Single-channel recordings of two types of calcium channels in rat pancreatic beta-cells.

Using the cell-attached configuration of the patch clamp technique, we have identified two different types of Ca channels in rat pancreatic beta-cell membranes. The two channels differ in single channel conductance, voltage dependence, and inactivation properties. The single-channel conductance, measured with 100 mM Ba2+ in the pipette, was 21.8 pS for the large channel and 6.4 pS for the small channel. The large-conductance channel is similar to the fast deactivating or L-type Ca channel described in other preparations. It is voltage dependent, has a threshold for activation around -30 mV, and can be activated from a holding potential of -40 mV. On the other hand, the small-conductance Ca channel is similar to the SD or T type Ca channel; it has a lower activation threshold, around -50 mV, and it can be inactivated by holding the membrane potential at -40 mV.     

Calcium modulation of mitochondrial inner membrane channel activity.

Protocols were defined that enable patch-clamp studies of the approximately 107 pS voltage dependent channel and a class of activity we refer to as MCC (multiconductance channel) which is characterized by multiple levels and transitions as high as 1 to 1.5 nS. If free calcium was kept at 10(-7) M or lower during mitochondrial isolation, no activity was observed at low voltage (+/- 60 mV). If free calcium levels were higher, MCC activity was observed in about 96% of the patches. The observation of approximately 107 pS channel was enhanced from 2% to 68% of patches by washing isolated mitoplasts (mitochondria stripped of outer membrane) with EGTA. Increasing matrix calcium from 10(-9) to 10(-5) M decreased the probability of opening for the MCC and approximately 107 pS activities.     

Kinetic study of N-type calcium current modulation by delta-opioid receptor activation in the mammalian cell line NG108-15

The voltage-dependent inhibition of N-type Ca2+ channel current by the delta-opioid agonist [D-pen2, D-pen5]-enkephalin (DPDPE) was investigated in the mammalian cell line NG108-15 with 10 microM nifedipine to block L-type channels, with whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells DPDPE reversibly decreased omega-conotoxin GVIA-sensitive Ba2+ currents in a concentration-dependent way. Inhibition was maximal with 1 microM DPDPE (66% at 0 mV) and was characterized by a slowing of Ba2+ current activation at low test potentials. Both inhibition and kinetic slowing were attenuated at more positive potentials and could be relieved up to 90% by strong conditioning depolarizations. The kinetics of removal of inhibition (de-inhibition) and of its retrieval (re-inhibition) were also voltage dependent. Both de-inhibition and re-inhibition were single exponentials and, in the voltage range from -20 to +10 mV, had significantly different time constants at a given membrane potential, the time course of re-inhibition being faster than that of de-inhibition. The kinetics of de-inhibition at -20 mV and of reinhibition at -40 mV were also concentration dependent, both processes becoming slower at lower agonist concentrations. The rate of de-inhibition at +80/+120 mV was similar to that of Ca2+ channel activation at the same potentials measured during application of DPDPE (approximately 7 ms), both processes being much slower than channel activation in controls (<1 ms). Moreover, the amplitude but not the time course of tail currents changed as the depolarization to +80/+120 mV was made longer. The state-dependent properties of DPDPE Ca2+ channel inhibition could be simulated by a model that assumes that inhibition by DPDPE results from voltage- and concentration-dependent binding of an inhibitory molecule to the N-type channel.     

Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.     

Recording of single K+ channels in the membrane of cytoplasmic drop ofChara australis

Summary The cytoplasmic drop formed of effused cytoplasm fromChara internodes is enclosed by a membrane. Patch clamp experiments have been carried out on this membrane, revealing a K⁺ channel as the most frequently detected ion translocator. The K⁺ channel is saturated at a level of about 20 pA inward and 10 pA outward current. The channel conductance is dependent on the accessability of K⁺ ions, its maximum value amounts to about 165 pS. The discrimination of Na⁺ and Cl^– is significant, permeability ratios P_Na/P_K and P_Cl/P_K were estimated to be 0.01 either. Binding experiments with the fluorescent probe concanavalin A/FITC suggest that the membrane is derived from the tonoplast.Abbreviations E_K K⁺ equilibrium potential - FITC fluorescein isothiocyanat - V_m membrane voltage - V_pip pipette clamp voltage - V_r reversal voltage     

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

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

Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide

A COOH-terminal tryptic fragment (Mr approximately equal to 20,000) of colicin E1 has been proposed to contain the membrane channel-forming domain of the colicin molecule. A comparison is made of the conductance properties of colicin E1 and its COOH-terminal fragment in planar bilayer membranes. The macroscopic and single channel properties of colicin E1 and its COOH-terminal tryptic fragment are very similar, if not indistinguishable, implying that the NH2-terminal, two-thirds of the colicin E1 molecule, does not significantly influence its channel properties. The channel-forming activity of both polypeptides is dependent upon the presence of a membrane potential, negative on the trans side of the membrane. The average single channel conductance of colicin E1 and the COOH-terminal fragment is 20.9 +/- 3.9 and 19.1 +/- 2.9 picosiemens, respectively. The rate at which both proteins form conducting channels increases as the pH is lowered from 7 to 5. Both molecules require negatively charged lipids for activity to be expressed, exhibit the same ion selectivity, and rectify the current to the same extent. Both polypeptides associate irreversibly with the membrane in the absence of voltage, but subsequent formation of conducting channels requires a negative membrane potential.     

Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria

Summary We have incorporated into planar lipid bilayer membranes a voltage-dependent, anion-selective channel (VDAC) obtained fromParamecium aurelia. VDAC-containing membranes have the following properties: (1) The steady-state conductance of a many-channel membrane is maximal when the transmembrane potential is zero and decreases as a steep function of both positive and negative voltage. (2) The fraction of time that an individual channel stays open is strongly voltage dependent in a manner that parallels the voltage dependence of a many-channel membrane. (3) The conductance of the open channel is about 500 pmho in 0.1 to 1.0m salt solutions and is ohmic. (4) The channel is about 7 times more permeable to Cl^– than to K⁺ and is impermeable to Ca⁺⁺. The procedure for obtaining VDAC and the properties of the channel are highly reproducible.VDAC activity was found, upon fractionation of the paramecium membranes, to come from the mitochondria. We note that the published data on mitochondrial Cl^– permeability suggest that there may indeed be a voltage-dependent Cl^– permeability in mitochondria.The method of incorporating VDAC into planar lipid bilayers may be generally useful for reconstituting biological transport systems in these membranes.     

Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.     

Reconstitution of ionic channels from inner and outer membranes of mammalian cardiac nuclei.

Recent reports suggest that the nuclear envelope possesses specific ion transport mechanisms that regulate the electrolyte concentrations within the nucleoplasm and perinuclear space. In this work, intact nuclei were isolated from sheep cardiac cells. After chromatin digestion, the nuclear envelopes were sonicated and four nuclear vesicle populations were separated by sucrose step gradients (SF1-SF4). These fractions were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their protein content was analyzed by Western blot, using lamin and SEC 61 antibodies. The lamins, which are associated with the inner nuclear membrane, were present in three fractions, SF2, SF3, and SF4, with a lower amount in SF2. The SEC 61 protein, a marker of the rough endoplasmic reticulum, was detected in small amounts in SF1 and SF2. Upon fusion of vesicles into bilayers, the activities of nuclear ionic channels were recorded in 50 mM trans/250 mM cis KCl or CsCl, pH 7.2. Two types of Cl- selective channels were recorded: a large conducting 150-180-pS channel displaying substates, and a low conducting channel of 30 pS. They were both spontaneously active into bilayers, and their open probability was poorly voltage dependent at negative voltages. Retinoic acid (10(-8) M) increases the po of the large Cl- conducting channel, whereas ATP modifies the kinetics of the low conductance anion selective channel. Our data also suggest that this anionic channel is mainly present in the SF3 and SF4 population. The presence of a 181 +/- 10 pS cation-selective channel was consistently observed in the SF2 population. The behavior of this channel was voltage dependent in the voltage range -80 to +60 mV. Furthermore, we report for the first time the activity of a channel exclusively present in the SF3 and SF4 fractions, shown to contain mainly inner membrane vesicles. This cation selective channel displays a 75-pS conductance in 50 mM trans/250 mM cis K-gluconate. It is concluded that the bilayer reconstitution technique is an attractive approach to studying the electrophysiological properties of the inner and outer membranes of the nuclear envelope.     

Statistical analysis of single sodium channels. Effects of N-bromoacetamide

Currents were obtained from single sodium channels in outside-out excised patches of membrane from the cell line GH3. The currents were examined in control patches and in patches treated with N- bromoacetamide ( NBA ) to remove inactivation. The single-channel current-voltage relationship was linear over the range -60 to + 10 mV, and was unaffected by NBA . The slope conductance at 9.3 degrees C was 12 pS, and the Q10 for single channel currents was about 1.35. The currents in both control and NBA -treated patches showed evidence of a slow process similar to desensitization in acetylcholine-receptor channels. This process was especially apparent at rapid rates of stimulation (5 Hz), where openings occurred in clusters of records. The clustering of records with and without openings was analyzed by runs analysis, which showed a statistically significant trend toward nonrandom ordering in the responses of channels to voltage pulses. NBA made this nonrandom pattern more apparent. The probability that an individual channel was "hibernating" during an activating depolarization was estimated by a maximum likelihood method. The lifetime of the open state was also estimated by a maximum likelihood method, and was examined as a function of voltage. In control patches the open time was mildly voltage-dependent, showing a maximum at about -50 mV. In NBA -treated patches the open time was greater than in the control case and increased monotonically with depolarization; it asymptotically approached that of the control patches at hyperpolarized potentials. By comparing channel open times in control and NBA -treated patches, we determined beta A and beta I, the rate constants for closing activation gates and fast inactivation gates. Beta I was an exponential function of voltage, increasing e-fold for 34 mV. beta A had the opposite voltage dependence. The probability of an open channel closing its fast inactivation gate, rather than its activation gate, increased linearly with depolarization from -60 to -10 mV. These results indicate that inactivation is inherently voltage dependent.     

Comparison of excitatory currents activated by different transmitters on crustacean muscle. I. Acetylcholine-activated channels

The properties of acetylcholine-activated excitatory currents on the gm1 muscle of three marine decapod crustaceans, the spiny lobsters Panulirus argus and interruptus, and the crab Cancer borealis, were examined using either noise analysis, analysis of synaptic current decays, or analysis of the voltage dependence of ionophoretically activated cholinergic conductance increases. The apparent mean channel open time (tau n) obtained from noise analysis at -80 mV and 12 degrees C was approximately 13 ms; tau n was prolonged e-fold for about every 100-mV hyperpolarization in membrane potential; tau n was prolonged e- fold for every 10 degrees C decrease in temperature. Gamma, the single- channel conductance, at 12 degrees C was approximately 18 pS and was not affected by voltage; gamma was increased approximately 2.5-fold for every 10 degrees C increase in temperature. Synaptic currents decayed with a single exponential time course, and at -80 mV and 12 degrees C, the time constant of decay of synaptic currents, tau ejc, was approximately 14-15 ms and was prolonged e-fold about every 140-mV hyperpolarization; tau ejc was prolonged about e-fold for every 10 degrees C decrease in temperature. The voltage dependence of the amplitude of steady-state cholinergic currents suggests that the total conductance increase produced by cholinergic agonists is increased with hyperpolarization. Compared with glutamate channels found on similar decapod muscles (see the following article), the acetylcholine channels stay open longer, conduct ions more slowly, and are more sensitive to changes in the membrane potential.     

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).     

An inwardly rectifying chloride channel in ragweed-sensitized canine tracheal epithelial cells

The single channel inside-out patch clamp technique was used to characterize ion channels in the apical membranes of ragweed-sensitized and control canine tracheal epithelial cells maintained in primary culture. Patches were obtained from single isolated cells or from cells at the edges of confluent sheets. A new type of chloride channel was seen in sensitized cells but not in control cells. The channel showed inward rectification in symmetric chloride solutions with conductance varying from 95 pS to 52 pS over the range of –60 mV to 60 mV membrane potential. Channel gating was voltage dependent with maximal opening at about –30 mV Kinetic analysis showed that distributions of closed and open times could both be well fitted by the sums of three exponential components. Rate constants for transitions between the states of a linear kinetic model were calculated, with only one rate being significantly voltage dependent. The possible significance of this channel is discussed. Offprint requests to: A. S. French     

Multiple conductance channel activity of wild-type and voltage-dependent anion-selective channel (VDAC)-less yeast mitochondria.

Yeast mitoplasts (mitochondria with the outer membrane stripped away) exhibit multiple conductance channel activity (MCC) in patch-clamp experiments that is very similar to the activity previously described in mammalian mitoplasts. The possible involvement of the voltage-dependent anion-selective channel (VDAC) of the outer membrane in MCC activity was explored by comparing the channel activity in wild-type yeast mitoplasts with that of a VDAC-deletion mutant. The channel activity recorded from the mutant is essentially the same as that of the wild-type in the voltage range of -40 to 30 mV. These observations indicate that VDAC is not required for MCC activity. Interestingly, the channel activity of the VDAC-less yeast mitoplasts exhibits altered gating properties at transmembrane potentials above and below this range. We conclude that the deletion of VDAC somehow results in a modification of MCC's voltage dependence. In fact, the voltage profile recorded from the VDAC-less mutant resembles that of VDAC.     

Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.

Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.     

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

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

Voltage-dependent inactivation of T-tubular skeletal calcium channels in planar lipid bilayers

Single-channel properties of dihydropyridine (DHP)-sensitive calcium channels isolated from transverse tubular (T-tube) membrane of skeletal muscle were explored. Single-channel activity was recorded in planar lipid bilayers after fusion of highly purified rabbit T-tube microsomes. Two populations of DHP-sensitive calcium channels were identified. One type of channel (noninactivating) was active (2 microM +/- Bay K 8644) at steady-state membrane potentials and has been studied in other laboratories. The second type of channel (inactivating) was transiently activated during voltage pulses and had a very low open probability (Po) at steady-state membrane potentials. Inactivating channel activity was observed in 47.3% of the experiments (n = 84 bilayers). The nonstationary kinetics of this channel was determined using a standard voltage pulse (HP = -50 mV, pulse to 0 mV). The time constant (tau) of channel activation was 23 ms. During the mV). The time constant (tau) of channel activation was 23 ms. During the pulse, channel activity decayed (inactivated) with a tau of 3.7 s. Noninactivating single-channel activity was well described by a model with two open and two closed states. Inactivating channel activity was described by the same model with the addition of an inactivated state as proposed for cardiac muscle. The single-channel properties were compared with the kinetics of DHP-sensitive inward calcium currents (ICa) measured at the cellular level. Our results support the hypothesis that voltage-dependent inactivation of single DHP-sensitive channels contributes to the decay of ICa.     

Electro-pharmacological profile of a mitochondrial inner membrane big-potassium channel from rat brain

Recent studies have indicated a calcium-activated large conductance potassium channel in rat brain mitochondrial inner membrane (mitoBK channel). Accordingly, we have characterized the functional and pharmacological profile of a BK channel from rat brain mitochondria in the present study. Brain mitochondrial inner membrane preparations were subjected to SDS-PAGE analysis and channel protein reconstitution into planar lipid bilayers. Western blotting and antibodies directed against various cellular proteins revealed that mitochondrial inner membrane fractions did not contain specific proteins of the other subcellular compartments except a very small fraction of endoplasmic reticulum. Channel incorporation into planar lipid bilayers revealed a voltage dependent 211 pS potassium channel with a voltage for half activation (V(1/2)) of 11.4±1.1mV and an effective gating charge z(d) of 4.7±0.9. Gating and conducting behaviors of this channel were unaffected by the addition of 2.5mM ATP, and 500 nM charybdotoxin (ChTx), but the channel appeared sensitive to 100 nM iberiotoxin (IbTx). Adding 10mM TEA at positive potentials and 10mM 4-AP at negative or positive voltages inhibited the channel activities. These results demonstrate that the mitoBK channel, present in brain mitochondrial inner membrane, displays different pharmacological properties than those classically described for plasma membrane, especially in regard to its sensitivity to iberiotoxin and charybdotoxin sensitivity.     

A long-lasting electrically mediated block, due to the egg membrane hyperpolarization at fertilization, ensures physiological monospermy in eggs of the crab Maia squinado

In response to fertilization, the membrane potential (Em) of the crab egg hyperpolarizes from about -50 mV to about -80 mV in 400 msec. To establish whether this fast hyperpolarization is correlated with physiological polyspermy or conversely mediates an electrical block to polyspermy, we examined the morphological and electrophysiological characteristics of eggs from the crab Maia squinado. Fertilized naturally spawned eggs were found to be physiologically monospermic and their average Em was constant at -77 +/- 0.5 mV. To examine a possible electrical block ensuring this monospermy, unfertilized eggs were voltage clamped at various Em values ranging from +20 to -90 mV, inseminated, and examined morphologically. All eggs clamped at +20 to -65 mV responded by developing a fertilization current, If. It consisted of an outwardly directed K+ current in one or several steps, each caused by a single spermatozoon interacting with the egg membrane. The percentage of eggs clamped at values more negative than -65 mV, which responded at insemination by developing an If, decreased and dropped to 0 at -80 mV. This indicated that the membrane processes occurring during the contact between gametes and eliciting an electrical response by the egg membrane are voltage dependent. Further, the spermatozoon never penetrated into eggs voltage clamped at a Em between +20 and -60 mV and at voltages more negative than -75 mV. Em values between -65 and -75 mV were required for spermatozoon incorporation into the egg, indicating that sperm entry is also voltage dependent. It is proposed that the hyperpolarization of the egg membrane in response to fertilization constitutes a long-lasting electrical block to polyspermy in crab eggs.