Properties of single- and double-barreled Cl channels of shark rectal gland in planar bilayers

Chloride channels from the apical plasma membrane fraction of rectal gland of Squalus acanthias were characterized by incorporation into planar bilayers in the presence of cAMP-PK/ATP. In a total of 80 bilayer preparations, 21 Cl-selective channels were observed as single channels and 13 as pairs. This was a significantly greater number of double Cl channels than expected from a binomial distribution. The double Cl channels were divided into two groups based on kinetic and voltage-dependent behavior. One group had properties identical to the single channels (gb1) while the other was consistent with a double-barreled channel (gb2) with coordinated activity between proto-channels. The single-channel slope conductances of gb1 and gb2 from -60 to +20 mV with a 250/70 mM KCl gradient were 41 and 75 pS, respectively. With symmetrical 250 mM KCl, the I-V relation of gb1 showed outward rectification with 47.8 +/- 6.6 pS at cis negative potentials and 68.9 +/- 6.1 pS at cis positive potentials. gb1 was open from 70 to 95% at all electrochemical potentials from -80 to +40 mV. gb2 was steeply voltage dependent between -80 and -20 mV. Both gb1 and gb2 were insensitive to Ca (from 100 nm to 1 microM), blocked by 0.1 mM DIDS and highly selective for chloride. These data suggest that double-barreled Cl channels are related to the family of small, outwardly rectifying Cl channels of epithelial membranes.     

Heterogeneity of conductance states in calcium channels of skeletal muscle

The single channel conductance of the dihydropyridine (DHP)-sensitive calcium channel from rabbit skeletal muscle transverse tubules was analyzed in detail using the planar bilayer recording technique. With 0.1 M BaCl2 on both sides of the channel (symmetrical solutions), the most frequent conductance is 12 pS, which is independent of holding potential in the range of -80 to +80 mV. This conductance accounts for approximately 80% of all openings analyzed close to 0 mV. Two additional channels of conductance 9 and 3 pS are also present at all positive potentials, but their relative occurrence close to 0 mV is low. All channels depend on the presence of agonist Bay K 8644 and are inhibited by the antagonist nitrendipine. The relative occurrence of 9 and 3 pS can be increased, and that of 12 pS decreased, by several interventions such as external addition of cholesterol, lectin (wheat germ agglutinin), or calmodulin inhibitor R24571 (calmidazolium). The 9- and 3-pS channels are also conspicuous at positive potentials larger than +40 mV. We suggest that 9- and 3-pS channels are two elementary conductances of the same DHP-sensitive Ca channel. Under most circumstances, these two conductances are gated in a coupled way to generate a channel with a unitary conductance of 12 pS. Interventions tested, including large depolarizations, probably decompose or uncouple the 12-pS channel into 9 and 3 pS.     

Voltage-activated ionic channels and conductances in embryonic chick osteoblast cultures

Patch-clamp measurements were made on osteoblast-like cells isolated from embryonic chick calvaria. Cell-attached-patch measurements revealed two types of high conductance (100-250 pS) channels, which rapidly activated upon 50-100 mV depolarization. One type showed sustained and the other transient activation over a 10-sec period of depolarization. The single-channel conductances of these channel types were about 100 or 250 pS, depending on whether the pipettes were filled with a low K+ (3 mM) or high K+ (143 mM) saline, respectively. The different reversal potentials under these conditions were consistent with at least K+ conduction. Whole-cell measurements revealed the existence of two types of outward rectifying conductances. The first type conducts K+ ions and activates within 20-200 msec (depending on the stimulus) upon depolarizing voltage steps from less than -60 mV to greater than -30 mV. It inactivates almost completely with a time constant of 2-3 sec. Recovery from inactivation is biphasic with an initial rapid phase (1-2 sec) followed by a slow phase (greater than 20 sec). The second whole-cell conductance activates at positive membrane potentials of greater than +50 mV. It also rapidly turns on upon depolarizing voltage steps. Activation may partly disappear at the higher voltages. Its single channels of 140 pS conductance were identified in the whole cell and did conduct K+ ions but were not highly Cl- or Na+ selective. The results show that osteoblasts may express various types of voltage controlled ionic channels. We predict a role for such channels in mineral metabolism of bone tissue and its control by osteoblasts.     

Channel reconstitution in liposomes and planar bilayers with HPLC-purified MIP26 of bovine lens

Summary The major intrinsic protein (MIP26) of bovine lens membranes, purified by HPLC, was incorporated into liposomes and planar bilayers. Permeability of MIP26 channels was studied in liposomes by a spectrophotometric osmotic-swelling assay, and channel electrical properties were monitored in planar bilayers following liposome fusion. Particle formation in liposomes was determined by freeze fracture. MIP26 channels were permeable to KCl and sucrose. In planar bilayers, channel-conductance transitions were observed only after addition of liposomes to both chambers and with voltages greater than ±20 mV. Channel open probability decreased progressively as voltage increased, and an open probability of 50% was at 60–80 mV, indicating that the channels are voltage dependent. Histograms of single-channel current amplitudes at 80 mV showed a Gaussian distribution that peaked at 10 pA (120 pS), after subtraction of 1 pA baseline current. Frequency distributions of open and closed times at 80 mV were single exponential functions with time constants of 0.13 and 1.9 sec, respectively. Open time constants ranged from 0.1 to 0.3 sec, and closed time constants ranged from 1 to 7 sec. Cs⁺ did not decrease conductance, but reduced mean open time from 0.2 to 0.038 sec and mean closed time from 1.5 to 0.38 sec. The increase in channel flickering with Cs⁺ occurred in bursts. TEA affected neither conductance nor kinetics. Channel events were also observed in Na⁺ solutions (zero K⁺). These data indicate that MIP26 channels are not K⁺-selective channels. Channel characteristics such as: permeability to molecules larger than small ions, conductance greater than 100 pS, long open and closed time constants, etc., are similar to those of gap junction channels.     

Ca2+ currents in cerebral artery smooth muscle cells of rat at physiological Ca2+ concentrations

Single Ca2+ channel and whole cell currents were measured in smooth muscle cells dissociated from resistance-sized (100-microns diameter) rat cerebral arteries. We sought to quantify the magnitude of Ca2+ channel currents and activity under the putative physiological conditions of these cells: 2 mM [Ca2+]o, steady depolarizations to potentials between -50 and -20 mV, and (where possible) without extrinsic channel agonists. Single Ca2+ channel conductance was measured over a broad range of Ca2+ concentrations (0.5-80 mM). The saturating conductance ranged from 1.5 pS at 0.5 mM to 7.8 pS at 80 mM, with a value of 3.5 pS at 2 mM Ca (unitary currents of 0.18 pA at -40 mV). Both single channel and whole cell Ca2+ currents were measured during pulses and at steady holding potentials. Ca2+ channel open probability and the lower limit for the total number of channels per cell were estimated by dividing the whole-cell Ca2+ currents by the single channel current. We estimate that an average cell has at least 5,000 functional channels with open probabilities of 3.4 x 10(-4) and 2 x 10(-3) at -40 and -20 mV, respectively. An average of 1-10 (-40 mV and -20 mV, respectively) Ca2+ channels are thus open at physiological potentials, carrying approximately 0.5 pA steady Ca2+ current at -30 mV. We also observed a very slow reduction in open probability during steady test potentials when compared with peak pulse responses. This 4- 10-fold reduction in activity could not be accounted for by the channel's normal inactivation at our recording potentials between -50 and -20 mV, implying that an additional slow inactivation process may be important in regulating Ca2+ channel activity during steady depolarization.     

A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers

A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris or 10 mM HEPES buffer, but substitution of 100 mM NaCl with 100 mM tetraethylammonium chloride on the cis side results in increased rectification with a 40% reduction in current at negative voltages. The gating of the channel is weakly voltage dependent with an open-state probability of 0.23 at -75 mV and 0.64 at +75 mV. Channel gating is sensitive to cis pH with an increased opening probability observed for a pH change of 7.4 to 11 and nearly complete inhibition for a pH change of 7.4 to 6.0. The lobster Cl- channel is reversibly blocked by the anion transport inhibitors, SITS (4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid). Many of these characteristics are similar to those previously described for small conductance Cl- channels in various vertebrate cells, including epithelia. These functional comparisons suggest that this invertebrate Cl- channel is an evolutionary prototype of a widely distributed class of small conductance anion channels.     

Two types of single inward rectifying potassium channels in rat myocardial cells

The patch-clamp method was used to examine inward rectifying potassium channels in the membrane of rat ventricular myocytes. Two types of inward rectifying channels strongly selective for K+ ions and with different conductance and kinetics coexist in rat myocardial cells. When the concentration of K+ was 140 mmol/l on the extracellular side of the patch, the conductance was 38.9 pS for type I channels and 25.7 pS for the type II. The type II channels had a detectable conductance (4 pS) at potentials positive than the potassium equilibrium potential. The mean open time was 18 ms at -60 mV patch membrane potential and 12 ms at -100 mV for type I channels, and 1.3 s at -60 mV and 0.94 s at -105 mV for type II channels, respectively. The opening probability of type II channels decreased with hyperpolarization. The type II channels can adopt several (about 10 or more) conductance states, which can occur either within one opening or as individual events.     

Calcium- and voltage-activated potassium channels in human macrophages.

Single calcium-activated potassium channel currents were recorded in intact and excised membrane patches from cultured human macrophages. Channel conductance was 240 pS in symmetrical 145 mM K+ and 130 pS in 5 mM external K+. Lower conductance current fluctuations (40% of the larger channels) with the same reversal potential as the higher conductance channels were noted in some patches. Ion substitution experiments indicated that the channel is permeable to potassium and relatively impermeable to sodium. The frequency of channel opening increased with depolarization and intracellular calcium concentration. At 10(-7) M (Ca++)i, channel activity was evident only at potentials of +40 mV or more depolarized, while at 10(-5) M, channels were open at all voltages tested (-40 to +60 mV). In intact patches, channels were seen at depolarized patch potentials of +50 mV or greater, indicating that the ionized calcium concentration in the macrophage is probably less than 10(-7) M.     

Voltage dependent calcium channels in cerebellar granule cell primary cultures

Voltage activated calcium channels were studied in rat cerebellar granule cells in primary culture. Macroscopic currents, carried by 20mM Ba2+, were measured in the whole-cell configuration. Slowly inactivating macroscopic currents, with a maximum value at a membrane potential around 5 mV, were recorded between the 1st and the 4th day in culture. These currents were completely blocked by 5mM Co2+, partially blocked by 10 microM nifedipine, and increased by 2 to 5 microM BAY K-8644. Two types of channels, in the presence of 80 mM Ba2+, were identified by single channel recording in cell-attached patches. The first type, which was dihydropyridine agonist sensitive, had a conductance of 18 pS, a half activation potential of more than 10 mV and did not inactivate. This type of channel was the only type found during the first four days in culture, although it was also present up to the 11th day. The second type of channel was dihydropyridine insensitive, had a conductance of 10 pS, a half activation potential less than -15 mV, and displayed voltage dependent inactivation. This second type of channel was found in cells for more than four days in culture.     

A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus

Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

K channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

By averaging the current that passes through cell-attached patches on beating heart cells, while measuring action potentials with a whole-cell electrode, we were able to study K channels during beating. In 7-d chick ventricle in 1.3 mM K physiological solutions at room temperature, delayed-rectifier channels have three linear conductance states: 60, 30, and 15 pS. The 60 and 15 pS conductances can exist alone, but all three states may appear in the same patch as interconverting conductance levels. The delayed-rectifier conductance states have low densities (less than 10 channels per 10-microns diam cell), and all have a reversal potential near -75 mV and the same average kinetics. Outward K current through delayed-rectifier channels follows the upstroke without appreciable delay and lasts throughout the action potential. No inward current flows through delayed-rectifier channels during beating. The early outward channel has a nonlinear conductance of 18-9 pS depending on the potential. It also turns on immediately after the upstroke of the action potential and lasts on average only 50 ms. The early outward channel has an extrapolated reversal potential near -30 mV; no inward current flows during beating. The inward-rectifier has an extrapolated conductance and reversal potential of 2-3 pS and -80 mV in 1.3 mM K. Channel kinetics are independent of external K between 10 and 120 mM, and the channel conducts current only during the late repolarization and diastolic phases of the action potential. No outward current flows through inward-rectifier channels during beating. This work parallels a previous study of Na channels using similar techniques (Mazzanti, M., and L. J. DeFelice. 1987, Biophys. J. 52:95-100).     

Characterization of ion channels on the surface membrane of adult rat skeletal muscle.

The channels present on the surface membrane of isolated rat flexor digitorum brevis muscle fibers were surveyed using the patch clamp technique. 85 out of 139 fibers had a novel channel which excluded the anions chloride, sulfate, and isethionate with a permeability ratio of chloride to sodium of less than 0.05. The selectivity sequence for cations was Na+ = K+ = Cs+ greater than Ca++ = Mg++ greater than N-Methyl-D-Glucamine. The channel remained closed for long periods, and had a large conductance of approximately 320 pS with several subconductance states at approximately 34 pS levels. Channel activity was not voltage dependent and the reversal potential for cations in muscle fibers of approximately 0 mV results in the channel's behaving as a physiological leakage conductance. Voltage activated potassium channels were present in 65 of the cell attached patches and had conductances of mostly 6, 12, and 25 pS. The voltage sensitivity of the potassium channels was consistent with that of the delayed rectifier current. Only three patches contained chloride channels. The scarcity of chloride channels despite the known high chloride conductance of skeletal muscle suggests that most of the chloride channels must be located in the transverse tubular system.     

The frog interosseal muscle fiber as a new model for patch clamp studies of chemosensitive- and voltage-sensitive ion channels: actions of acetylcholine and batrachotoxin

The patch clamp technique was used to record the currents flowing through single ion channels in isolated frog muscle fibers. The majority of the acetylcholine (ACh)-activated channels had a conductance of 32 pS, although 20 pS channels were also occasionally observed. Lifetimes of ACh-activated channels increased with the transmembrane potential in the range from - 30 mV to - 105 mV. In these same fibers we also observed channels which were activated by low concentrations of batrachotoxin (BTX; 10 nM). These channels, presumed to be Na channels, had a conductance of 19 pS and opened at potentials at which Na channels would not normally open. A notable feature of these BTX-activated channels was that they opened and closed repeatedly. Therefore, it appears that the toxin, in addition to activating Na channels, also blocks the inactivation process. The physiological properties of these channels reveal significant differences between the ion channels of tissue-cultured and mature tissues.     

A calcium and ATP sensitive nonselective cation channel in the antiluminal membrane of rat cerebral capillary endothelial cells.

The patch-clamp technique was applied to the antiluminal membrane of freshly isolated capillaries of rat brain (blood-brain barrier). With 1.3 mM Ca2+ in the bath, excision of membrane patches evoked ion channels, which could not be observed in cell-attached mode. The channel was about equally permeable to Na+ and K+ ions, but not measurable permeable to Cl- and the divalent ions Ca2+ and Ba2+. The current-voltage curve was linear in the investigated voltage range (-80 mV to +80 mV), and the single-channel conductance was 31 +/- 2 pS (n = 22). The channel open probability was not dependent on the applied potential. Lowering of Ca2+ to 1 microM or below on the cytosolic side inactivated the channels, whereas addition of cytosolic ATP (1 mM) inhibited channel activity completely and reversibly. The channel was blocked by the inhibitor of nonselective cation channels in rat exocrine pancreas 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10 microM) and by the antiinflammatory drugs flufenamic acid (greater than 10 microM) and tenidap (100 microM), as well as by gadolinium (10 microM). Thus, these nonselective cation channels have many properties in common with similar channels observed in fluid secreting epithelia. The channel could be involved in the transport of K+ ions from brain to blood side.     

Multiple types of voltage-dependent Ca2+-activated K+ channels of large conductance in rat brain synaptosomal membranes

K+-selective ion channels from a mammalian brain synaptosomal membrane preparation were inserted into planar phospholipid bilayers on the tips of patch-clamp pipettes, and single-channel currents were measured. Multiple distinct classes of K+ channels were observed. We have characterized and described the properties of several types of voltage-dependent, Ca2+-activated K+ channels of large single-channel conductance (greater than 50 pS in symmetrical KCl solutions). One class of channels (Type I) has a 200-250-pS single-channel conductance. It is activated by internal calcium concentrations greater than 10(-7) M, and its probability of opening is increased by membrane depolarization. This channel is blocked by 1-3 mM internal concentrations of tetraethylammonium (TEA). These channels are similar to the BK channel described in a variety of tissues. A second novel group of voltage-dependent, Ca2+-activated K+ channels was also studied. These channels were more sensitive to internal calcium, but less sensitive to voltage than the large (Type I) channel. These channels were minimally affected by internal TEA concentrations of 10 mM, but were blocked by a 50 mM concentration. In this class of channels we found a wide range of relatively large unitary channel conductances (65-140 pS). Within this group we have characterized two types (75-80 pS and 120-125 pS) that also differ in gating kinetics. The various types of voltage-dependent, Ca2+-activated K+ channels described here were blocked by charybdotoxin added to the external side of the channel. The activity of these channels was increased by exposure to nanomolar concentrations of the catalytic subunit of cAMP-dependent protein kinase. These results indicate that voltage-dependent, charybdotoxin-sensitive Ca2+-activated K+ channels comprise a class of related, but distinguishable channel types. Although the Ca2+-activated (Type I and II) K+ channels can be distinguished by their single-channel properties, both could contribute to the voltage-dependent Ca2+-activated macroscopic K+ current (IC) that has been observed in several neuronal somata preparations, as well as in other cells. Some of the properties reported here may serve to distinguish which type contributes in each case. A third class of smaller (40-50 pS) channels was also studied. These channels were independent of calcium over the concentration range examined (10(-7)-10(-3) M), and were also independent of voltage over the range of pipette potentials of -60 to +60 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nonselective cation channels in intact human T lymphocytes.

Nonselective cation channels were found in single channel recordings from cell-attached patches on human T lymphocytes. These channels were active under conditions that should lead to cell swelling (hypotonic bath solutions with NaCl or KCl); however, a definite dependence of activity on cell swelling has not been proven. Under these conditions similar channels were found in 20 of 23 patches from 11 different blood donors. The current-voltage relation was approximately linear for outward current (11-14 pS) and inwardly rectifying (to 23 pS) when the intact cells were depolarized with high KCl in the bath. The voltage dependence of channel activity is consistent with closing at hyperpolarized membrane potentials (Vm less than or equal to -50 mV) and block of open channels at strongly depolarized membrane potentials (Vm greater than 0 mV). Reversal potentials under all ionic gradients tested are consistent with a channel that is poorly selective between Na+ and K+ ions. Active channels in cell-attached patches were rapidly blocked by bath addition of the membrane-permeant inhibitor quinine. Channels that were active in cell-attached became quiescent after patch excision; however, two patches remained active long enough to obtain current-voltage relations. These were linear with a slope conductance for outward current of 8-11 pS. Because of the clustering of single-channel openings, detailed voltage dependence of kinetics and probability of opening were not studied.     

Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells

Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.     

Development of ionic channels during mouse neuronal differentiation

Using a mouse embryonal teratocarcinoma (E.C.) cell line, it was possible to follow the sequence of development of ionic channels during neuronal differentiation, with patch-clamp techniques. 1003 E.C. cells were induced to differentiate into neurons by culturing them in defined medium without foetal calf serum (DARMON et al., 1981). Non-differentiated cells were not excitable and presented mainly 2 types of K+ channels: a Ca2+ activated K+ channel (220 pS in symmetrical K+) and a delayed rectifier (30 pS in symmetrical K+). When the cells start to grow neurites, a low threshold calcium current can be recorded, only if the cell is held at hyperpolarized potentials (-70 to -80 mV). Fully differentiated cells with long neurites presented a complete repertoire of ionic channels: voltage dependent Na+ and Ca2+ channels, Ca2+ activated K+ channel and K+ delayed rectifier.     

Single chloride channels in cultured rat neurones

Single-channel currents through chloride channels were recorded in cultured hippocampal neurones from rats using the patch-clamp method. The channel is active at voltages between -80 and +80 mV, and the time spent in the open state increases with depolarization (almost fourfold for 120 mV). The channel conductance is 62 pS in symmetrical 150 mM NaCl saline. In salt gradient conditions the channel was measurably permeable to Na+. Substitution of NO3- and Br- for Cl- gave higher single-channel currents, meaning a higher permeability of the channel toward the two anions than to Cl-. SO4(2-) ions were poorer charge carriers, yet contributed measurable inward current at negative voltages. Channel activity appeared independent of intracellular Ca2+ concentration. Taken together, these features would suggest for this channel a role in stabilizing resting membrane potential and in maintaining normal cell excitability.     

Cadmium block of calcium current in frog sympathetic neurons.

Cd2+ blocks whole-cell calcium currents in frog sympathetic neurons by 50% at approximately 300 nM. Strong depolarizations rapidly reverse that blockade (tau = 1.3 ms at +120 mV). Reblocking follows bimolecular kinetics (rate = 1.2 x 10(8) M-1 s-1) at voltages where channels are mostly open (0 to +30 mV). The unblocking rate is approximately 50 s-1, so the dissociation constant calculated from the rate constants is approximately 400 nM. Steady-state block is strong at -80 mV, so closed channels can also be blocked. However, reblocking is extremely slow (tau = 1-2 s) at voltages where the channels are mostly closed. The rates for Cd2+ entry and exit are greater than 100-fold lower for closed channels than for open channels, and closed channels appear to be closed at both ends.