Quinine inhibits chloride and nonselective cation channels in isolated rat distal colon cells.

Isolated cells from rat distal colon were investigated with the patch-clamp technique. In cell-attached and cell-excised patches (inside-out) single chloride channels with outward-rectifying properties were observed. In excised patches the single-channel conductance g was 47 +/- 5 pS at positive and 22 +/- 2 pS at negative clamp potentials (n = 6). The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10 microM) induced fast closing events, whereas 10 microM of 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) had no effect when applied to the cytosolic side. Quinine in the bath inhibited the Cl- channel by reducing its single-channel amplitude and increased open channel noise. With 0.1 mM the current amplitude decreased by 54% and with 1 mM quinine by 67%. Ca2(+)-dependent nonselective cation channels where observed after excision of the membrane patch. This channel was completely and reversibly inhibited by 100 microM DCDPC. Application of 1 mM quinine to the bath induced flickering and reduced the open-state probability from 0.94 to 0.44. In summary, besides its well established effects on K+ channels, quinine also inhibits nonselective cation channels and chloride channels by inducing fast closing events.     

Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.

Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.     

A class of non-selective cation channels in human fibroblasts

Non-selective cation channels were detected in membrane patches of cultured human fibroblasts. The channels had a unitary conductance which ranged from 14 to 25 pS in symmetrical 130 mM NaCl and were permeable to both sodium and potassium ions. Open channel probability was dependent either on the membrane potential and the Ca2+ concentration on the intracellular side of the membrane. High Ca2+ concentrations in the millimolar range were needed to keep the channel active.     

Adenosine 5'-monophosphate is a selective inhibitor of the brown adipocyte nonselective cation channel

Calcium-activated nonselective cation channels (NSC(Ca)) in brown adipocytes are inhibited by several nucleotides acting on the cytosolic side of the membrane. We used excised inside-out patches from rat brown adipocytes to identify important nucleotide structures for NSC-channel inhibition. We found that 100 microM 5'-AMP inhibited NSC-channel activity more than did ATP or ADP. Adenosine was a weak inhibitor, whereas adenine and ribose-5-phosphate had no effect. The channel activity was effectively blocked by 10 microM AMP, but it was unaffected by 10 microM cAMP, CMP, GMP, IMP, TMP or UMP. Dose-response studies yielded IC(50)-values of 4 microM for AMP and 32 microM for cAMP. dAMP was as effective as AMP, but all 5'-phosphate group modifications on AMP dramatically lowered the inhibitory effect. 10 microM of the AMP precursor adenylosuccinate weakly inhibited the channel activity. An increase in AMP concentration from 1 to 10 microM shifted the EC(50) for Ca(2+) activation almost 1 order of magnitude; a Schild plot analysis yielded a K(B) value of 0.3 microM for AMP. We conclude that AMP is the most efficacious endogenous nucleotide inhibitor of the brown adipocyte nonselective cation channel (NSC(Ca/AMP)) yet identified and that there is functional competition between Ca(2+) and AMP. The brown adipocyte NSC(Ca/AMP) thus appears to be functionally different from the NSC(Ca,PKA) in the exocrine pancreas and the NSC-(Ca,cAMP) in the endocrine pancreas, but similar to the NSC(Ca/AMP) in the endocrine pancreas.     

Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel.

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosolic (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ryanodine receptors) were examined using the planar lipid bilayer method. Single channels were recorded in symmetric 0.25 M KCl media with K+ as the major current carrier. With nanomolar [Ca2+] in both bilayer chambers, the addition of 2 mM cytosolic ATP greatly increased the number of short channel openings. As lumenal [Ca2+] was increased from < 0.1 microM to approximately 250 microM, increasing channel activities and events with long open time constants were seen at negative holding potentials. Channel activity remained low at positive holding potentials. Further increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decrease in channel activities at negative holding potentials and increased activities at positive holding potentials. A voltage-dependent activation by 50 microM lumenal Ca2+ was also observed when the channel was minimally activated by < 1 microM cytosolic Ca2+ in the absence of ATP. With microM cytosolic Ca2+ in the presence or absence of 2 mM ATP, single-channel activities showed no or only a weak voltage dependence. Other divalent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the contrary, cytosolic ATP-activated channel activities were decreased as lumenal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2 or MgCl2 to the lumenal side, which contained 50 microM Ca2+. An increase in [KCl] from 0.25 M to 1 M also reduced single-channel activities. Addition of the "fast" Ca2+ buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cls chamber increased cytosolic ATP-, lumenal Ca(2+)-activated channel activities to a nearly maximum level. These results suggested that lumenal Ca2+ flowing through the skeletal muscle Ca2+ release channel may regulate channel activity by having access to cytosolic Ca2+ activation and Ca2+ inactivation sites that are located in "BAPTA-inaccessible" and "BAPTA-accessible" spaces, respectively.     

Modulation of the activity of Ca(2+)-activated K+ channels by internal Mg2+ in cultured kidney cells vero.

The inside-out mode of the patch-clamp method was used to study the effects of internal Mg2+ on single large-conductance (193+/-7 pS) Ca(2+)-activated K+ channels in cultured kidney cells. In the absence of Ca2+, Mg2+ (1 to 10 mM) did not activate the channels but modified the activating effect of Ca2+ ions: it decreased the Hill coefficient (n), reduced the apparent dissociation constant (K0.5), and modified the channel open and closed times. K0.5 was found to be a voltage-dependent parameter. In the absence of Mg2+, it averaged 600 microM at -20 mV and 27 microM at +30 mV (22 degrees C, pH 6.8). Mg2+ at saturating concentrations (5 to 10 mM) decreased K0.5 to 50 microM at -20 mV and to 15 microM at +30 mV. Irrespective of the membrane potential, K0.5 tended to its limit value of about 12.6 microM. Thus, the effects of membrane depolarization and Mg2+ exhibited a non-additive, competitive relationship. Mg2+ perturbed the exponential shape of the voltage dependences of K0.5. The Hill coefficient characterizing the interaction of Ca2+ ions with the channels was found to be voltage-dependent. In the absence of Mg2+, it increased rather sharply from approx. 2 to 3.5 when the membrane potential was raised from -10 to 0 mV. Mg2+ increased n in a dose-dependent manner; however, about a twofold increase of n occurred within a narrow concentration range (2 to 3 mM). The action of Mg2+ on n was, apparently, voltage-independent, and the effects of Mg2+ and voltage on n were seemingly additive.     

Properties of a new calcium-permeable single channel from tracheal microsomes

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.     

Cyclopiazonic acid activates a Ca2+-permeable, nonselective cation conductance in porcine and bovine tracheal smooth muscle.

Capacitative Ca2+ entry has been examined in several tissues and, in some, appears to be mediated by nonselective cation channels collectively referred to as "store-operated" cation channels; however, relatively little is known about the electrophysiological properties of these channels in airway smooth muscle. Consequently we examined the electrophysiological characteristics and changes in intracellular Ca2+ concentration associated with a cyclopiazonic acid (CPA)-evoked current in porcine and bovine airway smooth muscle using patch-clamp and Ca2+-fluorescence techniques. In bovine tracheal myocytes, CPA induced an elevation of intracellular Ca2+ that was dependent on extracellular Ca2+ and was insensitive to nifedipine (an L-type voltage-gated Ca2+ channel inhibitor). Using patch-clamp techniques and conditions that block both K+ and Cl- currents, we found that CPA rapidly activated a membrane conductance (I(CPA)) in porcine and bovine tracheal myocytes that exhibits a linear current-voltage relationship with a reversal potential around 0 mV. Replacement of extracellular Na+ resulted in a marked reduction of I(CPA) at physiological membrane potentials (i.e., -60 mV) that was accompanied by a shift in the reversal potential for I(CPA) toward more negative membrane potentials. In addition, I(CPA) was markedly inhibited by 10 microM Gd3+ and La3+ but was largely insensitive to 1 microM nifedipine. We conclude that CPA induces capacitative Ca2+ entry in porcine and bovine tracheal smooth muscle via a Gd3+- and La3+-sensitive, nonselective cation conductance.     

Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites.

The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca2+ was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca2+ (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca2+ on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low trans Ca2+ (20 microM), increasing cis Ca2+ from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating cis Ca2+ above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca2+] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca2+ enhanced the sensitivity of the channel to activating cytosolic Ca2+, and it essentially reversed the inhibition of the channel by high cytosolic Ca2+. Potentiation of Po by increased luminal Ca2+ occurred irrespective of whether the electrochemical gradient for Ca2+ supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca2+ through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca2+ acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca2+-sensitive site(s) at the luminal face of the channel or associated protein.     

Inhibition by cytoplasmic nucleotides of a new cation channel in freshly isolated human and rat type II pneumocytes

Here we report a 26- to 29-pS cation channel abundantly expressed in freshly isolated and primary cultured type II cells from rat or healthy human lungs. The channel was never spontaneously active in cell-attached patches but could be activated by cell permeabilization with beta-escin. Excised patch-clamp experiments revealed activation by Ca(2+) concentrations at the cytoplasmic side in the micromolar range. High concentrations of amiloride (>10 microM) at the extracellular side did not inhibit. The channel was equally permeable for K(+) and Na(+) but was essentially impermeable for Cl(-), Ca(2+), and Mg(2+). It was blocked by adenosine nucleotides (cytoplasmic side) with the following order of potency: AMP approximately ADP (EC(50) ATP > adenosine > cyclic AMP. The blocking effect of ATP was reproduced by its nonhydrolyzable analogs AMPPNP or ATP-gamma-S. GTP did not inhibit. Cd(2+) blocked the channel with an EC(50) approximately 55.5 nM. We conclude that type II cells express a Ca(2+)-dependent, nucleotide-inhibited, nonselective, and Ca(2+)-impermeable cation channel (NSC(Ca/AMP)) with tonically suppressed activity. RT-PCR confirmed expression of TRPM4b, a channel with functional characteristics almost identical with NSC(Ca/AMP). Potential physiological roles are discussed.     

Caffeine activates a Ca(2+)-permeable, nonselective cation channel in smooth muscle cells

The effects of caffeine on cytoplasmic [Ca2+] ([Ca2+]i) and plasma membrane currents were studied in single gastric smooth muscle cells dissociated from the toad, Bufo marinus. Experiments were carried out using Fura-2 for measuring [Ca2+]i and tight-seal voltage-clamp techniques for recording membrane currents. When the membrane potential was held at -80 mV, in 15% of the cells studied caffeine increased [Ca2+]i without having any effect on membrane currents. In these cells ryanodine completely abolished any caffeine induced increase in [Ca2+]i. In the other cells caffeine caused both an increase in [Ca2+]i and activation of an 80-pS nonselective cation channel. In this group of cells ryanodine only partially blocked the increase in [Ca2+]i induced by caffeine; moreover, the change in [Ca2+]i that did occur was tightly coupled to the time course and magnitude of the cation current through these channels. In the presence of ryanodine, blockade of the 80-pS channel by GdCl3 or decreasing the driving force for Ca2+ influx through the plasma membrane by holding the membrane potential at +60 mV almost completely blocked the increase in [Ca2+]i induced by caffeine. Thus, the channel activated by caffeine appears to be permeable to Ca2+. Caffeine activated the cation channel even when [Ca2+]i was clamped to below 10 nM when the patch pipette contained 10 mM BAPTA suggesting that caffeine directly activates the channel and that it is not being activated by the increase in Ca2+ that occurs when caffeine is applied to the cell. Corroborating this suggestion were additional results showing that when the membrane was depolarized to activate voltage-gated Ca2+ channels or when Ca2+ was released from carbachol- sensitive internal Ca2+ stores, the 80-pS channel was not activated. Moreover, caffeine was able to activate the channel in the presence of ryanodine at both positive and negative potentials, both conditions preventing release of Ca2+ from stores and the former preventing its influx. In summary, in gastric smooth muscle cells caffeine transiently releases Ca2+ from a ryanodine-sensitive internal store and also increases Ca2+ influx through the plasma membrane by activating an 80- pS cation channel by a mechanism which does not seem to involve an elevation of [Ca2+]i.     

Fatty acid modifies Ca2+-dependent potassium channel activity in smooth muscle cells from the human aorta

By using the patch-clamp technique the effect of 2-decenoic acid (DA) on Ca2+-activated potassium (K+) channels in the membrane of smooth muscle cells from the human aorta was studied. In the presence of 0.5 microM Ca2+ and 2 mM Mg2+ on the cytoplasmic side of the membrane, a more than tenfold elevation in the probability of the channels being open (po) was observed under the effect of DA. With divalent cation concentrations of less than 1 nM DA caused a more than twofold elevation in po. In the DA-treated membranes Mg2+ ions, which normally fail to activate the channels, brought about a nearly threefold increase in the channel activity when applied to the inner membrane surface. Channel sensitivity to the activating effect of cytoplasmic Ca2+ ions did not increase with the application of DA. Single-channel conductance was unchanged by DA exposure. We suggest that DA alters the Ca2+-binding mechanism of the channel, increasing its sensitivity to Mg2+ ions, presumably owing to membrane fluidization.     

Ion channels in mammalian proximal renal tubules.

In the plasma membranes of mammalian proximal renal tubules single ion channels were investigated mainly in isolated tubules perfused on one side, in isolated nonperfused (collapsed) tubules and in primary cell cultures. With these techniques, the following results were obtained: in the luminal membrane of isolated one-sided perfused tubules of rabbit and mouse S3 segments, K(+)-selective channels with single-channel conductance (g) of 33 pS and 63 pS, respectively, were recorded. In primary cultures of rabbit S1 segments, a small-conductance (42 pS) as well as a large-conductance (200 pS) K+ channel were observed. The latter was Ca2(+)- and voltage-sensitive. In cultured cells a Ca2(+)-activated, nonselective cation channel with g = 25 pS was also recorded. On the other hand, an amiloride-sensitive channel with g = 12 pS, which was highly selective for Na+ over K+, was observed in the isolated perfused S3 segment. In the basolateral membrane of isolated perfused S3 segments, two types of K+ channels with g = 46 pS and 36 pS, respectively, were observed. The latter channel was not dependent on cytosolic Ca2+ in cell-excised patches. A K+ channel with g = 54 pS was recorded in isolated nonperfused S1 segments. This channel showed inward rectification and was more active at depolarizing potentials. In isolated perfused S3 segments, in addition to the K+ channels also a nonselective cation channel with g = 28 pS was observed. This channel was highly dependent on cytosolic Ca2+ in cell-free patches. It can be concluded that the K+ channels both in the luminal and contraluminal cell membrane are involved in the generation of the cell potential. Na+ channels in the luminal membrane may participate in Na+ reabsorption, whereas the function of a basolateral cation channel remains unclear. Recently, single anion-selective channels were recorded in membranes of endocytotic vesicles, isolated from rat proximal tubules. Vesicles were enlarged by the dehydration/rehydration method and investigated with the patch clamp technique. The Cl- channel had a conductance of 73 pS, the current-voltage curve was linear and the channel inactivated at high negative clamp potentials. It is suggested that this channel is responsible for charge neutrality during active H+ uptake into the endosomes.     

Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells.

We studied monovalent permeability of Ca2+ release-activated Ca2+ channels (ICRAC) in Jurkat T lymphocytes following depletion of calcium stores. When external free Ca2+ ([Ca2+]o) was reduced to micromolar levels in the absence of Mg2+, the inward current transiently decreased and then increased approximately sixfold, accompanied by visibly enhanced current noise. The monovalent currents showed a characteristically slow deactivation (tau = 3.8 and 21.6 s). The extent of Na+ current deactivation correlated with the instantaneous Ca2+ current upon readdition of [Ca2+]o. No conductance increase was seen when [Ca2+]o was reduced before activation of ICRAC. With Na+ outside and Cs+ inside, the current rectified inwardly without apparent reversal below 40 mV. The sequence of conductance determined from the inward current at -80 mV was Na+ > Li+ = K+ > Rb+ >> Cs+. Unitary inward conductance of the Na+ current was 2.6 pS, estimated from the ratios delta sigma2/delta Imean at different voltages. External Ca2+ blocked the Na+ current reversibly with an IC50 value of 4 microM. Na+ currents were also blocked by 3 mM Mg2+ or 10 microM La3+. We conclude that ICRAC channels become permeable to monovalent cations at low levels of external divalent ions. In contrast to voltage-activated Ca2+ channels, the monovalent conductance is highly selective for Na+ over Cs+. Na+ currents through ICRAC channels provide a means to study channel characteristics in an amplified current model.     

Homeostatic control of slow vacuolar channels by luminal cations and evaluation of the channel-mediated tonoplast Ca2+ fluxes in situ

Ca(2+), Mg(2+), and K(+) activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca(2+), by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca(2+) activity was approximately 0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca(2+) in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca(2+)) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca(2+) within a physiological range of concentrations (0.1-1.0 mM). Aggregation of the physiological vacuolar Na(+) (60 mM) and Mg(2+) (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca(2+) variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca(2+) activates the SV channel in a voltage-independent manner with K(d)=0.7-1.5 microM. Comparison of the vacuolar Ca(2+) fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 microM) cytosolic Ca(2+), only 0.5% of SV channels are open. This mediates a Ca(2+) release of only a few pA per vacuole (approximately 0.1 pA per single SV channel). Overall, our data suggest that the release of Ca(2+) through SV channels makes little contribution to a global cytosolic Ca(2+) signal.     

Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells

Ion channels, activated upon IgE-Fc epsilon receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc epsilon receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc epsilon receptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 microM Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 +/- 11 mV in the presence of sodium, 125 +/- 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the interaction of bovine pancreatic trypsin inhibitor with maxi Ca(2+)-activated K+ channels. A model system for analysis of peptide-induced subconductance states.

Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue basic peptide that is a representative member of a widely distributed class of serine protease inhibitors known as Kunitz inhibitors. BPTI is also homologous to dendrotoxin peptides from mamba snake venom that have been characterized as inhibitors of various types of voltage-dependent K+ channels. In this study we compared the effect of DTX-I, a dendrotoxin peptide, and BPTI on large conductance Ca(2+)-activated K+ channels from rat skeletal muscle using planar bilayer methodology. As previously found for DTX-I (1990. Neuron. 2:141-148), BPTI induces the appearance of distinct subconductance events when present on the internal side of maxi K(Ca) channels. The single channel kinetics of substate formation follow the predictions of reversible binding of the peptide to a single site or class of sites with a Kd of 4.6 microM at 0 mV and 50 mM symmetrical KCl. The apparent association rate of BPTI binding decreases approximately 1,000-fold per 10-fold increase in ionic strength, suggestive of a strong electrostatic interaction between the basic peptide and negative surface charge in the vicinity of the binding site. The equilibrium Kd for BPTI and DTX-I is also voltage dependent, decreasing e-fold per 30 mV of depolarization. The unitary subconductance current produced by BPTI binding exhibits strong inward rectification in the presence of symmetrical KCl, corresponding to 15% of open channel current at +60 mV and 70% of open state at -40 mV. In competition experiments, the internal pore-blocking ions, Ba2+ and TEA+, readily block the substate with the same affinity as that for blocking the normal open state. These results suggest that BPTI does not bind near the inner mouth of the channel so as to directly interfere with cation entry to the channel. Rather, the mechanism of substate production appears to involve a conformational change that affects the energetics of K+ permeation.     

Potassium channel activity recorded from the apical membrane of freshly isolated epithelial cells in rat caudal epididymis.

K+ channels were recorded in excised, inside-out patches from the apical membrane of the freshly isolated tubule of the caudal portion of the rat epididymis. With asymmetric K+ concentrations in bath and pipette (140 mM K+in/6 mM K+out), the channels had a slope conductance of 54.2 pS at 0 mV. The relative permeability of K+ over Na+ was about 171 to 1. The channels were activated by intracellular Ca2+ and by membrane depolarization. These channels belong to a class defined as "intermediate-conductance Ca2+-activated K+ channel. " External tetraethylammonium ions (TEA+) caused a flickery block of the channel with reduction in single-channel current amplitude measured at a range of holding membrane potentials (-40 to 60 mV). Activity of the K+ channels was inhibited by intracellular ATP (KD =1.188 mM). The channel activity was detected only occasionally in patches from the apical membrane (about 1 in 17 patches containing active channels). The presence of the intermediate-conductance Ca2+-activated K+ channels indicates that they could provide a route for K+ secretion in a Ca2+-dependent process responsible for a high luminal K+ concentration found in the epididymal duct of the rat.     

Purified IP3 receptor from smooth muscle forms an IP3 gated and heparin sensitive Ca2+ channel in planar bilayers.

The IP3 receptor of aortic smooth muscle, purified to near homogeneity, was incorporated into vesicle derived planar bilayers. The receptor forms channels which are gated by Ins(1,4,5)P3 (0.5 microM) and are permeable to Ca2+ (Ca2+ greater than K+ much greater than Cl-). Channel activation is specific for Ins(1,4,5)P3. Essentially no activation of channel currents was found for Ins(1,3,4)P3 or Ins(1,3,4,5)P4 at 10 microM. Heparin (25 micrograms/ml) blocked induced currents completely at all levels of activity while ATP (50 microM) increased mean current levels 2 to 4 fold. Ins(1,4,5)P3 activated mean currents increased non-linearly with voltage above about -40 mV applied voltage. Mean current levels could be reversibly adjusted by voltage to the single channel level (0 to -50 mV) or to macroscopic levels (-50 to -100 mV) over periods exceeding 1 h. Single channel events are characterized by fast transitions between predominantly non-resolved sublevels. Estimates of maximal single event currents yield a slope conductance of 32 +/- 4 pS (0 to -60 mV, 50 mM CaCl2). Thus, the purified IP3 receptor forms a channel with functional properties characteristic of IP3 triggered Ca2+ release.     

Cyclic nucleotide-gated channels of rat olfactory receptor cells: divalent cations control the sensitivity to cAMP

cAMP-gated channels were studied in inside-out membrane patches excised from the apical cellular pole of isolated olfactory receptor cells of the rat. In the absence of divalent cations the dose-response curve of activation of patch current by cAMP had a KM of 4.0 microM at -50 mV and of 2.5 microM at +50 mV. However, addition of 0.2 or 0.5 mM Ca2+ shifted the KM of cAMP reversibly to the higher cAMP concentrations of 33 or 90 microM, respectively, at -50 mV. Among divalent cations, the relative potency for inducing cAMP affinity shifts was: Ca2+ > Sr2+ > Mn2+ > Ba2+ > Mg2+, of which Mg2+ (up to 3 mM) did not shift the KM at all. This potency sequence corresponds closely to that required for the activation of calmodulin. However, the Ca(2+)-sensitivity is lower than expected for a calmodulin-mediated action. Brief (60 s) transient exposure to 3 mM Mg2+, in the absence of other divalent cations, had a protective effect in that following washout of Mg2+, subsequent exposure to 0.2 mM Ca2+ no longer caused affinity shifts. This protection effect did not occur in intact cells and was probably a consequence of patch excision, possibly representing ablation of a regulatory protein from the channel cyclic nucleotide binding site. Thus, the binding of divalent cations, probably via a regulatory protein, controls the sensitivity of the cAMP-gated channels to cAMP. The influx of Ca2+ through these channels during the odorant response may rise to a sufficiently high concentration at the intracellular membrane surface to contribute to the desensitization of the odorant- induced response. The results also indicate that divalent cation effects on cyclic nucleotide-gated channels may depend on the sequence of pre-exposure to other divalent cations.