Single sodium channels from the squid giant axon

Since the work of A. L. Hodgkin and A. F. Huxley (1952. J. Physiol. [Lond.].117:500-544) the squid giant axon has been considered the classical preparation for the study of voltage-dependent sodium and potassium channels. In this preparation much data have been gathered on macroscopic and gating currents but no single sodium channel data have been available. This paper reports patch clamp recording of single sodium channel events from the cut-open squid axon. It is shown that the single channel conductance in the absence of external divalent ions is approximately 14 pS, similar to sodium channels recorded from other preparations, and that their kinetic properties are consistent with previous results on gating and macroscopic currents obtained from the perfused squid axon preparation.     

Mechanism of blockage of amphotericin B channels in a lipid bilayer.

A number of organic compounds (non-electrolytes, tetraalkylammonia, etc.) with a molecular size of 6--8 angstrom decrease the conductance of ionic channels formed in the lipid bilayer by a polyene antibiotic amphotericin B. It is suggested that these compounds, upon entering the channel, block the passage of inorganic ions. The extent of conductance blockage by organic ions depends on the membrane potential and electrolyte concentration. In the presence of ionic blockers, for instance tetraethylammonium, amphotericin B-containing membranes assume some properties characteristic of excitable membranes, i.e. the current-voltage characteristic acquires the negative resistance region, and in response to a potential step activation followed by inactivation of conductance is observed. It is shown that the potential dependence of the blockage is due to interaction inside the channel of the blocker ion with penetrating ions, by a mechanism similar to that described by Armstrong ((1979) Q. Rev. Biophys. 7, 179--210) for blockage of squid axon potassium channels by ammonium derivatives.     

Survival of K+ permeability and gating currents in squid axons perfused with K+-free media

K+ currents were recorded in squid axons internally perfused with impermeant electrolyte. Total absence of permeant ions inside and out leads to an irreversible loss of potassium conductance with a time constant of approximately 11 min at 8 degrees C. Potassium channels can be protected against this effect by external K+, Cs+, NH4+, and Rb+ at concentrations of 100-440 mM. These experiments suggest that a K+ channel is normally occupied by one or more small cations, and becomes nonfunctional when these cations are removed. A large charge movement said to be related to K+ channel gating in frog skeletal muscle is absent in squid giant axons. However, deliberate destruction of K+ conductance by removal of permeant cations is accompanied by measurable loss in asymmetric charge movement. This missing charge component is large enough to contain a contribution from K+ gating charge movements of more than five elementary charges per channel.     

Analysis of sodium current fluctuations in the cut-open squid giant axon

Patch pipettes were used to record the current arising from small populations of sodium channels in voltage-clamped cut-open squid axons. The current fluctuations associated with the time-variant sodium conductance were analyzed with nonstationary statistical techniques in order to obtain an estimate for the conductance of a single sodium channel. The results presented support the notion that the open sodium channel in the squid axon has only one value of conductance, 3.5 pS.     

On the voltage-dependent action of tetrodotoxin.

The use of the maximum rate-of-rise of the action potential (Vmax) as a measure of the sodium conductance in excitable membranes is invalid. In the case of membrane action potentials, Vmax depends on the total ionic current across the membrane; drugs or conditions that alter the potassium or leak conductances will also affect Vmax. Likewise, long-term depolarization of the membrane lessens the fraction of total ionic current that passes through the sodium channels by increasing potassium conductance and inactivating the sodium conductance, and thereby reduces the effect of Vmax of drugs that specifically block sodium channels. The resultant artifact, an apparent voltage-dependent potency of such drugs, is theoretically simulated for the effects of tetrodotoxin on the Hodgkin-Huxley squid axon.     

Cholesterol influences voltage-gated calcium channels and BK-type potassium channels in auditory hair cells

The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitability are unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassium current by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (~30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.     

Ionic conductances of squid giant fiber lobe neurons

The cell bodies of the neurons in the giant fiber lobe (GFL) of the squid stellate ganglion give rise to axons that fuse and thereby form the third-order giant axon, whose initial portion functions as the postsynaptic element of the squid giant synapse. We have developed a preparation of dissociated, cultured cells from this lobe and have studied the voltage-dependent conductances using patch-clamp techniques. This system offers a unique opportunity for comparing the properties and regional differentiation of ionic channels in somatic and axonal membranes within the same cell. Some of these cells contain a small inward Na current which resembles that found in axon with respect to tetrodotoxin sensitivity, voltage dependence, and inactivation. More prominent is a macroscopic inward current, carried by Ca2+, which is likely to be the result of at least two kinetically distinct types of channels. These Ca channels differ in their closing kinetics, voltage range and time course of activation, and the extent to which their conductance inactivates. The dominant current in these GFL neurons is outward and is carried by K+. It can be accounted for by a single type of voltage-dependent channel. This conductance resembles the K conductance of the axon, except that it partially inactivates during relatively short depolarizations. Ensemble fluctuation analysis of K currents obtained from excised outside-out patches is consistent with a single type of K channel and yields estimates for the single channel conductance of approximately 13 pS, independently of membrane potential. A preliminary analysis of single channel data supports the conclusion that there is a single type of voltage-dependent, inactivating K channel in the GFL neurons.     

Chemically induced K+ conduction noise in squid axon

Internal perfusion of tetraethylammonium ions (TEA) in squid axons produces a significant high frequency noise component. Although internal TEA suppresses the potassium conductance (G _K) noise at relatively low frequencies, it induces high frequency noise which exceeds the intensity of the normal potassium and sodium noise. In addition, the induced noise is dependent on the presence of internal potassium ions (K⁺) suggesting that this source of noise arises from a modulation of the K⁺ conductance due to the blocking and unblocking of the K⁺ channel. The simplest model describing the TEA data is a two-step sequential pseudo-unimolecular reaction where TEA binds during an open conductance state. A unit channel conductance of 2 pS is estimated from the TEA data as well as noise induced by triethyldecylammonium (TEDA) ions. Thus, these data are consistent with the hypothesis that the channel is blocked whenever the quaternary ammonium ion binding site, located near or within the K⁺ channel, is occupied.     

Molecular identification of SqKv1A. A candidate for the delayed rectifier K channel in squid giant axon

We have cloned the cDNA for a squid Kvl potassium channel (SqKv1A). SqKv1A mRNA is selectively expressed in giant fiber lobe (GFL) neurons, the somata of the giant axons. Western blots detect two forms of SqKv1A in both GFL neuron and giant axon samples. Functional properties of SqKv1A currents expressed in Xenopus oocytes are very similar to macroscopic currents in GFL neurons and giant axons. Macroscopic K currents in GFL neuron cell bodies, giant axons, and in Xenopus oocytes expressing SqKv1A, activate rapidly and inactivate incompletely over a time course of several hundred ms. Oocytes injected with SqKv1A cRNA express channels of two conductance classes, estimated to be 13 and 20 pS in an internal solution containing 470 mM K. SqKv1A is thus a good candidate for the "20 pS" K channel that accounts for the majority of rapidly activating K conductance in both GFL neuron cell bodies and the giant axon.     

Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.     

Stretch-activated cation channels in human fibroblasts.

Nonconfluent fibroblasts are relatively depolarized when compared with confluent fibroblasts, and transient hyperpolarizations result from a range of external stimuli as well as internal cellular activities. This electrical activity ceases, along with growth and mitogenic activity, when the cells become confluent. A calcium-activated potassium conductance is thought to be responsible for these hyperpolarizations, but in human fibroblasts the large calcium-activated potassium channel is not stretch-activated. We report here the identification of single stretch-activated cation channels in human fibroblasts, using the cell-attached and inside-out patch clamp techniques. The most prominent channel had a conductance of approximately 60 pS (picoSeimens) in 140 mM potassium and was permeable to potassium and sodium. The channel showed significant adaptation of activity when stretch was maintained over a period of several seconds, but a static component persisted for much longer periods. Higher conductance channels were also observed in a few excised patches.     

Single channel studies of the ATP-regulated potassium channel in brain mitochondria

Mitochondrial potassium channels in the brain have been suggested to have an important role in neuroprotection. The single channel activity of mitochondrial potassium channels was measured after reconstitution of the purified inner membrane from rat brain mitochondria into a planar lipid bilayer. In addition to a large conductance potassium channel that was described previously, we identified a potassium channel that has a mean conductance of 219 ± 15 pS. The activity of this channel was inhibited by ATP/Mg²⁺ and activated by the potassium channel opener BMS191095. Channel activity was not influenced either by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-regulated potassium channels, or by the plasma membrane ATP-regulated potassium channel blocker HMR1098. Likewise, this mitochondrial potassium channel was unaffected by the large conductance potassium channel inhibitor iberiotoxin or by the voltage-dependent potassium channel inhibitor margatoxin. The amplitude of the conductance was lowered by magnesium ions, but the opening ability was unaffected. Immunological studies identified the Kir6.1 channel subunit in the inner membrane from rat brain mitochondria. Taken together, our results demonstrate for the first time the single channel activity and properties of an ATP-regulated potassium channel from rat brain mitochondria.     

Potassium conductance of the squid giant axon. Single-channel studies

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.     

Proton block of the pore underlies the inhibition of hERG cardiac K+ channels during acidosis

Human ether-a-go-go-related gene (hERG) potassium channels are critical determinants of cardiac repolarization. Loss of function of hERG channels is associated with Long QT Syndrome, arrhythmia, and sudden death. Acidosis occurring as a result of myocardial ischemia inhibits hERG channel function and may cause a predisposition to arrhythmias. Acidic pH inhibits hERG channel maximal conductance and accelerates deactivation, likely by different mechanisms. The mechanism underlying the loss of conductance has not been demonstrated and is the focus of the present study. The data presented demonstrate that, unlike in other voltage-gated potassium (Kv) channels, substitution of individual histidine residues did not abolish the pH dependence of hERG channel conductance. Abolition of inactivation, by the mutation S620T, also did not affect the proton sensitivity of channel conductance. Instead, voltage-dependent channel inhibition (δ = 0.18) indicative of pore block was observed. Consistent with a fast block of the pore, hERG S620T single channel data showed an apparent reduction of the single channel current amplitude at low pH. Furthermore, the effect of protons was relieved by elevating external K(+) or Na(+) and could be modified by charge introduction within the outer pore. Taken together, these data strongly suggest that extracellular protons inhibit hERG maximal conductance by blocking the external channel pore.     

A model for conductance changes in the squid giant axon. II. Channel kinetics

The kinetics of the sodium and potassium channels in voltage clamped squid giant axon following a relaxation of the membrane subunits are examined and compared with the Hodgkin-Huxley equations. Mechanisms are suggested for the turn-off of the sodium conductance and a set of kinetic states are proposed for the potassium channel which are consistent with the experimental observations. Determination of the rate constants for relaxation of the surface subunits which triggers the subsequent changes within the independent channels provide information on the equilibrium constant and free energy for this process. The free energy is observed to approach zero as the depolarizing voltage of the clamp approaches $\text{E}{}_{\mathrm{<mtext>Na</mtext>}}$, the voltage for zero sodium current in voltage clamped axons. Analysis of the final rate constants in the kinetic sequence for potassium indicates a symmetry of the channel when it is in its steady-state configuration during clamp in the absence of external gradients.     

Modification of potassium channel kinetics by histidine-specific reagents

We have examined the actions of histidine-specific reagents on potassium channels in squid giant axons. External application of 20-500 microM diethylpyrocarbonate (DEP) slowed the opening of potassium channels with little or no effect on closing rates. Sodium channels were not affected by these low external concentrations of DEP. Internal application of up to 2 mM DEP had no effect on potassium channel kinetics. Steady-state potassium channel currents were reduced in an apparently voltage-dependent manner by external treatment with this reagent. The shape of the instantaneous current-voltage relation was not altered. The voltage-dependent probability of channel opening was shifted toward more positive membrane potentials, thus accounting for the apparent voltage-dependent reduction of steady-state current. Histidine-specific photo-oxidation catalyzed by rose bengal produced alterations in potassium channel properties similar to those observed with DEP. The rate of action of DEP was consistent with a single kinetic class of histidine residues. In contrast to the effects on ionic currents, potassium channel gating currents were not modified by treatment with DEP. These results suggest the existence of a histidyl group (or groups) on the external surface of potassium channels important for a weakly voltage-dependent conformational transition. These effects can be reproduced by a simple kinetic model of potassium channels.     

Design and synthesis of hydroxy-alkynoic acids and their methyl esters as novel activators of BK channels

Physiological and pharmacological agents that activate large conductance, voltage-, and calcium-gated potassium (BK) channels located in the smooth muscle are effective vasodilators. Thus, activators of smooth muscle BK channels may be potential therapeutic tools to treat cardiovascular disease associated with vasoconstriction and/or impaired dilation, such as cerebrovascular spasm and constriction. We previously showed that lithocholic acid (LC) and other cholane derivatives activated smooth muscle BK channels and, thus, caused endothelium-independent cerebral artery dilation. However, clinical use of these cholane derivatives could be limited by the actions of these steroids, such as elevation of intracellular calcium and induction of apoptosis. Using LC as template, we designed and synthesized a series of hydroxy-alkynoic acids and corresponding methyl esters, as putative, non-steroid BK channel activators. Indeed, the newly synthesized compounds effectively and reversibly activated rat cerebrovascular myocyte BK channel at concentrations similar to those found effective with LC. Among all the novel compounds tested, C-10 hydroxy-alkynoic acid methyl ester appears to be the most effective activator of vascular myocyte BK channels.     

Voltage-Dependent Conductance Induced in Thin Lipid Membranes by Monazomycin

When present in micromolar amounts on one side of phospholipid bilayer membranes, monazomycin (a positively charged, polyene-like antibiotic) induces dramatic voltage-dependent conductance effects. Voltage clamp records are very similar in shape to those obtained from the potassium conductance system of the squid axon. The steady-state conductance is proportional to the 5th power of the monazomycin concentration and increases exponentially with positive voltage (monazomycin side positive); there is an e-fold change in conductance per 4–6 mv. The major current-carrying ions are univalent cations. For a lipid having no net charge, steady-state conductance increases linearly with KCl (or NaCl) concentration and is unaffected by Ca⁺⁺ or Mg⁺⁺. The current-voltage characteristic which is normally monotonic in symmetrical salt solutions is converted by a salt gradient to one with a negative slope-conductance region, although the conductance-voltage characteristic is unaffected. A membrane treated with both monazomycin and the polyene antibiotic nystatin (which alone creates anion-selective channels) displays bistability in the presence of a salt gradient. Thus monazomycin and nystatin channels can exist in parallel. We believe that many monazomycin monomers (within the membrane) cooperate to form a multimolecular conductance channel; the voltage control of conductance arises from the electric field driving monazomycin molecules at the membrane surface into the membrane and thus affecting the number of channels that are formed.     

Calcium- and voltage-activated potassium channels in human macrophages.

Single calcium-activated potassium channel currents were recorded in intact and excised membrane patches from cultured human macrophages. Channel conductance was 240 pS in symmetrical 145 mM K+ and 130 pS in 5 mM external K+. Lower conductance current fluctuations (40% of the larger channels) with the same reversal potential as the higher conductance channels were noted in some patches. Ion substitution experiments indicated that the channel is permeable to potassium and relatively impermeable to sodium. The frequency of channel opening increased with depolarization and intracellular calcium concentration. At 10(-7) M (Ca++)i, channel activity was evident only at potentials of +40 mV or more depolarized, while at 10(-5) M, channels were open at all voltages tested (-40 to +60 mV). In intact patches, channels were seen at depolarized patch potentials of +50 mV or greater, indicating that the ionized calcium concentration in the macrophage is probably less than 10(-7) M.