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.     

Properties and modulation of alpha human atrial natriuretic peptide (alpha-hANP)-formed ion channels

Using the lipid bilayer technique we have optimized recording conditions and confirmed that alpha human atrial natriuretic peptide [alpha-hANP(1-28)] forms single ion channels. The single channel currents recorded in 250/50 mM KCl cis/trans chambers show that the ANP-formed channels were heterogeneous, and differed in their conductance, kinetic, and pharmacological properties. The ANP-formed single channels were grouped as: (i) H202- and Ba2+-sensitive channel with fast kinetics; the nonlinear current-voltage (I-V) relationship of this channel had a reversal potential (Erev) of -28.2 mV, which is close to the equilibrium potential for K+ (EK = -35 mV) and a maximal slope conductance (gmax) of 68 pS at positive potentials. Sequential ionic substitution (KCl, K gluconate and choline Cl) of the cis solution suggests that the current was carried by cations. The fast channel had three modes (spike mode, burst mode, and open mode) that differed in their kinetics but not in their conductance properties. (ii) A large conductance channel possessing several subconductance levels that showed time-dependent inactivation at positive and negative membrane potentials (Vm). The inactivation ratio of the current at the end of the voltage step (Iss) to the initial current (Ii) activated immediately after the voltage step, (Iss/Ii), was voltage dependent and described by a bell-shaped curve. The maximal current-voltage (I-V) relationship of this channel, which had an Erev of +17.2 mV, was nonlinear and the value of gmax was 273 pS at negative voltages. (iii) A transiently-activated channel: the nonlinear I-V relationship of this channel had an Erev of -29.8 mV and the value of gmax was 160 pS at positive voltages. We propose that the voltage-dependence of the ionic currents and the kinetic parameters of these channel types indicate that if they were formed in vivo and activated by cytosolic factors they could change the membrane potential and the electrolyte homeostasis of the cell.     

Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic beta-cells

The contribution of Ca2(+)-activated and delayed rectifying K+ channels to the voltage-dependent outward current involved in spike repolarization in mouse pancreatic beta-cells (Rorsman, P., and G. Trube. 1986. J. Physiol. 374:531-550) was assessed using patch-clamp techniques. A Ca2(+)-dependent component could be identified by its rapid inactivation and sensitivity to the Ca2+ channel blocker Cd2+. This current showed the same voltage dependence as the voltage-activated (Cd2(+)-sensitive) Ca2+ current and contributed 10-20% to the total beta-cell delayed outward current. The single-channel events underlying the Ca2(+)-activated component were investigated in cell-attached patches. Increase of [Ca2+]i invariably induced a dramatic increase in the open state probability of a Ca2(+)-activated K+ channel. This channel had a single-channel conductance of 70 pS [( K+]o = 5.6 mM). The Ca2(+)-independent outward current (constituting greater than 80% of the total) reflected the activation of an 8 pS [( K+]o = 5.6 mM; [K+]i = 155 mM) K+ channel. This channel was the only type observed to be associated with action potentials in cell-attached patches. It is suggested that in mouse beta-cells spike repolarization results mainly from the opening of the 8-pS delayed rectifying K+ channel.     

Regulation of ROMK by extracellular cations

The effect of external potassium (K) and cesium (Cs) on the inwardly rectifying K channel ROMK2 (K(ir)1.1b) was studied in Xenopus oocytes. Elevating external K from 1 to 10 mM increased whole-cell outward conductance by a factor of 3.4 +/- 0.4 in 15 min and by a factor of 5.7 +/- 0.9 in 30 min (n = 22). Replacing external Na by Cs blocked inward conductance but increased whole-cell conductance by a factor of 4.5 +/- 0.5 over a period of 40 min (n = 15). In addition to this slow increase in conductance, there was also a small, rapid increase in conductance that occurred as soon as ROMK was exposed to external cesium or 10 mM K. This rapid increase could be explained by the observed increase in ROMK single-channel conductance from 6.4 +/- 0.8 pS to 11.1 +/- 0.8 pS (10 mM K, n = 8) or 11.7 +/- 1.2 pS (Cs, n = 8). There was no effect of either 10 mM K or cesium on the high open probability (P(o) = 0.97 +/- 0.01; n = 12) of ROMK outward currents. In patch-clamp recordings, the number of active channels increased when the K concentration at the outside surface was raised from 1 to 50 mM K. In cell-attached patches, exposure to 50 mM external K produced one or more additional channels in 9/16 patches. No change in channel number was observed in patches continuously exposed to 50 mM external K. Hence, the slow increase in whole-cell conductance is interpreted as activation of pre-existing ROMK channels that had been inactivated by low external K. This type of time-dependent channel activation was not seen with IRK1 (K(ir)2.1) or in ROMK2 mutants in which any one of 6 residues, F129, Q133, E132, V121, L117, or K61, were replaced by their respective IRK1 homologs. These results are consistent with a model in which ROMK can exist in either an activated mode or an inactivated mode. Within the activated mode, individual channels undergo rapid transitions between open and closed states. High (10 mM) external K or Cs stabilizes the activated mode, and low external K stabilizes the inactivated mode. Mutation of a pH-sensing site (ROMK2-K61) prevents transitions from activated to inactivated modes. This is consistent with a direct effect of external K or Cs on the gating of ROMK by internal pH.     

The synaptic vesicle protein synaptophysin: purification and characterization of its channel activity

The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a relatively low concentration of Triton X-100 (0.2%) and was highly purified (above 95%) using a rapid single chromatography step on hydroxyapatite/celite resin. Purified synaptophysin was reconstituted into a planar lipid bilayer and the channel activity of synaptophysin was characterized. In asymmetric KCl solutions (cis 300 mM/trans 100 mM), synaptophysin formed a fast-fluctuating channel with a conductance of 414 +/- 13 pS at +60 mV. The open probability of synaptophysin channels was decreased upon depolarization, and channels were found to be cation-selective. Synaptophysin channels showed higher selectivity for K(+) over Cl(-) (P(K(+))/P(Cl(-)) > 8) and preferred K(+) over Li(+), Na(+), Rb(+), Cs(+), or choline(+). The synaptophysin channel is impermeable to Ca(2+), which has no effect on its channel activity. This study is the second demonstration of purified synaptophysin channel activity, but the first biophysical characterization of its channel properties. The availability of large amounts of purified synaptophysin and of its characteristic channel properties might help to establish the role of synaptophysin in synaptic transmission.     

Mechanisms of Cs+ blockade in a Ca2+-activated K+ channel from smooth muscle.

Large unitary conductance Ca2+-activated K+ channels from smooth muscle membrane were incorporated into phospholipid planar bilayers, and the blockade induced by internally and externally applied Cs+ was characterized. Internal Cs+ blockade is voltage dependent and can be explained on the basis of a Cs+ binding to a site that senses 54% of the applied voltage, with an apparent dissociation constant, Kd(0), of 70 mM. On the other hand, external Cs+ blocks the channel in micromolar amounts, and the voltage dependence of blockade is a function of Cs+ concentration. The fractional electrical distance can be as large as 1.4 at 10 mM Cs+. This last result suggests that the channel behaves as a multi-ion pore. At large negative voltages the I-V relationships in the presence of external Cs+ show an upturn, indicating relief of Cs+ block. External Cs+ blockade is relieved by increasing the internal K+ concentration, but can be enhanced by increasing the external K+. All the characteristics of external Cs+ block can be explained by a model that incorporates a "knock-on" of Cs+ by K+.     

Molecular properties of Kcv, a virus encoded K+ channel

The miniature viral K+ channel Kcv represents the pore module of all K+ channels. A synthetic gene of Kcv with an elevated GC content compared to that of the wild-type gene was expressed heterologously in Pichia pastoris, and the purified protein was functionally reconstituted into liposomes. Biochemical assays reveal a remarkable cation selective stability of the channel tetramer via SDS-PAGE. Only cations, which permeate Kcv, were able to protect the oligomer against disassembly into monomers at high temperatures. Electrophysiological characterization of the single Kcv channel reveals a saturating conductance (lambda(max)) of 360 pS; the single-channel current-voltage relation was strongly rectifying with a negative slope conductance at extreme voltages. The channel was highly selective for K+ and was blocked by Ba2+ and in a side specific manner by Na+ and Cs+ also. The channel conducted Rb+, but as a consequence, the channel was shifted into a hyperactive state. We conclude that specific binding interactions of cations in the conductive pathway are an important determinant of channel stability and function.     

The K+ channel of sarcoplasmic reticulum. A new look at Cs+ block.

K+-selective ion channels from mammalian sarcoplasmic reticulum were inserted into planar phospholipid bilayers, and single-channel currents measured in solutions containing Cs+. Current through this channel can be observed in symmetrical solutions containing only Cs+ salts. At zero voltage, the Cs+ conductance is approximately 15-fold lower than the corresponding K+ conductance. The open channel rectifies strongly in symmetrical Cs+ solutions, and the Cs+ currents are independent of Cs+ concentration in the range 18-600 mM. Biionic (Cs+/K+) reversal potentials are only 10 mV, showing that Cs+ is nearly as permeant as K+, though much less conductive. Addition of Cs+ to symmetrical K+ solutions reduces current through the channel in a voltage-dependent way. The results can be explained by a free energy profile in which the channel's selectivity filter acts in two ways: to provide binding sites for the conducting ions and to serve as a major rate-determining structure. According to this picture, the main difference between high-conductance K+ and low-conductance Cs+ is that Cs+ binds to an asymmetrically positioned site approximately 20-fold more tightly than does K+.     

Evidence for KCNQ1 K+ channel expression in rat zymogen granule membranes and involvement in cholecystokinin-induced pancreatic acinar secretion

Secretion of enzymes and fluid induced by Ca(2+) in pancreatic acini is not completely understood and may involve activation of ion conductive pathways in zymogen granule (ZG) membranes. We hypothesized that a chromanol 293B-sensitive K(+) conductance carried by a KCNQ1 protein is expressed in ZG membranes (ZGM). In suspensions of rat pancreatic ZG, ion flux was determined by ionophore-induced osmotic lysis of ZG suspended in isotonic salts. The KCNQ1 blocker 293B selectively blocked K(+) permeability (IC(50) of approximately 10 microM). After incorporation of ZGM into planar bilayer membranes, cation channels were detected in 645/150 mM potassium gluconate cis/trans solutions. Channels had linear current-voltage relationships, a reversal potential (E(rev)) of -20.9 +/- 0.9 mV, and a single-channel K(+) conductance (g(K)) of 265.8 +/- 44.0 pS (n = 39). Replacement of cis 500 mM K(+) by 500 mM Na(+) shifted E(rev) to -2.4 +/- 3.6 mV (n = 3), indicating K(+) selectivity. Single-channel analysis identified several K(+) channel groups with distinct channel behaviors. K(+) channels with a g(K) of 651.8 +/- 88.0 pS, E(rev) of -22.9 +/- 2.2 mV, and open probability (P(open)) of 0.43 +/- 0.06 at 0 mV (n = 6) and channels with a g(K) of 155.0 +/- 11.4 pS, E(rev) of -18.3 +/- 1.8 mV, and P(open) of 0.80 +/- 0.03 at 0 mV (n = 3) were inhibited by 100 microM 293B or by the more selective inhibitor HMR-1556 but not by the maxi-Ca(2+)-activated K(+) channel (BK channel) inhibitor charybdotoxin (5 nM). KCNQ1 protein was demonstrated by immunoperoxidase labeling of pancreatic tissue, immunogold labeling of ZG, and immunoblotting of ZGM. 293B also inhibited cholecystokinin-induced amylase secretion of permeabilized acini (IC(50) of approximately 10 microM). Thus KCNQ1 may account for ZG K(+) conductance and contribute to pancreatic hormone-stimulated enzyme and fluid secretion.     

Ion channels in the thick ascending limb of Henle's loop.

The thick ascending limb of Henle's loop (TAL) is polarized with respect to its conductances. The luminal membrane contains a K+ conductance which is made up by the synchronous operation of 60- to 80-pS K+ channels. The basolateral membrane contains a chloride conductance. This conductance corresponds most likely to a 30- to 60-pS Cl- channel present in this membrane. Our knowledge on the properties of the K+ channels of these cells has been increased rapidly by patch clamp studies: these K+ channels are inwardly rectifying. They are highly selective for K+ over Na+, Li+ and many other cations. They do not conduct Rb+, Cs+, NH+4 or other larger cations. In fact, all these three cations as well as choline, tetraethylammonium, lidocaine, verapamil, diltiazem, quinine, quinidine and Ba2+ inhibit these K+ channels. As apparent from kinetic studies the mechanisms of inhibition are different for the various blockers. The TAL K+ channels are downregulated by increasing cytosolic Ca2+ activity. Cytosolic adenosine trisphosphate (ATP) has a similar effect. This ATP inhibition is Ca2+ dependent. The affinity to ATP is augmented by increasing Ca2+. Cytosolic alkalinity increases the open probability of these channels, and cytosolic acidification has the opposite effect. This pH dependence is very marked. A change by 0.2 pH units leads to a more than twofold change in the open-channel probability. The basolateral chloride conductance reflects the properties of an outwardly rectifying 30- to 60-pS Cl- channel. This channel behaves, in many respects, like the Cl- channels of a multitude of Cl- transporting epithelia. It is characterized by two open and two closed states. It is highly selective for Cl- as compared with larger anions, and it is inhibited reversibly by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate.     

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.     

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).     

Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells.

Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch-clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage-dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two-electrode voltage-clamp and patch-clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage-dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range -160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)-selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage-dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells.     

Patch clamp recordings from membranes which contain gap junction channels.

The septal membranes of the median and lateral giant axons of earthworm, which contain gap junctions, were exposed by cutting one segment of the cord. Patch recordings were obtained from the exposed cytoplasmic side of the septum. Seal resistances ranged from 2 to 15 G omega. The patch could be excised (detached) or left attached to the whole cell. Two types of channels were observed. One type was blocked by tetraethylammonium (TEA) or Cs+ and had a unitary conductance of 30-40 pS. It appears to be a K+ channel. The other channel type had a unitary conductance of 90-110 pS and was unaffected by TEA+ or Cs+. In the detached configuration the channel was shown to conduct Cs+, K+, Na+, TMA+, Cl- and TEA+ even in the presence of 2 mM Zn2+, 1 mM Ni2+, 1 mM Co2+, and 4 mM 4-aminopyridine. The conductance ratios relative to K+ were 1.0 for Cs+, 0.84 for Na+, 0.64 for TMA+, 0.52 for Cl- and 0.2 for TEA+. The channel appears to be voltage insensitive whether monitored in detached or attached recording mode. Both H+ and Ca2+ reduce the probability of opening. Thus, the 100 pS channel has many of the properties expected of a gap junction channel.     

Further characterization of the cation channel of a yeast vacuolar membrane in a planar lipid bilayer

A voltage-dependent and Ca2(+)-activated cation channel recently found in the vacuolar membrane of the yeast Saccharomyces cerevisiae was incorporated into planar lipid bilayers and further characterized in macroscopic and single channel levels. Single channel conductances for various cations were in the order: NH4+ greater than K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+, and were nearly consistent with the order of permeability ratio estimated from reversal potentials determined by macroscopic measurement. Up to 6 mM of Ca2+ added to the cis (cytoplasmic) side opened the channel, but higher concentrations closed the channel without affecting the single channel conductance. Ba2+ closed the channel without affecting the single channel conductance. Ba2+ closed the channel from the cis side. In addition to the above channel, a small cation-selective channel of about 40 pS was found.     

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.     

A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid

Mechanosensitive ion channels have been described in many types of cells. These channels are believed to transduce pressure signals into intracellular biochemical and physiological events. In this study, the patch-clamp technique was used to identify and characterize a mechanosensitive ion channel in rat atrial cells. In cell-attached patches, negative pressure in the pipette activated an ion channel in a pressure-dependent manner. The pressure to induce half-maximal activation was 12 +/- 3 mmHg at +40 mV, and nearly full activation was observed at approximately 20 mmHg. The probability of opening was voltage dependent, with greater channel activity at depolarized potentials. The mechanosensitive channel was identical to the K+ channel previously shown to be activated by arachidonic acid and other lipophilic compounds, as judged by the outwardly rectifying current-voltage relation, single channel amplitude, mean open time (1.4 +/- 0.3 ms), bursty openings, K+ selectivity, insensitivity to any known organic inhibitors of ion channels, and pH sensitivity. In symmetrical 140 mM KCl, the slope conductance was 94 +/- 11 pS at +60 mV and 64 +/- 8 pS at -60 mV. Anions and cations such as Cl-, glutamate, Na+, Cs+, Li+, Ca2+, and Ba2+ were not permeant. Extracellular Ba2+ (1 mM) blocked the inward K+ current completely. GdCl3 (100 microM) or CaCl2 (100 microM) did not alter the K+ channel activity or amplitude. Lowering of intracellular pH increased the pressure sensitivity of the channel. The K+ channel could be activated in the presence of 5 mM intracellular [ATP] or 10 microM glybenclamide in inside-out patches. In the absence of ATP, when the ATP-sensitive K+ channel was active, the mechanosensitive channel could further be activated by pressure, suggesting that they were two separate channels. The ATP-sensitive K+ channel was not mechanosensitive. Pressure activated the K+ channel in the presence of albumin, a fatty acid binding protein, suggesting that pressure and arachidonic acid activate the K+ channel via separate pathways.     

Gadolinium-sensitive, voltage-dependent calcium release channels in the endoplasmic reticulum of a higher plant mechanoreceptor organ.

The lipid bilayer technique was adapted to the functional reconstitution of ion channels from the endoplasmic reticulum of a higher plant. This was obtained at high purity from touch-sensitive tendrils of Bryonia dioica. In this preparation, a calcium-selective strongly rectifying channel is prevailing whose single-channel properties have been characterized. The single-channel conductance is 29 pS in 50 mM CaCl2. The Ca2+: K+ selectivity was determined to be approximately 6.6. The channel is voltage-gated and, more importantly, the gating voltage is strongly shifted towards more negative voltages when a transmembrane Ca2+ gradient is applied. Thus, at physiological voltages across the endoplasmic reticulum membrane, the channel's open probability will be governed largely by the chemical potential gradient of Ca2+, generated by the Ca(2+)-ATPase in that same membrane. The calcium release channel described here is effectively blocked by Gd3+ which also completely suppresses a tendril's reaction to touch, suggesting that this channel could be a key element of calcium signaling in higher plant mechanotransduction. Its molecular characteristics and inhibitor data show it to be the first known member of a hitherto unrecognized class of calcium channels.     

Ca2+ channels from the sea urchin sperm plasma membrane

Ca2+ influx across the sea urchin sperm plasma membrane is a necessary step during the egg jelly-induced acrosome reaction. There is pharmacological evidence for the involvement of Ca2+ channels in this influx, but their presence has not been directly demonstrated because of the small size of this cell. Sea urchin sperm Ca2+ channels are being studied by fusing isolated plasma membranes into planar lipid bilayers. With this strategy, a Ca2+ channel has been detected with the following characteristics: (a) the channel exhibits a high mainstate conductance (gamma MS) of 172 pS in 50 mM CaCl2 solutions with voltage-dependent decaying to smaller conductance states at negative Em; (b) the channel is blocked by millimolar concentrations of Cd2+, Co2+, and La3+, which also inhibit the egg jelly-induced acrosome reaction; (c) the gamma MS conductance sequence for the tested divalent cations is the following: Ba2+ greater than Sr2+ greater than Ca2+; and (d) the channel discriminates poorly for divalent over monovalent cations (PCa/PNa = 5.9). The sperm Ca2+ channel gamma MS rectifies in symmetrical 10 mM CaCl2, having a maximal slope conductance value of 94 pS at +100 mV applied to the cis side of the bilayer. Under these conditions, a different single-channel activity of lesser conductance became apparent above the gamma MS current at positive membrane potentials. Also in 10 mM Ca2+ solutions, Mg2+ permeates through the main channel when added to the cis side with a PCa/PMg = 2.9, while it blocks when added to the trans side. In 50 mM Ca2+ solutions, the gamma MS open probability has values of 1.0 at voltages more positive than -40 mV and decreases at more negatives potentials, following a Boltzmann function with an E0.5 = -72 mV and an apparent gating charge value of 3.9. These results describe a novel Ca2(+)-selective channel, and suggest that the main channel works as a single multipore assembly.