Cytoplasmic Determinants of Piperidine Blocking Affinity for N-type Calcium Channels

Piperidines are a relatively novel class of calcium channel blockers which act at a unique receptor site associated with the calcium channel α₁ subunit. Calcium channel blocking affinities ranging from subnanomolar to several hundred micromolar have been reported in the literature, suggesting that piperidine block is highly sensitive to the cellular environment experienced by the channel. Here, I have investigated some of the cytoplasmic determinants of haloperidol block of N-type calcium channels expressed in human embryonic kidney cells. In perforated patch clamp recordings, haloperidol blocks N-type calcium channels with an inhibition constant of 120 μM. Upon internal dialysis with chloride containing pipette solution, the blocking affinity increases by 40-fold. This effect could be attributed in part to the presence of internal chloride ions, as replacement of intracellular chloride with methanesulfonate reduced haloperidol blocking affinity by almost one order of magnitude. Tonic inhibition of N-type channels by G_βγ subunits further enhanced the blocking effects of haloperidol, suggesting the possibility of direct effects of G_βγ binding on the local environment of the piperidine receptor site. Overall, depending on the cytoplasmic environment experienced by the channel, the blocking affinity of N-type calcium channels for haloperidol may vary by more than two orders of magnitude. Thus, absolute blocking affinities at the piperidine receptor site must be interpreted cautiously and in the context of the particular experimental setting. Received: 23 July 1998/Revised: 19 October 1998     

Activators of Epithelial Na+ Channels Inhibit Cytosolic Feedback Control. Evidence for the Existence of a G Protein-Coupled Receptor for Cytosolic Na+

We have previously shown that epithelial Na⁺ channels in mouse mandibular gland duct cells are controlled by cytosolic Na⁺ and Cl⁻, acting, respectively, via G _o and G _i proteins. Since we found no evidence for control of epithelial Na⁺ channels by extracellular Na⁺ ([Na⁺] _o ), our findings conflicted with the long-held belief that Na⁺ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues, like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel site which otherwise inhibits channel activity in response to increasing [Na⁺] _o . Instead, we now show that PCMPS acts by rendering epithelial Na⁺ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na⁺ and Cl⁻ on Na⁺ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane, block feedback inhibition of Na⁺ channels by cytosolic Na⁺, while leaving inhibition by cytosolic Cl⁻ unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated α-subunit of G _o in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na⁺ receptor. Received: 1 October 1997/Revised: 24 December 1997     

Characterization of Lonidamine and AF2785 Blockade of the Cyclic AMP-Activated Chloride Current in Rat Epididymal Cells

It has been shown previously that the antifertility agents Lonidamine and its analogue AF2785, [1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid] are potent inhibitors of the cAMP-activated chloride channel (CFTR) in rat epididymal cells. In this study, we further characterized the blocking actions of these two compounds and compared them with the known chloride channel blocker diphenylamine-2-carboxylate (DPC). Results show that the order of potency in blocking the cAMP-activated current is AF2785 > Lonidamine > DPC. All three compounds shared similar blocking characteristics. Firstly, their blockade of the current exhibited voltage dependence; all three agents blocked the current more markedly at negative than at positive membrane potentials. Secondly, they blocked the channels from the outside of the cell. Thirdly, their blocking efficacies were maximal at low extracellular pH. Lastly, the time course of the block by AF2785 and DPC appeared to be more rapid than that of Lonidamine. It is hoped that further studies with other indazole compounds will add knowledge to the physiology and pharmacology of CFTR in the epididymis. Such information will be of great importance to our quest for novel male contraceptives. Received: 8 February 2000/Revised: 25 September 2000     

Competition for Binding Between Veratridine and KIFMK: An Open Channel Blocking Peptide of the RIIA Sodium Channel

Veratridine, an alkaloid isolated from the rhizome of V. album, binds and slows the inactivation of the brain sodium channels. The synthetic pentapeptide KIFMK causes a voltage- and use-dependent open-channel block of the RIIA (rat brain type IIA) sodium channel (Eaholtz, Scheuer & Catterall, 1994). Our studies on the RIIA sodium channel expressed in CHO cells reveal that the fraction of veratridine modified sodium channels decreases linearly with increasing KIFMK concentration. However, the time constant for dissociation of veratridine from the channel remains unchanged in the presence of a high concentration of KIFMK, as opposed to that in the presence of QX314 where the dissociation appears to be more complex. These data are consistent with mutually exclusive binding of the open channel blocking peptide and veratridine to the brain sodium channel. Received: 19 November 1996/Revised: 31 July 1997     

Increased Resistance to Extracellular Cation Block by Mutation of the Pore Domain of the Arabidopsis Inward-rectifying K+ Channel KAT1

Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K⁺ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K⁺ channel blockers are potent tools for studying the physiological functions and structural properties of K⁺ channels. In the present study the structural and biophysical mechanisms of Cs⁺ and TEA⁺ block of a cloned Arabidopsis inward-rectifying K⁺ channel (KAT1) were analyzed. Effects of the channel blockers Cs⁺ and TEA⁺ were characterized both extracellularly and intracellularly. Both external Cs⁺ and TEA⁺ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs⁺ and TEA⁺ than the wild-type K⁺ channel. Internal TEA⁺ and Cs⁺ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K⁺ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K⁺ channels. Received: 31 July 1995/Revised: 5 January 1996     

Ethanol Activates Maxi Ca2+-activated K+ Channels of Clonal Pituitary (GH3) Cells

The effect of ethanol on maxi Ca²⁺-activated K⁺ channels (BK channels) in GH3 pituitary tumor cells was investigated using single-channel recordings and focusing on intracellular signal transduction. In outside-out patches, ethanol caused a transient concentration-dependent increase of BK-channel activity. 30 mm (1.4‰) ethanol significantly increased mean channel open time and channel open probability by 26.3 ± 9% and 78.8 ± 10%, respectively; single-channel current amplitude was not affected by ethanol. The augmenting effect of ethanol was blocked in the presence of protein kinase C (PKC) inhibitors staurosporine, bisindolylmaleimide, and PKC (19–31) pseudosubstrate inhibitor as well as by AMP-PNP (5′-adenylylimidodiphosphate), a nonhydrolyzable ATP-analogue, but not by the phospholipase C blocker U-73122. Phosphatase inhibitors microcystin-LR and okadaic acid promoted the ethanol effect. The blocking effect was released at higher concentrations of ethanol (100 mm) suggesting a second site of action or a competition between blockers and ethanol. Our results suggest that the effect of ethanol on BK-channels is mediated by PKC stimulation and phosphorylation of the channels which increases channel activity and hence may influence action potentials duration and hormone secretion. Received: 24 July 1996/Revised: 27 December 1996     

Evidence for Negative Charge in the Conduction Pathway of the Cardiac Ryanodine Receptor Channel Provided by the Interaction of K+ Channel N-type Inactivation Peptides

We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH₂-terminal domain of the Shaker K⁺ channel with purified, ryanodine-modified, cardiac Ca²⁺-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec⁻¹μm ⁻¹ for ShB and 2000 ± 200 sec⁻¹μm ⁻¹ for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K_off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K⁺ channels, this is mediated by a large increase in K_on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel. Received: 15 December 1997/Revised: 13 March 1998     

Nonselective Block by La3+ of Arabidopsis Ion Channels Involved in Signal Transduction

Lanthanide ions such as La³⁺ are frequently used as blockers to test the involvement of calcium channels in plant and animal signal transduction pathways. For example, the large rise in cytoplasmic Ca²⁺ concentration triggered by cold shock in Arabidopsis seedlings is effectively blocked by 10 mm La³⁺ and we show here that the simultaneous large membrane depolarization is similarly blocked. However, a pharmacological tool is only as useful as it is selective and the specificity of La³⁺ for calcium channels was brought into question by our finding that it also blocked a blue light (BL)-induced depolarization that results from anion channel activation and believed not to involve calcium channels. This unexpected inhibitory effect of La³⁺ on the BL-induced depolarization is explained by our finding that 10 mm La³⁺ directly and completely blocked the BL-activated anion channel when applied to excised patches. We have investigated the ability of La³⁺ to block noncalcium channels in Arabidopsis. In addition to the BL-activated anion channel, 10 mm La³⁺ blocked a cation channel and a stretch-activated channel in patches of plasma membrane excised from hypocotyl cells. In root cells, 10 mm La³⁺ inhibited the activity of an outward-rectifying potassium channel at the whole cell and single-channel level by 47% and 58%, respectively. We conclude that La³⁺ is a nonspecific blocker of multiple ionic conductances in Arabidopsis and may disrupt signal transduction processes independently of any effect on Ca²⁺ channels. Received: 28 July 1997/Revised: 13 November 1997     

A Novel Approach to Study the Geometry of the Water Lumen of Ion Channels: Colicin Ia Channels in Planar Lipid Bilayers

This paper describes a new approach to evaluate the inner structure (including a main constriction and its localization) of the water lumen of an ion channel. The method is based on the determination of channel filling by different nonelectrolyte molecules through each side of an ion channel. The method has two characteristic features that make its use attractive: (i) the possibility to ascertain the existence, localization and size of a narrow part inside an ion channel water lumen and (ii) the chances to determine the maximal size of both entrances of an ion channel and to obtain additional information about the geometry of its water lumen at the same time. Determinations were made on colicin Ia ion channels inserted into planar lipid bilayers. This channel was chosen because there is an apparent contradiction between its low single channel conductance and the large diameter of its water lumen. Our results show that the water lumen of the colicin Ia channel has a funnel-like structure with a small trans-entrance, with a diameter of about 1.0 nm, and a large cis-entrance, with a diameter of approximately 1.8 nm. A constriction with a diameter of approximately 0.7 nm is shown to be located close to the trans-entrance of the channel. The method can also be applied to patch clamp studies of single ion channels. Received: 20 February 1997/Revised: 19 August 1997     

Effects of Mechano-Gated Cation Channel Blockers on Xenopus Oocyte Growth and Development

The putative role(s) of a mechanically gated (MG) cation channel in Xenopus oocyte growth, maturation, fertilization and embryogenesis has been examined. Using a pharmacological approach, we have tested the effects of the MG channel blockers, gadolinium, gentamicin and amiloride on the above developmental events. Our results indicate that oocyte maturation, fertilization and early embryogenesis (up to the free-swimming stage 45) can proceed normally in the presence of concentrations of agents that either completely abolish (i.e., ≥10 μm Gd³⁺) or partially block (i.e., 1 mm gentamicin) single MG channel activity as measured by patch-clamp recording. However, we also find that higher concentrations of Gd³⁺ (≥50 μm) can lead to an increased percentage (>20%) of axis-perturbed embryos compared with control (<1%) and that amiloride (0.5 mm) reduces the success of fertilization (from 100% to <50%) and increases mortality (by ∼75%) in developing embryos. Furthermore, we find that all three agents inhibit oocyte growth in vitro. However, their order of effectiveness (amiloride > gentamicin > Gd³⁺) is opposite to their order for blocking MG channels (Gd³⁺≫ gentamicin > amiloride). These discrepancies indicated that the drugs effects occur by mechanisms other than, or in addition to, MG channel block. Our results provide no compelling evidence for the idea that MG channel activity is critical for development in Xenopus. This could mean that there are other mechanisms in the oocyte that can compensate when MG channel activity is blocked or that the protein that forms the channel can undergo additional interactions that result in a function insensitive to MG channel blockers. Received: 27 March 1998/Revised: 10 June 1998     

The influence of surface charges on quaternary ammonium block of shaker K+ channels

Block of K⁺ channels can be influenced by the ability of charged residues on the protein surface to accumulate cationic blocking ions to concentrations greater than those in bulk solution. We examined the ionic strength dependence of extracellular block of Shaker K⁺ channels by tetraethylammonium ions (TEA⁺) and by a trivalent quaternary ammonium ion, gallamine³⁺. Wild-type and mutant channels were expressed in Xenopus oocytes and currents recorded with the cut-open oocyte technique. Channel block by both compounds was substantially increased when the bathing electrolyte ionic strength was lowered, but with a much larger effect for trivalent gallamine. These data were quantitatively well described by a simple electrostatic model, accounting for accumulation of blocking ions near the pore of the channel by surface charges. The surface charge density of the wild-type channel consistent with the results was −0.1 e nm⁻². Shaker channels with T449Y mutations have an increased affinity for both TEA and gallamine but the ionic strength dependence of block was described with the same surface charge density as wild-type channels. Much of the increased sensitivity of Shaker K⁺ channels to gallamine may be due to a larger local accumulation of the trivalent ion. The negative charge at position 431 contributes to the sensitivity of channels to TEA (MacKinnon & Yellen, 1990). A charge reversal mutation at this location had little effect on the ionic strength dependence of quaternary ammonium ion block, suggesting that the charge on this amino acid may directly affect binding affinity but not local ion accumulation. Received: 7 December 2000/Revised: 27 April 2001     

The Ach-evoked Ca2+-activated K+ Current in Mouse Mandibular Secretory Cells. Single Channel Studies

Although acetylcholine (ACh) is able to activate voltage- and Ca²⁺-sensitive K⁺ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole cell K⁺ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K⁺ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant channel type activated by ACh (1 μmol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was sensitive to quinine and it conducted Rb⁺ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels. When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K⁺ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K⁺ current is not carried substantially by BK channels and show that it is carried by a small-conductance K⁺ channel with quite different properties. Received: 28 September 1995/Revised: 26 December 1995     

Transmembrane insertion of the Colicin Ia hydrophobic hairpin

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an α-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia. Received: 12 November 1996/Revised: 23 January 1997     

Extra- and Intracellular Proton-Binding Sites of Volume-Regulated Anion Channels

We have investigated the effects of extracellular and intracellular pH on single channel and macroscopic (macropatches) currents through volume-regulated anion channels (VRAC) in endothelial cells. Protonation of extracellular binding sites with an apparent pK of 4.6 increased voltage independent of the single-channel amplitude. Cytosolic acidification had a dual effect on VRAC currents: on the one hand, it increased single channel conductance by ∼20% due to protonation of a group with an apparent pK of 6.5 and a Hill coefficient of 2. On the other hand, it reduced channel activity due to protonation of a group with an apparent pK of 6.3 and a Hill coefficient of 2.1. This dual effect enhances the macroscopic current at a slightly acidic pH but inhibits it at more acidic pH. Cytosolic alkalization also reduced channel activity with a pK of 8.4 and a Hill coefficient of 1.9, but apparently did not affect single-channel conductance. These data show that VRAC channels are maintained in an active state in a narrow pH range around the normal physiological pH and shut down outside this range. They also show that HEPES-buffered pipette solutions do not effectively buffer pH in the vicinity of the VRAC channels. Received: 31 January 2000/Revised: 21 April 2000     

Voltage-Dependent Anion Channel of Arabidopsis Hypocotyls: Nucleotide Regulation and Pharmacological Properties

Plasma membrane anion channels are thought to play important roles in osmoregulation and signal transduction in higher plant cells. Knowledge of their pharmacology and regulation is of importance to unravel their physiological functions. In this study, we explore the pharmacological properties and the nucleotide regulation of the voltage-dependent anion channel of Arabidopsis hypocotyls. The pharmacological profile of this channel is characterized by a low sensitivity to most anion channel blockers. It is inhibited by niflumic acid with an IC₅₀ of 80 μm, but poorly sensitive to IAA-94 and NPPB and insensitive to 9-AC and DIDS. Nucleotides alter the amplitude, the kinetics and the voltage-dependence of the channel. The main effect of nucleotides is a shift of the voltage-dependent gate of the channel toward depolarized potentials leading to a strong reduction of the current amplitude. This regulation does not require ATP hydrolysis as nonhydrolyzable ATP analogues—AMPPNP and ATPγS—also regulate the anion current. This suggests that a nucleotide binding site is involved in the regulation. The study of the properties of this putative nucleotide binding site reveals that (i) ATP regulates the channel with an EC₅₀ of 0.7 mm, (ii) adenyl nucleotides modulate the channel with the following order of effectiveness: ATP > ADP ≫ AMP, and (iii) thiophosphate nucleotide analogues are the most potent agonists with EC₅₀ in the range of 80 μm. The hypothesis that this regulation may couple the electrical properties of the membrane with the metabolic status of the cell is discussed. Received: 26 December 1996/Revised: 21 March 1997     

Characterization of Calcium-activated Chloride Channels in Patches Excised from the Dendritic Knob of Mammalian Olfactory Receptor Neurons

We investigated the properties of calcium-activated chloride channels in inside-out membrane patches from the dendritic knobs of acutely dissociated rat olfactory receptor neurons. Patches typically contained large calcium-activated currents, with total conductances in the range 30–75 nS. The dose response curve for calcium exhibited an EC₅₀ of about 26 μm. In symmetrical NaCl solutions, the current-voltage relationship reversed at 0 mV and was linear between −80 and +70 mV. When the intracellular NaCl concentration was progressively reduced from 150 to 25 mM, the reversal potential changed in a manner consistent with a chloride-selective conductance. Indeed, modeling these data with the Goldman-Hodgkin-Katz equation revealed a P_Na/P_Cl of 0.034. The halide permeability sequence was P_Cl > P_F > P_I > P_Br indicating that permeation through the channel was dominated by ion binding sites with a high field strength. The channels were also permeable to the large organic anions, SCN⁻, acetate⁻, and gluconate⁻, with the permeability sequence P_Cl > P_SCN > P_acetate > P_gluconate. Significant permeation to gluconate ions suggested that the channel pore had a minimum diameter of at least 5.8 \A. Received: 16 April 1997/Revised: 3 October 1997     

Verapamil Block of Large-Conductance Ca-Activated K Channels in Rat Aortic Myocytes

The effects of verapamil on the large conductance Ca-activated K (BK) channel from rat aortic smooth muscle cells were examined at the single channel level. Micromolar concentrations of verapamil produced a reversible flickering block of the BK channel activity. Kinetic analysis showed that verapamil decreased markedly the time constants of the open states, without any significant change in the time constants of the closed states. The appearance of an additional closed state — specifically, a nonconducting, open-blocked state — was also observed, whose time constant would reflect the mean residence time of verapamil on the channel. These observations are indicative of a state-dependent, open-channel block mechanism. Dedicated kinetic (group) analysis confirmed the state-dependent block exerted by verapamil. D600 (gallopamil), the methoxy derivative of verapamil, was also tested and found to exert a similar type of block, but with a higher affinity than verapamil. The permanently charged and membrane impermeant verapamil analogue D890 was used to address other important features of verapamil block, such as the sidedness of action and the location of the binding site on the channel protein. D890 induced a flickering block of BK channels similar to that observed with verapamil only when applied to the internal side of the membrane, indicating that D890 binds to a site accessible from the cytoplasmic side. Finally, the voltage dependence of D890 block was assessed. The experimental data fitted with a Langmuir equation incorporating the Woodhull model for charged blockers confirms that the D890-binding site is accessed from the internal mouth of the BK channel, and locates it approximately 40% of the membrane voltage drop along the permeation pathway. Received: 11 April 2000/Revised: 17 October 2000     

Architecture of the Neuronal Nicotinic Acetylcholine Receptor Ion Channel at the Binding Site of bis-Ammonium Blockers

Structure-activity relationships of 56 pentamethylenbis-ammonium compounds, the blockers of the neuronal nicotinic acetylcholine receptor (nAChR) ion channel, have been studied to estimate the cross-sectional dimensions of the channel pore. The cat superior cervical sympathetic ganglion in situ and isolated guinea pig ileum were used to evaluate the potency of the compounds to block ganglionic transmission. Minimum-energy conformations of each compound were calculated by the molecular mechanics method. A topographic model of the binding site of the blockers was proposed. It incorporates two narrowings, a large and a small one. The small narrowing is located between the large one and the cytoplasmic end of the pore. The cross-sectional dimensions of the large and small narrowings estimated from the dimensions of the blockers are 6.1 × 8.3 ? and 5.5 × 6.4 ?, respectively, the distance between the narrowings along the pore being approximately 7 ?. Most potent blockers would occlude the pore via binding to the channel at the levels of both narrowings. Less potent blockers are either too large or too small to bind to both narrowings simultaneously: large blockers would occlude the pore at the level of large narrowing, while small blockers would pass the large narrowing and occlude the pore at the level of small narrowing only. A comparison of the topographic model with a molecular five-helix bundle model of nAChR pore predicts Serine and Threonine rings to be the most probable candidates for the large and small narrowings, respectively. Received: 6 September 1995/Revised: 12 March 1996     

Nickel Block of a Family of Neuronal Calcium Channels: Subtype- and Subunit-Dependent Action at Multiple Sites

Nickel ions have been reported to exhibit differential effects on distinct subtypes of voltage-activated calcium channels. To more precisely determine the effects of nickel, we have investigated the action of nickel on four classes of cloned neuronal calcium channels (α_1A, α_1B, α_1C, and α_1E) transiently expressed in Xenopus oocytes. Nickel caused two major effects: (i) block detected as a reduction of the maximum slope conductance and (ii) a shift in the current-voltage relation towards more depolarized potentials which was paralleled by a decrease in the slope of the activation-curve. Block followed 1:1 kinetics and was most pronounced for α_1C, followed by α_1E > α_1A > α_1B channels. In contrast, the change in activation-gating was most dramatic with α_1E, with the remaining channel subtypes significantly less affected. The current-voltage shift was well described by a simple model in which nickel binding to a saturable site resulted in altered gating behavior. The affinity for both the blocking site and the putative gating site were reduced with increasing concentration of external permeant ion. Replacement of barium with calcium reduced both the degree of nickel block and the maximal effect on gating for α_1A channels, but increased the nickel blocking affinity for α_1E channels. The coexpression of Ca channel β subunits was found to differentially influence nickel effects on α_1A, as coexpression with β_2a or with β₄ resulted in larger current-voltage shifts than those observed in the presence of β_1b, while elimination of the β subunit almost completely abolished the gating shifts. In contrast, block was similar for the three β subunits tested, while complete removal of the β subunit resulted in an increase in blocking affinity. Our data suggest that the effect of nickel on calcium channels is complex, cannot be described by a single site of action, and differs qualitatively and quantitatively among individual subtypes and subunit combinations. Received: 12 October 1995/Revised: 17 January 1996     

An ion''s view of the potassium channel. The structure of the permeation pathway as sensed by a variety of blocking ions

We have studied the block of potassium channels in voltage-clamped squid giant axons by nine organic and alkali cations, in order to learn how the channel selects among entering ions. When added to the internal solution, all of the ions blocked the channels, with inside-positive voltages enhancing the block. Cesium blocked the channels from the outside as well, with inside-negative voltages favoring block. We compared the depths to which different ions entered the channel by estimating the "apparent electrical distance" to the blocking site. Simulations with a three-barrier, double-occupancy model showed that the "apparent electrical distance," expressed as a fraction of the total transmembrane voltage, appears to be less than the actual value if the blocking ion can pass completely through the channel. These calculations strengthen our conclusion that sodium and cesium block at sites further into the channel than those occupied by lithium and the organic blockers. Our results, considered together with earlier work, demonstrate that the depth to which an ion can readily penetrate into the potassium channel depends both on its size and on the specific chemical groups on its molecular surface. The addition of hydroxyl groups to alkyl chains on a quaternary ammonium ion can both decrease the strength of binding and allow deeper penetration into the channel. For alkali cations, the degree of hydration is probably crucial in determining how far an ion penetrates. Lithium, the most strongly hydrated, appeared not to penetrate as far as sodium and cesium. Our data suggest that there are, minimally, four ion binding sites in the permeation pathway of the potassium channel, with simultaneous occupancy of at least two.