Gating of acid-sensitive ion channel-1: release of Ca2+ block vs. allosteric mechanism

The acid-sensitive ion channels (ASICs) are a family of voltage-insensitive sodium channels activated by external protons. A previous study proposed that the mechanism underlying activation of ASIC consists of the removal of a Ca2+ ion from the channel pore (Immke and McCleskey, 2003). In this work we have revisited this issue by examining single channel recordings of ASIC1 from toadfish (fASIC1). We demonstrate that increases in the concentration of external protons or decreases in the concentration of external Ca2+ activate fASIC1 by progressively opening more channels and by increasing the rate of channel opening. Both maneuvers produced similar effects in channel kinetics, consistent with the former notion that protons displace a Ca2+ ion from a high-affinity binding site. However, we did not observe any of the predictions expected from the release of an open-channel blocker: decrease in the amplitude of the unitary currents, shortening of the mean open time, or a constant delay for the first opening when the concentration of external Ca2+ was decreased. Together, the results favor changes in allosteric conformations rather than unblocking of the pore as the mechanism gating fASIC1. At high concentrations, Ca2+ has an additional effect that consists of voltage-dependent decrease in the amplitude of unitary currents (EC50 of 10 mM at -60 mV and pH 6.0). This phenomenon is consistent with voltage-dependent block of the pore but it occurs at concentrations much higher than those required for gating.     

Divalent cation selectivity for external block of voltage-dependent Na+ channels prolonged by batrachotoxin. Zn2+ induces discrete substates in cardiac Na+ channels

The mechanism of block of voltage-dependent Na+ channels by extracellular divalent cations was investigated in a quantitative comparison of two distinct Na+ channel subtypes incorporated into planar bilayers in the presence of batrachotoxin. External Ca2+ and other divalent cations induced a fast voltage-dependent block observed as a reduction in unitary current for tetrodotoxin-sensitive Na+ channels of rat skeletal muscle and tetrodotoxin-insensitive Na+ channels of canine heart ventricular muscle. Using a simple model of voltage-dependent binding to a single site, these two distinct Na+ channel subtypes exhibited virtually the same affinity and voltage dependence for fast block by Ca2+ and a number of other divalent cations. This group of divalent cations exhibited an affinity sequence of Co congruent to Ni greater than Mn greater than Ca greater than Mg greater than Sr greater than Ba, following an inverse correlation between binding affinity and ionic radius. The voltage dependence of fast Ca2+ block was essentially independent of CaCl2 concentration; however, at constant voltage the Ca2+ concentration dependence of fast block deviated from a Langmuir isotherm in the manner expected for an effect of negative surface charge. Titration curves for fast Ca2+ block were fit to a simplified model based on a single Ca2+ binding site and the Gouy-Chapman theory of surface charge. This model gave similar estimates of negative surface charge density in the vicinity of the Ca2+ blocking site for muscle and heart Na+ channels. In contrast to other divalent cations listed above, Cd2+ and Zn2+ are more potent blockers of heart Na+ channels than muscle Na+ channels. Cd2+ induced a fast, voltage-dependent block in both Na+ channel subtypes with a 46-fold higher affinity at 0 mV for heart (KB = 0.37 mM) vs. muscle (KB = 17 mM). Zn2+ induced a fast, voltage-dependent block of muscle Na+ channels with low affinity (KB = 7.5 mM at 0 mV). In contrast, micromolar Zn2+ induced brief closures of heart Na+ channels that were resolved as discrete substate events at the single-channel level with an apparent blocking affinity of KB = 0.067 mM at 0 mV, or 110-fold higher affinity for Zn2+ compared with the muscle channel. High-affinity block of the heart channel by Cd2+ and Zn2+ exhibited approximately the same voltage dependence (e-fold per 60 mV) as low affinity block of the muscle subtype (e-fold per 54 mV), suggesting that the block occurs at structurally analogous sites in the two Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: implications for channel gating

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 approximately 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.     

Extracellular Ba2+ and voltage interact to gate Ca2+ channels at the plasma membrane of stomatal guard cells

Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] ([Ca2+](i)) that regulate K+ and Cl- channels for stomatal closure in higher-plant leaves. Under voltage clamp, the initial rate of increase in [Ca2+](i) in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating. To test this idea, we recorded single-channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages. We found that the open probability (P(o)) increased strongly with external Ba2+ concentration, consistent with a 4-fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state. Dwell time analyses indicated the presence of a single open state and at least three closed states of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state. Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage. We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate.     

Permeation properties of a Ca(2+)-blockable monovalent cation channel in the ectoderm of the chick embryo: pore size and multioccupancy probed with organic cations and Ca2+

A Ca(2+)-blockable monovalent cation channel is present in the apical membrane of the ectoderm of the gastrulating chick embryo. We used the patch clamp technique to study several single-channel permeation properties of this channel. In symmetrical conditions without Ca2+, the Na+ current carried by the channel rectifies inwardly. The channel has an apparent dissociation constant for extracellular Na+ of 115 mM at 0 mV and a low density of negative surface charge (-0.03 e/nm2) at its extracellular entrance. The minimal pore diameter is approximately 5.8 A, as calculated from the relative permeabilities of 10 small organic cations. Extracellular application of six large organic cations decreased the inward Na+ current in a voltage-dependent manner, which strongly suggests an intrachannel block. The presence of at least two ion binding sites inside the pore is inferred from the Na+ dependence of the block by the organic cations. This hypothesis is strengthened by the fact that the extracellular Ca2+ block is also modified by the Na+ concentration. In particular, the rise of the unblocking rate with increased Na+ concentrations clearly suggests the presence of an interaction between Ca2+ and Na+ inside the channel. A low probability of double occupancy at physiological ionic conditions is implied from the absence of an anomalous mole fraction effect with mixtures of extracellular Li+ and K+. Finally, the absence of inward current at very strong hyperpolarizations and in the presence of 10 mM extracellular Ca2+ demonstrates the absence of significant Ca2+ current through this channel. It is argued that this embryonic epithelial Ca(2+)-blockable monovalent cation channel is related to both L-type Ca2+ channel and cyclic nucleotide-gated channels.     

Calcium dependent shifts of Na+ channel activation correlated with the state dependence of calcium-binding to the pore

Calcium ions block the open configuration and antagonise the tonic binding of TTX to the closed state of sodium channels in very different ranges of extracellular concentration, [Ca]_O. We measured the open-state block in channels expressed in Xenopus oocytes by α-subunits from rat brain (rBIIa) or adult rat skeletal muscle (rSkM1). Recordings of instantaneous tail-currents from cell-attached macro patches show that the binding of Ca²⁺ to the blocking site has a dissociation constant of about 20 mM at 0 mV and senses about 30% of the membrane potential drop, whereas the concentration of half-inhibition of TTX-binding is less than 1 mM and voltage-insensitive. Assuming that both effects involve a single binding site, a simple model predicts that the state-dependency of the dissociation constant entails positive shifts of activation and faster kinetics of deactivation at increasing [Ca]_O. The shifts of activation measured for rBIIA and rSkM1 channels are comparable in size to those predicted by the model, which accounts also for the observed larger shifts of the rBIIA-mutant K226Q as a consequence of its reduced voltage-sensitivity. Shifts attributable to surface-charge screening effects seem smaller in the oocyte than in native cell-membranes. The experimental [Ca]_O-dependence of deactivation kinetics is also consistent with the model and with the idea that Ca²⁺-binding changes to the same extent, but in opposite directions, the activation free-energies of both opening and closing transitions. Received: 1 December 1997 / Revised version: 25 March 1998 / Accepted: 27 March 1998     

Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. Interactions between toxin binding and channel gating

Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ.     

Molecular identification of a TTX-sensitive Ca(2+) current

The TTX-sensitive Ca(2+) current [I(Ca(TTX))] observed in cardiac myocytes under Na(+)-free conditions was investigated using patch-clamp and Ca(2+)-imaging methods. Cs(+) and Ca(2+) were found to contribute to I(Ca(TTX)), but TEA(+) and N-methyl-D-glucamine (NMDG(+)) did not. HEK-293 cells transfected with cardiac Na(+) channels exhibited a current that resembled I(Ca(TTX)) in cardiac myocytes with regard to voltage dependence, inactivation kinetics, and ion selectivity, suggesting that the cardiac Na(+) channel itself gives rise to I(Ca(TTX)). Furthermore, repeated activation of I(Ca(TTX)) led to a 60% increase in intracellular Ca(2+) concentration, confirming Ca(2+) entry through this current. Ba(2+) permeation of I(Ca(TTX)), reported by others, did not occur in rat myocytes or in HEK-293 cells expressing cardiac Na(+) channels under our experimental conditions. The report of block of I(Ca(TTX)) in guinea pig heart by mibefradil (10 microM) was supported in transfected HEK-293 cells, but Na(+) current was also blocked (half-block at 0.45 microM). We conclude that I(Ca(TTX)) reflects current through cardiac Na(+) channels in Na(+)-free (or "null") conditions. We suggest that the current be renamed I(Na(null)) to more accurately reflect the molecular identity of the channel and the conditions needed for its activation. The relationship between I(Na(null)) and Ca(2+) flux through slip-mode conductance of cardiac Na(+) channels is discussed in the context of ion channel biophysics and "permeation plasticity."     

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods

The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.     

Extracellular Ca2+ modulates the effects of protons on gating and conduction properties of the T-type Ca2+ channel alpha1G (CaV3.1)

Since Ca2+ is a major competitor of protons for the modulation of high voltage-activated Ca2+ channels, we have studied the modulation by extracellular Ca2+ of the effects of proton on the T-type Ca2+ channel alpha1G (CaV3.1) expressed in HEK293 cells. At 2 mM extracellular Ca2+ concentration, extracellular acidification in the pH range from 9.1 to 6.2 induced a positive shift of the activation curve and increased its slope factor. Both effects were significantly reduced if the concentration was increased to 20 mM or enhanced in the absence of Ca2+. Extracellular protons shifted the voltage dependence of the time constant of activation and decreased its voltage sensitivity, which excludes a voltage-dependent open pore block by protons as the mechanism modifying the activation curve. Changes in the extracellular pH altered the voltage dependence of steady-state inactivation and deactivation kinetics in a Ca2+-dependent manner, but these effects were not strictly correlated with those on activation. Model simulations suggest that protons interact with intermediate closed states in the activation pathway, decreasing the gating charge and shifting the equilibrium between these states to less negative potentials, with these effects being inhibited by extracellular Ca2+. Extracellular acidification also induced an open pore block and a shift in selectivity toward monovalent cations, which were both modulated by extracellular Ca2+ and Na+. Mutation of the EEDD pore locus altered the Ca2+-dependent proton effects on channel selectivity and permeation. We conclude that Ca2+ modulates T-type channel function by competing with protons for binding to surface charges, by counteracting a proton-induced modification of channel activation and by competing with protons for binding to the selectivity filter of the channel.     

Sensing of extracellular calcium by neurones

Transient changes in the intracellular concentration of Ca2+ provide a major signal for the regulation of many ion channels and enzymes in central neurones. In contrast, changes in extracellular Ca2+ are thought to play little or no signaling role. However, concentrations of extracellular calcium in the central nervous system do change dramatically during intense physiological and pathological stimulation, and recent studies have identified a number of membrane proteins that can sense and respond to changes in extracellular Ca2+. These include the recently cloned Ca(2+)-sensing receptor, hemi-gap-junction channels, and a potential Ca(2+)-sensing cation channel. Lowering extracellular Ca2+ strongly depolarizes and excites cultured hippocampal neurones. The excitation can be detected with decreases from physiological concentrations of as little as 100 microM. The depolarization results from activation of a nonselective cation current, which is sensitive to block by divalent and polyvalent cations. In outside-out patches, lowering Ca2+ induces a single-channel current with a conductance of 36 pS. Activation of this cation channel, in response to decreases in extracellular Ca2+, likely plays a key role in a positive feedback system of excessive neuronal depolarization, which accompanies intense excitatory activity in the hippocampus.     

Modulation of Neuronal Voltage-Activated Calcium and Sodium Channels by Polyamines and pH

The endogenous polyamines spermine, spermidine and putrescine are present at high concentrations inside neurons and can be released into the extracellular space where they have been shown to modulate ion channels. Here, we have examined polyamine modulation of voltage-activated Ca2+ channels (VACCs) and voltage-activated Na+ channels (VANCs) in rat superior cervical ganglion neurons using whole-cell voltage-clamp at physiological divalent concentrations. Polyamines inhibited VACCs in a concentration-dependent manner with IC50s for spermine, spermidine, and putrescine of 4.7 ± 0.7, 11.2 ± 1.4, and 90 ± 36 mM, respectively. Polyamines caused inhibition by shifting the VACC half-activation voltage (V0.5) to depolarized potentials and by reducing total VACC permeability. The shift was described by Gouy-Chapman-Stern theory with a surface charge density of 0.120 ± 0.005 e- nm-2 and a surface potential of -19 mV. Attenuation of spermidine and spermine inhibition of VACC at decreased pH was explained by H+ titration of surface charge. Polyamine-mediated effects also decreased at elevated pH due to the inhibitors having lower valence and being less effective at screening surface charge. Polyamines affected VANC currents indirectly by reducing TTX inhibition of VANCs at high pH. This may reflect surface charge induced decreases in the local TTX concentration or polyamine-TTX interactions. In conclusion, polyamines inhibit neuronal VACCs via complex interactions with extracellular H+ and Ca. Many of the observed effects can be explained by a model incorporating polyamine binding, H+ binding and surface charge screening.     

Channels produced by spider venoms in bilayer lipid membrane: mechanisms of ion transport and toxic action

The selectivity of ion channels produced by latrotoxin obtained from a black widow spider venom and by venom from the spider Steatoda paykulliana in bilayer phospholipid membrane was studied. Experimental current-voltage curves of these channels were used for the estimation of parameters of a two barrier model of their energy profiles. Selectivities of both types of channels are similar. Alkaline earth cations are permeable, the permeability increasing in the order Mg2+ less than Ca2+ less than Sr2+ less than Ba2+. In contrast transition metal cations block the channel, their efficiency decreases in the order: Cd2+ greater than or equal to Ni2+ greater than Zn2+ greater than Co2+ greater than Mn2+ (Steatoda paykulliana spider venom) and Cd2+ greater than Co2+ greater than Ni2+ greater than Zn2+ greater than Mn2+ (latrotoxin). Amplitudes of current carried by corresponding ions are mainly determined by the depth of the potential well for this ion, i.e., by its affinity to the cation binding site in the channel. The channels are also permeable to monovalent cations but they do not bind them. Selectivity for monovalent cations depends on Ca2+ concentration at the cis-side of membrane in the micromolar range. However, the addition of Ca2+ to the trans-side up to 10 mM does not affect currents carried by monovalent ions. It is suggested that venom-induced calcium channels have two conformational states with different selectivities which interconvert upon binding one calcium ion. Possible general schemes for the organisation of calcium channels in excitable membranes are also discussed. Finally, using a mathematical model of synaptic transmission, possible mechanisms of toxic action of spider venoms are considered.     

Open sodium channel properties of single canine cardiac Purkinje cells.

Open channel properties of canine cardiac Purkinje cell Na+ channels were studied with single channel cell-attached recording and with whole cell macroscopic current recording in internally perfused cells. Single channel currents and membrane currents increased with an increase in Na+ concentration, but showed evidence of saturation. Assuming first-order binding, the Km for Na+ was 370 mM. PCs/PNa was 0.020 and PK/PNa was 0.094. The current-voltage relationship for single channels showed prominent flattening in the hyperpolarizing direction. This flattening was accentuated by 10 mM Ca2+ and was greatly reduced in O mM Ca2+, indicating that the rectification was a consequence of Ca2+ block of the Na+ channels. A similar instantaneous current-voltage relationship was seen for the whole cell membrane currents. These results demonstrate that the cardiac channel shows substantial Ca2+ block, although it is relatively insensitive to tetrodotoxin. The Na+ and Ca2+ binding properties could be modeled by the four-barrier Eyring rate theory model, with similar values to those reported for the neuroblastoma Na+ channel (Yamamoto, D.,J.Z. Yeh, and T. Narahashi, 1984, Biophys J., 45:337-344).     

Mechanism of tetrodotoxin block and resistance in sodium channels

Tetrodotoxin (TTX) has been used for many decades to characterize the structure and function of biological ion channels. Yet, the precise mechanism by which TTX blocks voltage-gated sodium (Na_V) channels is not fully understood. Here molecular dynamics simulations are used to elucidate how TTX blocks mammalian voltage-gated sodium (Nav) channels and why it fails to be effective for the bacterial sodium channel, Na_VAb. We find that, in Na_VAb, a sodium ion competes with TTX for the binding site at the extracellular end of the filter, thus reducing the blocking efficacy of TTX. Using a model of the skeletal muscle channel, Na_V1.4, we show that the conduction properties of the channel observed experimentally are faithfully reproduced. We find that TTX occludes the entrance of Na_V1.4 by forming a network of hydrogen-bonds at the outer lumen of the selectivity filter. The guanidine group of TTX adopts a lateral orientation, rather than pointing into the filter as proposed previously. The acidic residues just above the selectivity filter are important in stabilizing the hydrogen-bond network between TTX and Na_V1.4. The effect of two single mutations of a critical tyrosine residue in the filter of Na_V1.4 on TTX binding observed experimentally is reproduced using computational mutagenesis.     

Inhibition of the olfactory cyclic nucleotide gated ion channel by intracellular calcium.

When olfactory receptor neurons are exposed to sustained application of odours, the elicited ionic current is transient. This adaptation-like effect appears to require the influx of Ca2+ through the odour-sensitive conductance; in the absence of extracellular Ca2+ the current remains sustained. Odour transduction proceeds through a G-protein-based second messenger system, resulting finally in the direct activation of an ion channel by cyclic AMP. This channel is one possible site for a negative feedback loop using Ca2+ as a messenger. In recordings of single cyclic AMP gated channels from olfactory receptor neurons, the open probability of the channel in saturating cAMP concentrations was dependent on the concentration of intracellular Ca2+. It could be reduced from 0.6 in 100 nm Ca2+ to 0.09 in 3 microM Ca2+. However, as neither the single channel conductance nor the mean open time were affected by Ca+ concentration, this does not appear to be a mechanism of simple channel block. Rather, these results suggest that intracellular Ca2+ acts allosterically to stabilize a closed state of the channel.     

Inwardly rectifying current-voltage relationship of small-conductance Ca2+-activated K+ channels rendered by intracellular divalent cation blockade

Small conductance Ca2+-activated K+ channels (SK(Ca) channels) are a group of K+-selective ion channels activated by submicromolar concentrations of intracellular Ca2+ independent of membrane voltages. We expressed a cloned SK(Ca) channel, rSK2, in Xenopus oocytes and investigated the effects of intracellular divalent cations on the current-voltage (I-V) relationship of the channels. Both Mg2+ and Ca2+ reduced the rSK2 channel currents in voltage-dependent manners from the intracellular side and thus rectified the I-V relationship at physiological concentration ranges. The apparent affinity of Mg2+ was changed as a function of both transmembrane voltage and intracellular Ca2+ concentration. Extracellular K+ altered the voltage dependence as well as the apparent affinities of Mg2+ binding from intracellular side. Thus, the inwardly rectifying I-V relationship of SK(Ca) channels is likely due to the voltage-dependent blockade of intracellular divalent cations and that the binding site is located within the ion-conducting pathway. Therefore, intracellular Ca2+ modulates the permeation characteristics of SK(Ca) channels by altering the I-V relationship as well as activates the channel by interacting with the gating machinery, calmodulin, and SK(Ca) channels can be considered as Ca2+-activated inward rectifier K+ channels.     

Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation.

A T-type Ca2+ channel is expressed during differentiation of the male germ lineage in the mouse and is retained in sperm, where is it activated by contact with the the egg's extracellular matrix and controls sperm acrosomal exocytosis. Here, we examine the regulation of this Ca2+ channel in dissociated spermatogenic cells from the mouse using the whole-cell patch-clamp technique. T currents were enhanced, or facilitated, after strong depolarizations or high frequency stimulation. Voltage-dependent facilitation increased the Ca2+ current by an average of 50%. The same facilitation is produced by antagonists of protein tyrosine kinase activity. Conversely, antagonists of tyrosine phosphatase activity block voltage-dependent facilitation of the current. These data are consistent with the presence of a two-state model, in which T channels are maintained in a low (or zero) conductance state by tonic tyrosine phosphorylation and can be activated to a high conductance state by a tyrosine phosphatase activity. The positive and negative modulation of this channel by the tyrosine phosphorylation state provides a plausible mechanism for the control of sperm activity during the early stages of mammalian fertilization.     

Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures

The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.     

Block of cardiac ATP-sensitive K+ channels by external divalent cations is modulated by intracellular ATP. Evidence for allosteric regulation of the channel protein

We have investigated the interactions between extracellular divalent cations and the ATP-sensitive potassium channel in single guinea pig ventricular cells and found that, under whole-cell patch clamp recording conditions, extracellularly applied Co2+, Cd2+, and Zn2+ block current through the ATP-sensitive K channel (IKATP). The respective Kd's for block of IKATP by Cd2+ and Zn2+ are 28 and 0.46 microM. The Kd for Co2+ is > 200 microM. Extracellular Ca2+ and Mg2+ appear to have no effect at concentrations up to 1 and 2 mM, respectively. Block of IKATP by extracellular cations is not voltage dependent, and both onset and recovery from block occur within seconds. Single-channel experiments using the inside-out patch configuration show that internally applied Cd2+ and Zn2+ are not effective blockers of IKATP. Experiments in the outside-out patch configuration confirm that the divalent cations interact directly with IKATP channel activity. Our study also shows that this block of IKATP is dependent on intracellular ATP concentrations. Under whole-cell conditions, when cells are dialyzed with [ATP]pipette = 0, the degree of cation block is reduced. This dependence on intracellular ATP was confirmed at the single-channel level by experiments in excised, inside-out patch configurations. Our results show that some, but not all, divalent cations inhibit current through IKATP channels by binding to sites that are not within the transmembrane electric field, but are on the extracellular membrane surface. The interdependence of internal ATP and external divalent cation binding is consistent with an allosteric interaction between two binding sites and is highly suggestive of a modulatory mechanism involving conformational change of the channel protein.