Interaction Between Store-Operated Non-Selective Cation Channels and the Na+-Ca2+ Exchanger During Secretion in the Rat Colon

The properties of capacitative Ca(2+) influx were studied using the whole-cell patch-clamp technique in crypts isolated from rat distal colon. Store-operated cation influx was evoked by increasing the intracellular buffering capacity for Ca(2+) in the pipette solution; contamination by Cl(-) currents was reduced by the use of NMDG gluconate as the main electrolyte in the pipette solution. The permeability of the non-selective cation conductance stimulated by store depletion had the following sequence for monovalent cations: Cs(+) > Na(+) > or = Li(+). The store-operated conductance is permeable to Na(+) and Ca(2+), but in contrast to Na(+), Ca(2+) also exerts a (feedback) inhibition on its own influx. Other divalent cations shared this inhibitory action with the sequence: Ca(2+) > or = Mg(2+) > or = Ba(2+) > or = Sr(2+). Fura-2 experiments revealed that replacement of extracellular Na(+) by NMDG(+) induced an increase in the intracellular Ca(2+) concentration, which was suppressed by the Na(+)-Ca(2+) exchange inhibitor, dichlorobenzamil, indicating the presence of a Na(+)-Ca(2+) exchanger within the colonic crypt cells. In Ussing chamber experiments dichlorobenzamil induced an increase in short-circuit current (I(sc)) in the majority of tissues tested indicating that this exchanger acts as a Ca(2+)-extruding transporter under physiological conditions. When Ca(2+)-dependent anion secretion was stimulated by the acetylcholine analogue carbachol, dichlorobenzamil no longer evoked an increase in I(sc), indicating that after stimulation of the store-operated cation conductance the Na(+)-Ca(2+) exchanger is turned off. Therefore, it is concluded that the influx of Na(+) across the non-selective store-operated cation conductance serves to reduce the driving force for Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and thereby maintains the increase in the intracellular Ca(2+) concentration during induction of secretion. Experimental Physiology (2001) 86.4, 461-468.     

Surface potential reflected in both gating and permeation mechanisms of sodium and calcium channels of the tunicate egg cell membrane

1. Threshold changes of Na and Ca currents due to various polyvalent cations (stabilizing cations) or H(+) ions were studied in the egg cell membrane of a tunicate, Halocynthia roretzi, by using the voltage-clamp technique.2. With an increase in [Ca](o) or a decrease in pH in the external solution, the current-voltage (I-V) relations for the peak of the Na and Ca currents shifted along the voltage axis in the positive direction. These voltage shifts in the I-V relations, measured at a potential of V((1/2)) where inward current attains its half-maximum, were shown to be identical to shifts in voltage-dependence of the time courses of Na and Ca currents, and also identical to shifts in the inactivation curves of Na current along the voltage axis.3. The shifts in V((1/2)) produced by various polyvalent cations or H(+) ions were analysed by the Gouy-Chapman equation for the diffuse double layer, by assuming that a change in V((1/2)) directly corresponds to a change in the surface double layer potential.4. The V((1/2))-divalent cation concentration relations of Na current were exactly described by the predictions of the theory with a constant value of the surface charge density of 1e(-)/(9 A)(2). The weak stabilizing effects of Mg(2+), Sr(2+) and Ba(2+) were quite similar to each other and were explained in terms of a ;screening' effect. Other divalent cations, such as Ca(2+), Mn(2+) and Ni(2+), showed various different stabilizing effects which were explained in terms of a ;binding' effect. The binding constants (K(1)'s) for Ca(2+), Mn(2+) and Ni(2+) were 0.21, 0.45 and 0.94 M(-1), respectively.5. H(+) ions showed a powerful stabilizing effect upon the Na current with a K(H) of 6 x 10(4)M(-1). This value indicates that the acidic sites around Na channels have a pK(a) of 4.78. La(3+) ions also acted as a strong stabilizer upon the Na current with a K(La) of 15 M(-1). For both H(+) and La(3+), the V((1/2))-concentration relations were also exactly described by the Gouy-Chapman equation with the same charge density of 1e(-)/(9 A)(2) as estimated by varying divalent cations.6. The stabilizing effect of permeant cations such as Ca(2+), Sr(2+) and Ba(2+) on Ca channel currents was analysed. The effect of lowering pH was also studied. It was found that the surface charge density of 1e(-)/(9 A)(2) estimated by Na current is also applicable to the explanation for the V((1/2))-divalent cation concentration or - pH relationships. The estimated binding constants for H(+), Ca(2+) and Sr(2+) were 1.2x10(5), 0.58 and 0.035 M(-1), respectively. Ba(2+) does not bind to charged sites near to the Ca channels.7. It was noticed that a considerable reduction in the conductances of Na and Ca currents occurred in parallel with a stabilizing effect. This reduction was ascribed to a decrease in the concentration of permeant cations at the external surface of the cell membrane, as predicted by the theory of the diffuse double layer. The Goldman, Hodgkin-Katz equation for ionic currents was applied to explain the conductance suppression.8. The conductance suppressions of Na and Ca channel currents due to Ca(2+), Sr(2+) and Ba(2+) were found to be apparent ones, only reflecting decreases in the surface concentration of permeant cations without any changes in the permeability. After correction for the apparent suppression, the real permeability ratio among Ca(2+), Sr(2+) and Ba(2+) for Ca channels was determined as 1.00, 0.56 and 0.21 respectively.9. The conductance suppression of Na current by lowering pH was explained in terms of a real suppression or blocking which is superimposed on the apparent suppression. Considering the surface [Na](o), the plot of P(Na) against the surface pH yielded a blocking curve of Na channel by H(+) ions, which implies that two H(+) ions are necessary to block each Na channel. For Ca channels no real blockage was observed in acidic pH.10. It was concluded from the present experiment that there exists a surface potential capable of affecting both gating and permeation mechanisms of ionic channels in this tunicate egg cell membrane.     

Monovalent cation permeability and Ca2+ block of the store-operated Ca2+ current I CRAC in rat basophilic leukemia cells

Like voltage-operated Ca(2+) channels, store-operated CRAC channels become permeable to monovalent cations in the absence of external divalent cations. Using the whole-cell patch-clamp technique, we have characterized the permeation and selectivity properties of store-operated channels in the rat basophilic leukemia (RBL-1) cell line. Store depletion by dialysis with InsP(3) and 10 mM EGTA resulted in the rapid development of large inward currents in Na(+)- and Li(+)-based divalent-free solutions. Cs(+) permeated the channels poorly (P(Cs)/ P(Na)=0.01). Trimethylamine (TMA(+)), tetramethylammonium (TeMA(+)), tetraethylammonium (TEA(+)), N-methyl- D-glucamine (NMDG(+)) and TRIS(+) were not measurably permeant. NH(4)(+) was conducted well. We estimated the minimum pore diameter under divalent-free conditions to be between 0.32 nm and 0.55 nm. When cells were dialysed with buffered Ca(2+) solution and I(CRAC) activated by application of thapsigargin, P(Cs)/ P(Na) was still low (0.08). Outward currents through CRAC channels were carried by intracellular Na(+), K(+) and, to a much lesser extent, by Cs(+). Currents were unaffected by dialysis with Mg(2+)-free solution. The Na(+) current was inhibited by external Ca(2+) (half-maximal blocking concentration of 10 microM). This Ca(2+)-dependent block could be alleviated by hyperpolarization. The monovalent Na(+) current was voltage dependent, increasing as the holding potential depolarized above 0 mV. Our results suggest that CRAC channels in RBL-1 cells have a smaller pore diameter than voltage-operated Ca(2+) channels, discriminate between Group I cations, and differ markedly in their selectivity from CRAC channels reported in lymphocytes.     

T-Type calcium channel alpha1G and alpha1H subunits in human retinoblastoma cells and their loss after differentiation

Human retinoblastoma cells are multipotent retinal precursor cells capable of differentiating into photoreceptors, neurons, and glia. The current-voltage relation of the undifferentiated cells is dominated by a transient inward current that disappears shortly after differentiation. In 20 mM Ba(2+)-containing bath solutions, the current has an activation midpoint near -25 mV and appears to be fully inactivated at -20 mV. Sr(2+) and Ca(2+) are preferred charge carriers relative to Ba(2+), and the current vanishes in the absence of these divalent cations. Cd(2+) blocks the current with an IC(50) of 160 microM, and Ni(2+) blocks in a biphasic manner with IC(50)s of 22 and 352 microM. The current is unaffected when sodium is replaced with other monovalent cations, and it is insensitive to nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. RT-PCR revealed the presence of alpha 1G and alpha 1H mRNA in undifferentiated cells, but following differentiation, a striking reduction of both alpha 1G and alpha 1H mRNA was found, and this was paralleled by the loss of T-type Ca channel currents. alpha 1I subunit mRNA levels were low in undifferentiated and differentiated cells. These results suggest that T-type Ca channels could play a role in undifferentiated retinoblastoma cell physiology since alpha 1G and alpha 1H Ca channel subunit expression is reduced in cells that have differentiated and exited the cell cycle.     

Axonal surface charges: binding or screening by divalent cations governed by external pH

1. The effect of different extracellular alkaline-earth cations (Ca(2+), Mg(2+), Sr(2+), Ba(2+)) upon the threshold membrane potential for spike initiation in crayfish axon has been studied by means of intracellular micro-electrodes. This was done at the following pHs of the bathing saline: 6.00, 5.35, 5.00, and 4.65 +/- 0.05.2. At pH 6.00, the four alkaline earths had essentially the same effect upon the threshold membrane potential.3. At pH 5.35 or lower, it was found that equal concentrations of the alkaline-earth cations did not have the same effect on the threshold potential. The selectivity sequences observed at the different pHs were: pH 5.35, Ca(2+) > Mg(2+) > Sr(2+) >/= Ba(2+); pH 5.00, Ca(2+) approximately Ba(2+) > Sr(2+) >/= Mg(2+); pH 4.65, Ba(2+), Sr(2+) > Ca(2+) > Mg(2+).4. It is shown that the individual selectivity sequences are predicted rather closely by the equilibrium selectivity theory for alkaline-earth cations.5. It is concluded that the only difference between excitable cells which show screening and those which bind divalent cations is the net density of ionized, surface acidic groups in the region of the sodium gates.     

Electrophysiological characteristics of rat gustatory cyclic nucleotide--gated channel expressed in Xenopus oocytes

The complementary DNA encoding gustatory cyclic nucleotide--gated ion channel (or gustCNG channel) cloned from rat tongue epithelial tissue was expressed in Xenopus oocytes, and its electrophysiological characteristics were investigated using tight-seal patch-clamp recordings of single and macroscopic channel currents. Both cGMP and cAMP directly activated gustCNG channels but with markedly different affinities. No desensitization or inactivation of gustCNG channel currents was observed even in the prolonged application of the cyclic nucleotides. Single-channel conductance of gustCNG channel was estimated as 28 pS in 130 mM of symmetric Na(+). Single-channel current recordings revealed fast open-close transitions and longer lasting closure states. The distribution of both open and closed events could be well fitted with two exponential components and intracellular cGMP increased the open probability (P(o)) of gustCNG channels mainly by increasing the slower opening rate. Under bi-ionic conditions, the selectivity order of gustCNG channel among divalent cations was determined as Na(+) approximately K(+) > Rb(+) > Li(+) > Cs(+) with the permeability ratio of 1:0.95:0.74:0.63:0.49. Magnesium ion blocked Na(+) currents through gustCNG channels from both intracellular and extracellular sides in voltage-dependent manners. The inhibition constants (K(i)s) of intracellular Mg(2+) were determined as 360 +/- 40 microM at 70 mV and 8.2 +/- 1.5 mM at -70 mV with z delta value of 1.04, while K(i)s of extracellular Mg(2+) were as 1.1 +/- 0.3 mM at 70 mV and 20.0 +/- 0.1 microM at -70 mV with z delta of 0.94. Although 100 microM l-cis-diltiazem blocked significant portions of outward Na(+) currents through both bovine rod and rat olfactory CNG channels, the gustCNG channel currents were minimally affected by the same concentration of the drug.     

T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.     

The effect of metal ions and antidiuretic hormone on oxygen consumption in toad bladder

1. The sodium-dependent oxygen consumption of pieces of toad bladder (Bufo marinus) has been investigated using an oxygen electrode.2. The effect of polyvalent cations (Ca(2+), Sr(2+), Mg(2+), Eu(3+), La(3+) and Mn(2+)) on sodium-dependent oxygen consumption has been measured. All cations inhibited oxygen consumption, the order of effectiveness being Ca(2+) > Sr(2+) > Mg(2+) > Mn(2+) > Eu(3+) > La(3+).3. Treatment of bladder pieces with antidiuretic hormone (50 m-u./ml.) decreased the effectiveness of Ca(2+) and Sr(2+) as inhibitors of sodium-dependent oxygen consumption. Mn(2+), Eu(2+) and La(2+) were more effective after hormonal treatment, while the effectiveness of Mg(2+) was unaltered.4. The results have been interpreted in terms of a model in which sodium entry to the transporting mechanisms of the epithelium is controlled by Ca(2+), and in which antidiuretic hormone alters Ca(2+) binding and so affects sodium transport.     

Replacement of calcium for strontium in hamster sperm incubation media: effect on sperm function

Calcium (Ca(2+)) is an absolute requirement for a decisive sperm function event: the acrosome reaction (AR). Physiologically, sperm capacitation is a prerequisite for this specialized exocytosis and both events are intimately related. In an effort to separate capacitation from AR, we have been using a modified sperm incubation medium where Ca(2+) is replaced by Strontium (Sr(2+)). The aim of this report is to analyze with more detail the difference between sperm incubated with Ca(2+) or Sr(2+) in several events. We found that sperm undergo the capacitation-related changes in the chlortetracycline (CTC) pattern and tyrosine phosphorylation, and also bind to the zona pellucida (ZP) when using Sr(2+)-instead of Ca(2+)-containing media. However, the spontaneous AR typical of hamster sperm does not take place in Sr(2+)-medium, even if sperm are previously capacitated with Ca(2+). Nevertheless, Sr(2+) was able to sustain AR when cells were treated with thapsigargin or depolarized with K(+) in Na(+)-depleted medium. Considering that the absence of Na(+) increased spontaneous AR in Sr(2+)-medium, we tested whether Na(+)-transport systems could be involved in the inability of Sr(2+)-incubated sperm to undergo AR. We found that when sperm incubated in Sr(2+)-medium are treated with amiloride to inhibit epithelial Na(+) channel (ENaC), they are able to undergo spontaneous AR. The same result was obtained when analyzing AR on the ZP. On the contrary, addition of ouabain (a Na(+)/K(+)-ATPase inhibitor) or DIDS (a Na(+)/HCO3(-) co-transporter inhibitor) showed no effect. These results suggest that, differing from what happens in Ca(2+)-incubated sperm, cells incubated in Sr(2+)-modified medium would have an active ENaC.     

Zn(2+) slows down Ca(V)3.3 gating kinetics: implications for thalamocortical activity

We employed whole cell patch-clamp recordings to establish the effect of Zn(2+) on the gating the brain specific, T-type channel isoform Ca(V)3.3 expressed in HEK-293 cells. Zn(2+) (300 microM) modified the gating kinetics of this channel without influencing its steady-state properties. When inward Ca(2+) currents were elicited by step depolarizations at voltages above the threshold for channel opening, current inactivation was significantly slowed down while current activation was moderately affected. In addition, Zn(2+) slowed down channel deactivation but channel recovery from inactivation was only modestly changed. Zn(2+) also decreased whole cell Ca(2+) permeability to 45% of control values. In the presence of Zn(2+), Ca(2+) currents evoked by mock action potentials were more persistent than in its absence. Furthermore, computer simulation of action potential generation in thalamic reticular cells performed to model the gating effect of Zn(2+) on T-type channels (while leaving the kinetic parameters of voltage-gated Na(+) and K(+) unchanged) revealed that Zn(2+) increased the frequency and the duration of burst firing, which is known to depend on T-type channel activity. In line with this finding, we discovered that chelation of endogenous Zn(2+) decreased the frequency of occurrence of ictal-like epileptiform discharges in rat thalamocortical slices perfused with medium containing the convulsant 4-aminopyridine (50 microM). These data demonstrate that Zn(2+) modulates Ca(V)3.3 channel gating thus leading to increased neuronal excitability. We also propose that endogenous Zn(2+) may have a role in controlling thalamocortical oscillations.     

Divalent cations and the action potential of leech Retzius cells

Summary The effects of Sr, Ba, Mn, La and Co on the action potential of the leech Retzius cell were examined using intracellular recording techniques. A previous paper showed that these cells could fire Ca-dependent action potentials in Na-free solution provided TEA was present (Kleinhaus and Prichard, 1975). Under the same conditions Sr 1.5-20 mM was capable of substituting as a current carrier. Ba 2–25 mM added to normal Ringer prolonged the duration and increased the amplitude of the action potential of the Retzius cell, and supported action potentials without requiring TEA in Na-free solutions. The overshoots of the Sr- and Ba-dependent action potentials varied with a slope of 40 mM and 75 mV, respectively per 10-fold change in divalent cation concentration. Mn and La selectively blocked that portion of the action potential resulting from an inward movement of Ca, Sr or Ba without affecting the Na-dependent depolarization. The actions of Ca 1 mM on Sr-dependent action potentials were compatible with reversible competitive antagonism. In conclusion the findings: 1. support the proposition that outward K current must be blocked in order for divalent cations to dominate the Retzius cell's behavior during excitation. 2. characterize the divalent cation conductance channel as pharmacologically distinct from the Na conductance channel in the Retzius cell and similar to those described in several other excitable membranes. 3. suggest that the current carrying divalent cations probably flow through the same channel.     

T-type Ca2+ channels contribute to IBMX/forskolin- and K(+)-induced Ca(2+) transients in porcine olfactory receptor neurons

T-type Ca(2+) channels are low-voltage-activated Ca(2+) channels that control Ca(2+) entry in excitable cells during small depolarization above resting potentials. Using Ca(2+) imaging with a laser scanning confocal microscope we investigated the involvement of T-type Ca(2+) channels in IBMX/forskolin- and sparingly elevated extracellular K(+)-induced Ca(2+) transients in freshly isolated porcine olfactory receptor neurons (ORNs). In the presence of mibefradil (10microM) or Ni(2+) (100microM), the selective T-type Ca(2+) channel inhibitors, IBMX/forskolin-induced Ca(2+) transients in the soma were either strongly (>60%) inhibited or abolished completely. However, the Ca(2+) transients in the knob were only partially (<60%) inhibited. Ca(2+) transients induced by 30mM K(+) were also partially ( approximately 60%) inhibited at both the knob and soma. Furthermore, ORNs responded to as little as a 2.5mM increase in the extracellular K(+) concentration (7.5mM K(+)), and such responses were completely inhibited by mibefradil or Ni(2+). These results reveal functional expression of T-type Ca(2+) channels in porcine ORNs, and suggest a role for these channels in the spread Ca(2+) transients from the knob to the soma during activation of the cAMP cascade following odorant binding to G-protein-coupled receptors on the cilia/knob of ORNs.     

Identical unitary current amplitude and Ca(2+) block of cardiac Na channel before and during beta-adrenergic stimulation

We examined the possibility of Ca(2+) permeation through cardiac Na channels ("slip mode conductance") by an analysis of the voltage-dependent block of Na channels by Ca(2+). A Ca(2+) block of Na channels was evident in rat and guinea pig ventricular myocytes during cell-attached single channel recordings with a physiological ionic environment (140 mM Na(+) and 1 to 10 mM Ca(2+) in the pipette solution). Increasing external Ca(2+) concentration ([Ca(2+)](o)) in the pipette solution reduced the unitary current amplitude predominantly at negative potentials. With [Ca(2+)](o) > 1 mM, unitary current amplitude did not increase at potentials negative to -40 mV in spite of augmented driving forces. The application of 5 microM isoproterenol potentiated the single channel activity elicited by depolarizing pulses from the holding potential of -120 mV, indicating that the channels in the patch under examination were modified by protein kinase A (PKA) stimulation. Increased activity was also confirmed with veratridine-modified Na channels, where channel openings were markedly prolonged. In either case, isoproterenol-induced potentiation neither reduced nor altered the properties of Ca(2+) block of cardiac Na channels, as evidenced by the stable unitary current amplitudes at potential levels from -60 to -20 mV. These results indicate that interactions among Na(+), Ca(2+), and the channel molecule were not modified with respect to permeation properties. They therefore argue against the "slip mode" concept of classical cardiac Na channel if a general concept of ion permeation through "multi-ion pores" is applicable to determine the ionic selectivity of Na channels.     

Two components of the calcium current in the egg cell membrane of the tunicate.

The Ca current of the egg cell membrane of a certain tunicate, Halocynthia roretzi Drasche, was studied by the voltage-clamp technique. 2. The Ca current in the standard artificial sea water (ASW) was produced at the critical membrane potential of -10 mV after inactivating the Na current by conditioning depolarization, -30 to -15 mV. The Ca current was abolished by replacing Ca in ASW with Mg2+ or Mn2+. The Ca current was not significantly influenced by replacing Na in ASW with choline or Cs. 3. The relation of Ca current to the external Ca concentration was a monotonously increasing function, but was not linear. The current tended to saturate above 50 mM-Ca. In 100 mM-Ca ASW, the maximum peak inward current of Ca ranged from 1 to 7 X 10(-9) A. 4. The kinetics of Ca current was accurately analysed because of the small contribution of K outward current and was found to be relatively slow in comparison with the Na current. The peak time and the half-decay time of the maximum Ca current at about 25 mV were about 25 and 100 msec respectively in 100 mM-Ca ASW at 15 degrees C. 5. Addition of 20 mM-Co2+ to 100 mM-Ca ASW reduced Ca current to one fourth and 1 mM-La3+ to 100 mM-Ca ASW abolished the current. 6. Sr and Ba could substitute for Ca in Ca channels. The selectivity ratios for the 'Ca channels's were Ca (1-00):Sr(1-17):Ba(0-71) at a potential level of +40 mV. The Ca current in the egg cell membrane appeared to be essentially the same as the Ca current in the common excitable membranes, such as the crustacean muscle fibre. 7. The polyvalent cations including Ca ion and monovalent H+ ion showed the stabilizing effect upon both Na and Ca currents, by shifting V-I relations along the voltage axis. From the prediction of a theory of the diffuse double layer, the shift in the V-I relation induced by those cations should be directly related to their binding powers to the membrane. Thus, the sequence of the binding powers was inferred as H+ greater than La3+ greater than Co2+ greater than Mn2+ greater than Ca2+ greater than Sr2+ larger than or equal to Ba2+ greater than Mg2+. 9. In Na-free ASW, such as isotonic Ca ASW, Ca current was composed of two components. The one component was the Ca current described in 1 to 6. The other was also dependent upon the external Ca concentration, but showed the more negative critical membrane potential and the faster kinetics. It was concluded that this component should be the Ca current through Na channels. 10. The selectivity among Ca, Sr and Ba for 'Ca' current through 'Na channels' was significantly different from that of 'Ca' current through 'Ca channels', being Ca greater than Sr larger than or equal to Ba = 0.     

Proton conductance of human transient receptor potential-vanilloid type-1 expressed in oocytes of Xenopus laevis and in Chinese hamster ovary cells

Transient receptor potential-vanilloid type-1 (TRPV1) is a ligand-gated cation channel with preference for divalent cations, especially Ca(2+) (sequence of conductances: Ca(2+)>Mg(2+)>Na(+) approximately/= K(+) approximately/= Cs(+)). In the present study, the two-electrode voltage-clamp technique was used on oocytes of Xenopus laevis expressing TRPV1 to evaluate whether human TRPV1 also conducts protons. In medium devoid of K(+), Na(+), Mg(2+), and Ca(2+), capsaicin 1 microM induced a significant inward current (62% of the current in physiological medium). The effects of capsaicin were abolished in the presence of capsazepine 3 microM. The capsaicin-induced currents in medium devoid of Na(+), K(+), Mg(2+), and Ca(2+) were dependent on pH, causing larger inward currents and less negative reversal potentials at low pH and vice versa. The same current was also demonstrated in Chinese hamster ovary cells expressing human TRPV1. We conclude that TRPV1 conducts protons, in addition to Na(+), K(+), Mg(2+), and Ca(2+). The proton conductance may help to initiate action potentials and to translocate H(+) dependent on TRPV1 activation and membrane potential.     

Sodium/calcium exchanger: influence of metabolic regulation on ion carrier interactions

The Na(+)/Ca(2+) exchanger's family of membrane transporters is widely distributed in cells and tissues of the animal kingdom and constitutes one of the most important mechanisms for extruding Ca(2+) from the cell. Two basic properties characterize them. 1) Their activity is not predicted by thermodynamic parameters of classical electrogenic countertransporters (dependence on ionic gradients and membrane potential), but is markedly regulated by transported (Na(+) and Ca(2+)) and nontransported ionic species (protons and other monovalent cations). These modulations take place at specific sites in the exchanger protein located at extra-, intra-, and transmembrane protein domains. 2) Exchange activity is also regulated by the metabolic state of the cell. The mammalian and invertebrate preparations share MgATP in that role; the squid has an additional compound, phosphoarginine. This review emphasizes the interrelationships between ionic and metabolic modulations of Na(+)/Ca(2+) exchange, focusing mainly in two preparations where most of the studies have been carried out: the mammalian heart and the squid giant axon. A surprising fact that emerges when comparing the MgATP-related pathways in these two systems is that although they are different (phosphatidylinositol bisphosphate in the cardiac and a soluble cytosolic regulatory protein in the squid), their final target effects are essentially similar: Na(+)-Ca(2+)-H(+) interactions with the exchanger. A model integrating both ionic and metabolic interactions in the regulation of the exchanger is discussed in detail as well as its relevance in cellular Ca(i)(2+) homeostasis.     

Properties of a Ca(2+)-activated large conductance K(+) channel with ATP sensitivity in human renal proximal tubule cells

The properties of a native Ca(2+)-activated large conductance K(+) channel (BK channel) present in the surface membrane of cultured human renal proximal tubule epithelial cells (RPTECs) were investigated by using the patch-clamp technique. The slope conductance of the BK channel was about 295 pS, and the channel was selective to K(+) over Na(+), with a selectivity ratio of about 12.2. The activity of the channel was almost maximally enhanced by 10(-4 )M or more Ca(2+) in the cytoplasmic surface of the patch membrane and was markedly diminished by reducing the cytoplasmic Ca(2+) to 10(-6) M at the membrane potential of about 0 mV. The depolarization of the patch membrane also enhanced the channel activity, and hyperpolarization lowered it. K(+) channel blockers, Ba(2+) (0.1-1 mM), tetraethylammonium (1 mM), and charybdotoxin (100 nM), were effective for the suppression of channel activity. A significant feature of the K(+) channel was that channel activity maintained by 10(-5)-10(-4 )M Ca(2+) in inside-out patches was inhibited by the addition of ATP (1-10 mM) to the bath solution. ATPgammaS, and a nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), also had inhibitory effects on channel activity. However, an inhibitor of ATP-sensitive K(+) channels, glibenclamide (0.1 mM), induced no appreciable change in channel activity from both intra- and extracellular sides. These results suggest that besides the common natures of the BK channel family such as regulation by cytoplasmic Ca(2+) and membrane potential, the BK channel in RPTECs is directly inhibited by intracellular ATP independent of phosphorylation processes and sulfonylurea receptor.     

Inactivation properties of human recombinant class E calcium channels

The electrophysiological and pharmacological properties of alpha(1E)-containing Ca(2+) channels were investigated by using the patch-clamp technique in the whole cell configuration, in HEK 293 cells stably expressing the human alpha(1E) together with alpha(2b) and beta(1b) accessory subunits. These channels had current-voltage (I-V) characteristics resembling those of high-voltage-activated (HVA) Ca(2+) channels (threshold at -30 mV and peak amplitude at +10 mV in 5 mM Ca(2+)). The currents activated and deactivated with a fast rate, in a time- and voltage-dependent manner. No difference was found in their relative permeability to Ca(2+) and Ba(2+). Inorganic Ca(2+) channel blockers (Cd(2+), Ni(2+)) blocked completely and potently the alpha(1E,)/alpha(2b)delta/beta(1b) mediated currents (IC(50) = 4 and 24.6 microM, respectively). alpha(1E)-mediated currents inactivated rapidly and mainly in a non-Ca(2+)-dependent manner, as evidenced by the fact that 1) decreasing extracellular Ca(2+) from 10 to 2 mM and 2) changing the intracellular concentration of the Ca(2+) chelator 1. 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), did not affect the inactivation characteristics; 3) there was no clear-cut bell-shaped relationship between test potential and inactivation, as would be expected from a Ca(2+)-dependent event. Although Ba(2+) substitution did not affect the inactivation of alpha(1E) channels, Na(+) substitution revealed a small but significant reduction in the extent and rate of inactivation, suggesting that besides the presence of dominant voltage-dependent inactivation, alpha(1E) channels are also affected by a divalent cation-dependent inactivation process. We have analyzed the Ca(2+) currents produced by a range of imposed action potential-like voltage protocols (APVPs). The amplitude and area of the current were dependent on the duration of the waveform employed and were relatively similar to those described for HVA calcium channels. However, the peak latency resembled that obtained for low-voltage-activated (LVA) calcium channels. Short bursts of APVPs applied at 100 Hz produced a depression of the Ca(2+) current amplitude, suggesting an accumulation of inactivation likely to be calcium dependent. The human alpha(1E) gene seems to participate to a Ca(2+) channel type with biophysical and pharmacological properties partly resembling those of LVA and those of HVA channels, with inactivation characteristics more complex than previously believed.