as far as we are aware, there are still no direct recordings of gap junction-mediated intercellular currents between human or rat detrusor myocytes. Northern and Western blots were used to identify connexin expression in frozen human bladder tissue and short-term cultured human detrusor myocytes. Double whole cell patch (DWCP) recording revealed that human detrusor myocyte cell pairs were well coupled with an average junctional conductance of 6.5 +/- 4.6 nS (ranging from 0.1 to 15 nS, n = 22 cell pairs). Macroscopic gap junction channel currents in human detrusor myocytes exhibited voltage dependence similar to homotypic connexin43. The normalized transjunctional conductance-voltage (G(j)-V(j)) relationship was symmetrical and well described by a two-state Boltzmann relation (G(min) approximately 0.33, V(0) = 63.6 mV, Z = 0.117 or equal to 2.95 gating charges), suggestive of a bilateral voltage-gated mechanism. In symmetric 165 mM CsCl, the measured single-channel slope conductance was approximately 120 pS for the fully open channel and approximately 26 pS for the major substate. Occasionally, other subconductance states were also observed. The single-channel mean open time declined with increasing V(j), accounting for the V(j)-dependent decline of macroscopic junctional current. Qualitatively similar electrophysiological characteristics were observed in DWCP of freshly isolated rat detrusor myocytes. These data confirm and extend previous observations and are consistent with reports in other smooth muscle cells types in which Cx43-mediated intercellular communication has been identified.

Properties of Mg2+‐dependent cation channels in human leukemia K562 cells

The endogenous Mg(2+)-inhibited cation (MIC) current was recently described in different cells of hematopoietic lineage and was implicated in the regulation of Mg2+ homeostasis. Here we present a single channel study of endogenously expressed Mg(2+)-dependent cation channels in the human myeloid leukemia K562 cells. Inwardly directed unitary currents were activated in cell-attached experiments in the absence of Ca2+ and Mg2+ in the pipette solution. The current-voltage (I-V) relationships displayed strong inward rectification and yielded a single channel slope conductance of approximately 30 pS at negative potentials. The I-V relationships were not altered by patch excision into divalent-free solution. Channel open probability (P(o)) and mean closed time constant (tau(C)) were strongly voltage-dependent, indicating that gating mechanisms may underlie current inward rectification. Millimolar concentrations of Ca2+ or Mg2+ applied to the cytoplasmic side of the membrane produced slow irreversible inhibition of channel activity. The Mg(2+)-dependent cation channels described in this study differ from the MIC channels described in human T-cells, Jurkat, and rat basophilic leukemia (RBL) cells in their I-V relationships, kinetic parameters and dependence on intracellular divalent cations. Our results suggested that endogenously expressed Mg(2+)-dependent cation channels in K562 cells and the MIC channels in other hematopoietic cells might be formed by different channel proteins.

Annexin 6 Modulates the Maxi-chloride Channel of the Apical Membrane of Syncytiotrophoblast Isolated from Human Placenta

The syncytiotrophoblast separates the maternal and fetal blood and constitutes the primary barrier for maternal-fetal transport. The Maxi-chloride channel from the apical membrane of the syncytiotrophoblast plays a role in the chloride conductance. Annexins can play an important role in the regulation of membrane events. In this study we evaluate the role of annexin 6 in the Maxichloride channel properties. The results showed that annexin 6 is bound in the apical placenta membranes in a calcium-dependent phospholipid-binding manner but also in a calcium-independent fashion. The neutralization of annexin 6 decreased the total current by 39 +/- 1.9% in the range of +/-80 mV, and the currents decrease with the time. The single-channel slope conductance was decreased from 253 +/- 7.4 pS (control) to 105 +/- 13 pS, and the amplitude decreased by 50%. The open probability was also affected when higher voltage steps were used, changes in either the positive or negative direction induced the channel to close, and the open probability (P(o)) did not decrease. In channels with neutralized annexin 6, it was maintained at 1 at +/-40 mV and at +/-80 mV. These results suggest that endogenous annexin 6 could regulate the Maxi-chloride channel. The results obtained with normal placentae, in which annexin 6 was neutralized, are similar to those described for the Maxi-chloride channel isolated from pre-eclamptic placenta. Together these data suggest that annexin 6 could play an important role in ion transport of the placenta.

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K+ channels.

Our objective was to identify and localize a K+ channel involved in gastric HCl secretion at the parietal cell secretory membrane and to characterize and compare the functional properties of native and recombinant gastric K+ channels. RT-PCR showed that mRNA for Kir2.1 was abundant in rabbit gastric mucosa with lesser amounts of Kir4.1 and Kir7.1, relative to beta-actin. Kir2.1 mRNA was localized to parietal cells of rabbit gastric glands by in situ RT-PCR. Resting and stimulated gastric vesicles contained Kir2.1 by Western blot analysis at approximately 50 kDa as observed with in vitro translation. Immunoconfocal microscopy showed that Kir2.1 was present in parietal cells, where it colocalized with H+ -K+ -ATPase and ClC-2 Cl- channels. Function of native K+ channels in rabbit resting and stimulated gastric mucosal vesicles was studied by reconstitution into planar lipid bilayers. Native gastric K+ channels exhibited a linear current-voltage relationship and a single-channel slope conductance of approximately 11 pS in 400 mM K2SO4. Channel open probability (Po) in stimulated vesicles was high, and that of resting vesicles was low. Reduction of extracellular pH plus PKA treatment increased resting channel Po to approximately 0.5 as measured in stimulated vesicles. Full-length rabbit Kir2.1 was cloned. When stably expressed in Chinese hamster ovary (CHO) cells, it was activated by reduced extracellular pH and forskolin/IBMX with no effects observed in nontransfected CHO cells. Cation selectivity was K+ = Rb+ >> Na+ = Cs+ = Li+ = NMDG+. These findings strongly suggest that the Kir2.1 K+ channel may be involved in regulated gastric acid secretion at the parietal cell secretory membrane.

The Effects of β3 Subunit Incorporation on the Pharmacology and Single Channel Properties of Oocyte-expressed Human α3β4 Neuronal Nicotinic Receptors

We compared the main properties of human recombinant alpha3beta4beta3 neuronal nicotinic receptors with those of alpha3beta4 receptors, expressed in Xenopus oocytes. beta3 incorporation decreased the channel mean open time (from 5.61 to 1.14 ms, after approximate correction for missed gaps) and burst length. There was also an increase in single channel slope conductance from 28.8 picosiemens (alpha3beta4) to 46.7 picosiemens (alpha3beta4beta3; in low divalent external solution). On the other hand, the calcium permeability (determined by a reversal potential method in chloride-depleted oocytes) and the pharmacological properties of beta3-containing receptors differed little from those of alpha3beta4. The main pharmacological difference in alpha3beta4beta3 "triplet" receptors was a 3-fold decrease in the potency of lobeline relative to acetylcholine. Nevertheless, there was no change in the rank order of potency for agonists (epibatidine >> lobeline > cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, nicotine > acetylcholine > carbachol for both receptors; measured at low agonist concentrations). Sensitivity to the competitive antagonists trimetaphan (0.2-1 microM) and dihydro-beta-erythroidine (30 microM) was similar for the two combinations, with a Schild KB for trimetaphan of 76 and 66 nM on alpha3beta4 and alpha3beta4beta3, respectively. The change in single channel conductance confirms that beta3 replaces a beta4 subunit in the pentamer. The absence of pronounced differences in the pharmacological profile of the triplet receptor argues against a role for the beta3 subunit in the formation of agonist binding sites, whereas the changes in channel kinetics suggest an important effect on receptor gating. The shortening of the burst length of beta3-containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics.

Analysis of the K+ current profile of mature rat oligodendrocytes in situ.

Previous studies have reported that mature oligodendrocytes (OLGs) in vitro display various voltage-dependent K+ currents while in situ OLGs show only voltage-independent K+ currents. Given this discrepancy and the lack of information on myelinating OLG ion channel expression in situ, we characterized mature OLG currents in myelinating corpus callosum slices from 17 to 36-day old rats. OLGs were recorded in cell-attached and whole-cell patch-clamp configurations, displayed morphology typical of mature OLGs, and stained positive for myelin basic protein. OLGs displayed large voltage-independent currents that decayed during the voltage pulse and small voltage-activated outward currents. The latter were blocked by TEA, activated between -40 and -50 mV, and decayed slowly. The former were composed of large voltage-independent, time-dependent Ba2+ (1 mM)-sensitive currents, and voltage-dependent Cs+ (5 mM) and Ba2+ (100 mM)-sensitive currents that reversed near the K+ equilibrium potential and inactivated at hyperpolarized potentials, identifying them as inwardly rectifying K+ currents. Inwardly rectifying single-channel K+currents could be recorded in the cell-attached configuration. The estimated single-channel slope conductance was 30 pS. The steady-state open probability was voltage-dependent and declined from 0.9 to 0.5 between -80 and -150 mV. Overall, mature OLGs in situ possess time- and also voltage-dependent K+ currents, which may facilitate clearance of K+ released during axonal firing.

Gap junction channel activity in cultured human bladder smooth muscle cell pairs: gating and unitary conductances

The apparent discordance between the absence of demonstrable gap junction plaques detected with light microscopy and the observation that the space constant for intercellular current flow in bladder strips is consistent with their presence has led to controversy concerning the putative presence and role of the intercellular pathway in modulating the contractility of the electrically excitable detrusor myocytes. To more directly address this issue, double whole cell patch recordings (DWCR), in conjunction with Northern and Western blot techniques were used to evaluate connexin43 (Cx43) expression and to characterize gap junction channel properties in human bladder smooth muscle cells. Northern and Western blots revealed the presence of Cx43 mRNA and protein, respectively, in cultured myocytes from human bladder, and Western blots confirmed the presence of Cx43 protein in situ as well. Consistent with the molecular observations, human bladder cell pairs were well coupled with average junctional conductances of 6.5 ± 4.6 picosiemens (pS) (ranging from 0 to 15 pS; n = 37). Macroscopic gap junction channel currents exhibited moderate voltage dependence, and the normalized Gj–Vj relation was both symmetrical on Vj polarity and, furthermore, well described by a 2-state Boltzmann relation (Gmin ≈0.33, V0 = 63.6 mV, Z = 0.117 or equal to 2.95 gating charges), indicative of a bilateral voltage-gated mechanism. The measured single channel slope conductance was 120 pS for the fully open channel and 26 pS for the substate state. Consistent with previous observations in other human smooth muscle cell types, the dwell time of the subconductance state was too small (≈10%) to be physiologically relevant to the macroscopic conductance. These data represent the first direct observation of gap junction channel activity in human bladder cell pairs. Moreover, in light of the known biophysical properties of Cx43-derived gap junction channels, these data clearly indicate that the absence of structural correlates of intercellular communication at the light microscopic level does not preclude an important role for gap junctions in the initiation, maintenance, and modulation of detrusor tone in the bladder.

Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons.

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV, and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+]i) with a Hill plot giving a half-saturating [Ca2+] (K0.5) of 1.35 microM and slope of approximately equals 3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 microM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+]i <100 nM and the increased channel activity evoked by ionomycin was consistent with a rise in [Ca2+]i to > or =0.3 microM. TEA (0.2-1 mM) increased the action potential duration approximately equals 1.5-fold and reduced the amplitude and duration of the afterhyperpolarization (AHP) by 26%. Charybdotoxin (100 nM) did not significantly alter the action potential duration or AHP amplitude but reduced the AHP duration by approximately equals 40%. Taken together, these data indicate that BK channel activation contributes to the action potential and AHP duration in rat intracardiac neurons.

Prostaglandin E1 activates the large-conductance KCa channel in human corporal smooth muscle cells.

The large conductance calcium-sensitive potassium channel (KCa or maxi-K) is an important modulator of human corporal smooth muscle tone, and therefore, erectile capacity. The goal of this investigation was to evaluate the actions of prostaglandin E1 (PGE1), the most widely used and effective drug for the treatment of impotence, on the activity of the KCa channel, a prominent K+ current present in human corporal smooth muscle. Whole-cell patch clamp studies conducted on short-term cultured and enzymatically dissociated human corporal smooth muscle cells, revealed mean resting potentials of -50.8 +/- 2.1 mV (n = 8) and -34 +/- 4 mV (n = 8), respectively. In the attached-patch configuration, the corresponding single-channel slope conductance values for the KCa channel subtype were 173 +/- 4 pS (n = 8) in cultured cells, and 190 +/- 13 pS (n = 3) in freshly isolated myocytes. Furthermore, voltage clamp experiments revealed that relative to control values, the application of PGE1 to cultured cells (3.3 or 33 microM) elicited an apparent increase in both the open probability (Po; ranging from 1.2-23 fold), and the mean open time (5-6 fold) of the KCa channel at membrane potentials of +90 mV and +110 mV. PGE1-induced alterations in KCa channel activity were also observed in freshly isolated corporal myocytes. In the whole cell-recording mode, statistically significant, Charybdotoxin-sensitive (100 nM) 2-3 fold increases in the outward K+ currents were observed in both cultured and freshly isolated corporal myocytes. The presence of a PKA inhibitor (fragment 6-22 amide; 10 microM) in the pipette tip was also associated with a nearly complete ablation of the observed PGE1-induced whole cell K+ currents. Taken together, these data confirm and extend our previous observations, and indicate that PGE1-induced relaxation of human corporal smooth muscle is related, at least in part, to activation of the KCa channel subtype resulting in cellular hyperpolarization.

Blockade of Ca2+-Activated K+ Channels Inhibits Proliferation of Human Endothelial Cells Induced by Basic Fibroblast Growth Factor

Basic fibroblast growth factor (bFGF) exerts angiogenic and mitogenic properties in human tissue. Since changes in ion currents modulate essential Ca²⁺-dependent intracellular pathways in endothelial cells, we have investigated a possible contribution of Ca²⁺-activated K⁺ channels (BK_Ca) on bFGF-induced endothelial cell proliferation. The patch-clamp technique was used to identify BK_Ca and to study their modulation by bFGF in cultured endothelial cells of human umbilical cord veins (HUVEC). Cell counts of HUVEC were carried out on different days to analyze bFGF-induced cell proliferation and its influence by the specific BK_Ca blocker iberiotoxin (IBX). Using single-channel recordings, we found characteristic BK_Ca with a single-channel slope conductance of 170.3 ± 2.1 pS (n = 7), half-maximal activation at internal pCa = 5.7 (n = 5; test potential: 80 mV), and dose-dependent block by IBX (25–100 nmol/l). In cell-attached patches bFGF (50 ng/ml) caused a significant increase in the open-state probability (NPo) after 6 min at test potentials of 80 and 100 mV (n = 28; p < 0.001), respectively, which lasted up to 30 min. After preincubation with pertussis toxin (100 ng/ml; 4 h) bFGF superfusion did not cause a significant increase in BK_Ca activity until 25 min had passed (n = 20; p < 0.01). Addition of 100 nmol/l IBX to the pipette solution caused a total block of BK_Ca within 2 min in cell-attached patches, whereas bFGF (50 ng/ml) was not able to activate BK_Ca. When incubated with IBX (25–100 nmol/l) every 2 days, bFGF-induced proliferation of HUVEC was significantly decreased by 50 (–41%) and 100 nmol/l (–50%) IBX (n = 5; p < 0.001) after 7 days. We conclude that activation of BK_Ca by bFGF may play an important role in bFGF-induced proliferation of human endothelial cells and thus might be important in the process of angiogenesis and vascular remodelling.

Activation kinetics and single channel properties of recombinant alpha1beta2gamma2L GABA(A) receptor channels.

Recombinant alpha1beta2gamma2L GABA(A) receptor channels, transiently expressed in HEK 293 cells, were investigated using the patch-clamp technique in combination with a device for ultra-fast solution exchange. The dose-response relationship revealed an EC50 of 11.6 +/- 0.9 microM and saturated with 3 mM GABA. The slope between 0.001 and 0.01 mM GABA was 2.2 +/- 0.4, indicating at least three binding sites for GABA. The rise time decreased from about 120 ms at 0.001 mM GABA to about 0.8 ms at 10 mM GABA. Single channel openings were grouped in bursts with an average duration of 10.3 +/- 3.0 ms. More than 95% of the current was represented by a single channel slope conductance of about 29 pS.

Modulation of voltage-dependent properties of a swelling-activated Cl- current.

We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl- current (ICl,swell) in BC3H1 myoblasts. This Cl- current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl- currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (approximately 60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10-80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg2+ concentration or reducing the Cl- concentration of the extracellular solution. Replacing extracellular Cl- by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN- > I- > NO3- > Cl- >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg2+, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of ICl,swell are strongly influenced by external pH, external divalent cations, and by the nature of the permeant anion.

Monovalent ion selectivity sequences of the rat connexin43 gap junction channel.

The relative permeability sequences of the rat connexin 43 (rCx43) gap junction channel to seven cations and chloride were examined by double whole cell patch clamp recording of single gap junction channel currents in rCx43 transfected neuroblastoma 2A (N2A) cell pairs. The measured maximal single channel slope conductances (gammaj, in pS) of the junctional current-voltage relationships in 115 mM XCI were RbC1 (103) > or = CsC1 (102) > KC1 (97) > NaC1 (79) > or = LiC1 (78) > TMAC1 (65) > TEAC1 (53) and for 115 mM KY were KBr (105) > KC1 (97) > Kacetate (77) > Kglutamate (61). The single channel conductance- aqueous mobility relationships for the test cations and anions were linear. However, the predicted minimum anionic and cationic conductances of these plots did not accurately predict the rCx43 channel conductance in 115 mM KC1. Instead, the conductance of the rCx43 channel in 115 mM KC1 was accurately predicted from cationic and anionic conductance-mobility plots by applying a mobility scaling factor Dx/Do, which depends upon the relative radii of the permeant ions to an estimated pore radius. Relative permeabilities were determined for all of the monovalent catious and anions tested from asymmetric salt reversal potential measurements and the Goldman-Hodgkin-Katz voltage equation. These experiments estimate the relative chloride to potassium permeability to be 0.13. The relationship between the relative cation permeability and hydrated radius was modeled using the hydrodynamic equation assuming a pore radius of 6.3 +/- 0.4 A. Our data quantitatively demonstrate that the rCx43 gap junction channel is permeable to monovalent atomic and organic cations and anions and the relative permeability sequences are consistent with an Eisenman sequence II or I, respectively. These predictions about the rCx43 channel pore provide a useful basis for future investigations into the structural determinants of the conductance and permeability properties of the connexin channel pore.

Capsaicin activates a nonselective cation channel in cultured neonatal rat dorsal root ganglion neurons

Capsaicin (CAP), a neurotoxin, has been reported to activate a nonselective cation current in dorsal root ganglion (DRG) neurons. In this paper, we identify and describe the properties of CAP-activated single channels in cultured neonatal rat DRG neurons. We first identified CAP-sensitive whole-cell currents that reversed near 0 mV in physiological solution. In solution containing 140 mM Na+, extracellular application of CAP to outside-out patches caused activation of an ion channel in a concentration-dependent manner (EC50 = 1.1 microM). The channel was blocked by the CAP antagonist capsazepine (10 microM). The channel was also activated by 2-10 nM resiniferatoxin, a potent analog of CAP. In symmetrical 140 mM Na+, the single-channel slope conductances were 45.3 +/- 1.0 and 80.0 +/- 4.2 pS at -60 and +60 mV, respectively, showing outward rectification (n = 9). The reversal potential did not shift significantly when Na+ was replaced by K+, Cs+, Rb+, or Li+, showing that the channel discriminated poorly among cations. The channel was also permeable to Ca2+. Although acid (pH < 6.2) was suggested to be an endogenous activator of the CAP receptor, an acid solution (pH 5.9-6.0) failed to activate the channels in outside-out patches. This is the first clear demonstration of the presence of the CAP-activated ion channel in DRG neuron. Opening of these ligand-gated, cation-selective channels gives rise to the whole-cell CAP-activated current in DRG neurons and may underlie the neurotoxic effects of CAP.

Arachidonic acid activation of a new family of K+ channels in cultured rat neuronal cells.

1. The presence and properties of K+ channels activated by arachidonic acid were studied in neuronal cells cultured from the mesencephalic and hypothalamic areas of rat brain. 2. Arachidonic acid produced a concentration-dependent (5-50 microM) and reversible activation of whole-cell currents. 3. In excised membrane patches, arachidonic acid applied to the cytoplasmic or extracellular side of the membrane caused opening of three types of channels whose current-voltage relationships were slightly outwardly rectifying, inwardly rectifying and linear, and whose single channel slope conductances at +60 mV were 143, 45 and 52 pS, respectively. 4. All three currents were K+ selective and blocked by 2 mM Ba2+ but not by other K+ channel blockers such as tetraethylammonium chloride, 4-aminopyridine and quinidine. The outwardly and inwardly rectifying currents were slightly voltage dependent with higher channel activity at more depolarized potentials. 5. Arachidonic acid activated the K+ channels in cells treated with cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and nordihydroguaiaretic acid), indicating that arachidonic acid itself can directly activate the channels. Alcohol and methyl ester derivatives of arachidonic acid failed to activate the K+ channels, indicating that the charged carboxyl group is important for activation. 6. Certain unsaturated fatty acids (linoleic, linolenic and docosahexaenoic acids), but not saturated fatty acids (myristic, palmitic, stearic acids), also reversibly activated all three types of K+ channel. 7. All three K+ channels were activated by pressure applied to the membrane (i.e. channels were stretch sensitive) with a half-maximal pressure of approximately 18 mmHg. The K+ channels were not blocked by 100 microM GdCl3. 8. A decrease in intracellular pH (over the range 5.6-7.2) caused a reversible, pH-dependent increase in channel activity whether the channel was initially activated by arachidonic acid or stretch. 9. Glutamate, a neurotransmitter reported to generate arachidonic acid in striatal neurons, did not cause activation of the K+ channels when applied extracellularly in cell-attached patches. 10. It is suggested that the K+ channels described here belong to a distinct family of ion channels that are activated by either fatty acids or membrane stretch. Although the physiological roles of these K+ channels are not yet known, they may be involved in cellular processes such as cell volume regulation and ischaemia-induced elevation of K+ loss.

Influence of extracellular Ca2+ on endogenous Cl- channels in Xenopus oocytes.

Removal of Ca2+ from the external bath solution evoked marked depolarization and large currents (up to several microamperes) in voltage-clamped defolliculated oocytes of Xenopus laevis. The resulting current was not carried by a cation influx but was due to a huge Cl- efflux, which could be strongly inhibited by the Cl- channel blockers flufenamic acid and niflumic acid. Removal of Mg2+ or Ba2+ from the solutions had the same effects as removing Ca2+. The reversal potential of -12 mV also indicated that Cl- channels were responsible for the large currents. Patch-clamp studies revealed a single-channel slope conductance of 90 pS. During oocyte maturation these channels remained active. The half-maximal Ca2+ concentration of about 20 microM showed that quite low doses of extracellular Ca2+ profoundly influence the electrical properties of the oocyte membrane.

Alterations in muscarinic K+ channel response to acetylcholine and to G protein-mediated activation in atrial myocytes isolated from failing human hearts.

A variety of previous studies have demonstrated reduced diastolic potential and electrical activity in atrial specimens from patients with heart disease. Although K+ channels play a major role in determining resting membrane potential and repolarization of the action potential, little is known about the effects of preexisting heart disease on human atrial K+ channel activity. We characterized the inwardly rectifying K+ channel (IKI) and the muscarinic K+ channel [IK(ACh)] in atrial myocytes isolated from patients with heart failure (HF) and compared electrophysiological characteristics with those from donors (control) by the patch-clamp technique. Resting membrane potentials of isolated atrial myocytes from HF were more depolarized (-51.1 +/- 9.7 mV, mean +/- SD, n = 30 patients) than those from donors (-73.0 +/- 7.2 mV, n = 4 patients, P < .001). The action potential duration in HF was longer than that in donors. Although acetylcholine (ACh) shortened the action potential, reduced the overshoot, and hyperpolarized the atrial cell membrane in HF, these effects were attenuated compared with those observed in donors. The whole-cell membrane current slope conductance in HF was small, the reversal potential was more positive, and the sensitivity to ACh was less compared with donors. In single-channel recordings from cell-attached patches, IK1 channel conductance and gating characteristics were the same in HF and donor atria. When ACh was included in the pipette solution, IK(ACh) was activated in both groups. Single-channel slope conductance of IK(ACh) averaged 42 +/- 3 pS (n = 28) in HF and 44 +/- 2 pS (n = 4) in donors, and mean open lifetime was 1.3 +/- 0.3 milliseconds (n = 24) in HF and 1.5 +/- 0.4 milliseconds (n = 4) in donors. These values were virtually identical in the two groups (not significantly different, NS), although both single IK1 and IK(ACh) channel densities were less in HF. Channel open probability of IK(ACh) was also less in HF (4.0 +/- 1.2%, n = 24) than in donors (6.8 +/- 1.1%, n = 3, P < .01). The concentration of ACh at half-maximal activation was 0.11 mumol/L in HF and 0.03 mumol/L in donors. In excised inside-out patches, IK(ACh) from HF required higher concentrations of GTP and GTP gamma S to activate the channel compared with donors. These results suggest a reduced IK(ACh) channel sensitivity to M2 cholinergic receptor-linked G protein (Gi) in HF compared with donors. Atrial myocytes isolated from failing human hearts exhibited a lower resting membrane potential and reduced sensitivity to ACh compared with donor atria. Whole-cell and single-channel measurements suggest that these alterations are caused by reduced IK1 and IK(ACh) channel density and reduced IK(ACh) channel sensitivity to Gi-mediated channel activation in HF.

Single channel recordings of Nt- and L-type Ca2+ currents in rat neurohypophysial terminals

1. Ca2+ currents through single channels in acutely dissociated nerve terminals from rat neurohypophyses were recorded using cell-attached patch recordings with 110 mM Ba2+ as the charge carrier. 2. One type (Nt, where the t denotes terminal) of single Ca2+ channel current was evoked only by depolarizing steps from holding potentials less negative than -50 mV. Because this channel opened primarily at the beginning of a 180-ms-long voltage pulse, the averaged ensemble current decayed rapidly (approximately 30 ms). Infrequently, the channel opened throughout such a long pulse, resulting in a long-lasting averaged ensemble current. The averaged channel open time constant (tau) was 0.34 ms and the two averaged closed time constants were 1.78 (tau 1) and 86.57 (tau 2) ms. The mean unitary slope conductance for this channel was 11 pS and its threshold for activation was approximately -10 mV. 3. The other type (L) of single Ca2+ channel current could be evoked in isolation by depolarizations from holding potentials more positive than or equal to -50 mV. This channel opened throughout an entire 180-ms-long voltage pulse. The averaged ensemble current, therefore, showed little inactivation. The averaged channel open-time constant was 0.49 ms and the two average closed time constants were 2.02 (tau 1) and 79.91 (tau 2) ms. The mean unitary slope conductance for this channel was 25 pS. 4. Bay K 8644 (5 microM), a dihydropyridine (DHP) Ca2+ channel agonist, increased the open probability of the larger-conductance L-type Ca2+ channel by prolonging the average duration (to 2.79 ms) of channel openings, but did not alter the single channel slope conductance. In contrast, the same concentration of Bay K 8644 did not affect the smaller-conductance Nt-type Ca2+ channel. The DHP Ca2+ channel antagonist nicardipine (5 microM), but not nifedipine (5 microM), reduced the open probability of the large-conductance L-type Ca2+ channel by shortening the duration (to 0.36 ms) of channel openings. 5. The voltage- and time-dependent properties of these two types of single Ca2+ channel currents are in close agreement with those of the two components of macroscopic Ca2+ currents previously reported using the "whole-terminal" recording method. Therefore these two types of single channels appear to underlie the macroscopic current. 6. Our studies suggest that the terminal Nt-type Ca2+ channel differs from the conventional somatic N- and T-type Ca2+ channels in some respects, and that the terminal L-type Ca2+ channel is similar to the conventional somatic L-type Ca2+ channel.(ABSTRACT TRUNCATED AT 400 WORDS)