Prion peptide fragment PrP[106-126] forms distinct cation channel types

Using the lipid bilayer technique we have optimized the recording conditions and confirmed that PrP[106-126] (KTNMKHMAGAAAAGAVVGGLG) forms single ion channels. Based on the conductance and kinetic parameters of the single channel currents recorded in 250/50 mM KCl cis/trans we have found that the PrP[106-126]-formed heterogeneous cation channels that differ in their conductance and kinetic properties. The most frequently observed PrP[106-126]-formed single cation channels were those of: (a) a GSSH- and TEA-sensitive channel with fast kinetics (n = 47). The current-voltage (I-V) relationship of this channel, that has a reversal potential E(rev) of -33 mV close to the equilibrium potential for K(+) (E(K) -35 mV), exhibited inward and outward rectification. The values of the maximal slope conductance (g(max)) were 138 and 141 pS at positive and negative potentials, respectively. The values of the permeability ratios were 1.0:0.87:0.72:0.49:0.41 for K(+) > Rb(+) > Na(+) > Cs(+) > Li(+) respectively. The probability of the channel being open (P(o)) and the frequency (F(o)) had bell-shaped curves with a peak at membrane potential (V(m)) between -10 and -5 mV whereas the mean open and closed times (T(o) and T(c)) had inverted bell-shaped curves; (b) a 4'-4'-dithiodipyridin (DTT)-sensitive channel with slow kinetics (n = 32). The I-V relationship of this channel that had an E(rev) of -35 mV and a g(max) of 41 pS at positive V(m) was non-linear. The parameter P(o) increased at positive V(m) to 0.6-0.7 at +80 mV. F(o) had an asymmetrical bell-shaped curve with a peak of 314 events/sec at -80 mV. The values of T(o) and T(c) were 312 and 164 msec at +120 mV, respectively; (c) a large channel (n = 24 channels) that had five equally spaced subconductances showed time-dependent fast and slow transitions at positive and negative V(m), respectively. The inactivation ratio I(ss)/I(i) was V(m) dependent and described by a bell-shape. The I-V relationship of this channel that had a E(rev) of -22 mV was non linear. The value of g(max) was 900 and 1444 pS at positive and negative V(m)s, respectively. The value of P(o) was 0.6 at negative V(m)s between -160 and -80 mV and 0.23 at +140 mV. F(o) increased from 22 events/sec at -160 mV to 80-100 events/sec at between +80 and +100 mV. T(o) decreased from 375 msec between -160 and -80 mV to 1-2 msec at V(m)s between 0 and +160 mV. In contrast, T(c) decreased from 160-240 msec at membrane voltages (V(m)s) between -160 and -80 mV. The biophysical properties of these channels indicate that they are capable of modifying cellular functions via modification of V(m) and electrolyte homeostasis of the cell.     

Inhibition of calcium currents in cultured myenteric neurons by neuropeptide Y: evidence for direct receptor/channel coupling.

Single channel recordings from rat myenteric plexus neurons demonstrated the presence of two categories of Ca2+ channels. One type of Ca channel had a slope conductance of 27 pS and was sensitive to dihydropyridines while the other channel type had a conductance of 14 pS and was dihydropyridine-insensitive. The 14 pS channel was mostly inactivated at a holding potential of -40 mV, while the 27 pS channel was much more resistant to depolarized holding potentials. A majority of whole-cell current was reprimed by the use of negative holding (-90 mV) potentials, when compared to that obtained at a holding potential of -40 mV. These properties are consistent with N- and L-type Ca channels previously described. In general, the inactivating part of the whole-cell Ca2+ current, selectively reprimed by negative holding potentials, was inhibited by neuropeptide Y (NPY). Depolarization-induced [Ca2+]i transients assessed using fura-2 showed that the inhibitory effects of nitrendipine and NPY were additive. The effects of NPY were abolished by pertussis toxin pretreatment. Single-channel experiments showed that neither the 14 nor the 27 pS Ca channel currents were inhibited by the addition of NPY outside the patch pipette. These results suggest that NPY modulates N-type Ca2+ channels selectively in these neurons and that an easily diffusible second messenger does not appear to participate in receptor/channel coupling.     

A single calcium channel with anomalous rectification in mollusk neurons

The patch clamp technique was used to examine the properties of an inward-rectifying potassium channel in the cell membrane of freshwater mollusc Planorbarius corneus neurons. Inward currents of single channels were observed at potentials more negative than potassium equilibrium potential (EK), when microelectrode contained potassium ions (50 mmol/l) and potassium channel blockers: tetraethylammonium, barium or cesium ions (10-20 mmol/l). The conductance of the single channel was equal to 81 +/- 12 pS at 50 mmol/l potassium ion concentration in the patch electrode. At potentials more positive than EK the conductance sharply decreased to 0 pS. The times of the open state of the closed one of the channel and probability of the open state existing for the ionic channel were estimated with various constant potentials. It was revealed that the channel openings were grouped in bursts. The lifetime of the open state and burst duration decreased with hypopolarization of the patch.     

Properties and rundown of sodium-activated potassium channels in rat olfactory bulb neurons.

We have used single-channel recording techniques to investigate the properties of sodium-activated potassium channels (KNa channels) in cultured rat olfactory bulb neurons, and in large neurons in the mitral cell layer of thin slices of olfactory bulb. Ion channels highly selective for potassium over sodium and chloride, and requiring 10-180 mM internal sodium (Nai) for their activation, were present in approximately 75% of inside-out membrane patches detached from cultured olfactory bulb neurons. Most of these patches contained several KNa channels. KNa channels were seen in cell-attached patches only when Nai was raised by including veratridine in the extracellular medium. Preincubation of the cell in TTX or removal of extracellular sodium prevented this effect of veratridine, confirming that the channels observed under these conditions were indeed KNa channels. Lithium did not substitute for Nai in activating these channels. With 150 mM potassium on both sides of the membrane, KNa channels had a single-channel conductance of 172 pS, and at least two subconducting states were observed in addition to this fully open state. Under these ionic conditions, the channels exhibited linear fully open channel current-voltage curves over the potential range of -100 to 0 mV. At voltages more positive than the potassium equilibrium potential, the single-channel currents exhibited inward rectification as a result of sodium block of outward potassium current. The channels opened in bursts, during which they fluctuated between the fully open and closed states, and the substates. Between bursts they sometimes entered a long-lived inactive state that could last for up to several minutes. In addition, KNa channels in the detached patches exhibited rundown, a progressive irreversible loss in activity, over a time course that varied from less than 1 min to longer than 1 hr. Rundown of KNa channel activity in cell-attached patches (in the presence of veratridine) did not occur, suggesting that some intracellular factor necessary for KNa channel activity is lost when the membrane patch is detached from the cell.     

Channel activity of deamidated isoforms of prion protein fragment 106-126 in planar lipid bilayers

Using the lipid bilayer technique, we have found that age-related derivatives, PrP[106-126] (L-Asp108) and PrP[106-126] (L-iso-Asp108), of the prion protein fragment 106-126 (PrP[106-126] (Asn108)) form heterogeneous ion channels. The deamidated isoforms, PrP[106-126] (L-Asp108) and PrP[106-126] (L-iso-Asp108), showed no enhanced propensity to form heterogeneous channels compared with PrP[106-126] (Asn108). One of the PrP[106-126] (L-Asp108)- and PrP[106-126] (L-iso-Asp108)-formed channels had three kinetic modes. The current-voltage (I-V) relationship of this channel, which had a reversal potential, E(rev), between -40 and -10 mV close to the equilibrium potential for K+ (E(K)-35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 62.5 pS at positive potentials between 0 and 140 mV. The probability (P(o)) and the frequency (F(o)) of the channel being open had inverted and bell-shaped curves, respectively, with a peak at membrane potential (V(m)) between -80 and +80 mV. The mean open and closed times (T(o) and T(c)) had inverted bell-shaped curves. The biophysical properties of PrP[106-126] (L-Asp108)- and PrP[106-126] (L-iso-Asp108)-formed channels and their response to Cu(2+) were similar to those of channels formed with PrP[106-126] (Asn108). Cu(2+) shifted the kinetics of the channel from being in the open state to a "burst state" in which rapid channel activities were separated by long durations of inactivity. The action of Cu(2+) on the open channel activity was both time-dependent and voltage-dependent. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in the conductance of the channel indicated that Cu(2+) binds at the mouth of the channel. Consistently with the hydrophilic and structural properties of PrP[106-126], the Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the Cu(2+) binding site could be located at M(109) and H(111) of this prion fragment.     

Mechanotransducing ion channels in astrocytes

Ion channels present on the soma of neonatal rat astrocytes in primary cell culture were studied using the single channel recording technique. Ion channels were activated by changing the pressure in the back of the pipette. The morphological structure of the patch membrane was examined while recording channel activity. One class of channel was activated by increasing the pipette pressure (curvature-sensitive or CS channels). CS channels were observed in 150 mM KCl, 150 mM NaCl, or 150 mM sodium gluconate. At constant pressure the closed times decreased with depolarization. CS channels had a conductance of 50 pS in 150 mM NaCl, and displayed an inwardly rectifying current-voltage relationship. CS channel activity was found only in cell-attached patches, and were active only when the patch membrane curved towards the soma. The other class of channel was found to be activated by both suction and pressure (stretch-activated or SA channels). Four SA conductance levels were found: 360, 230, 144, and 70 pS in 150 mM KCl. Each conductance displayed a linear current-voltage relationship. At negative membrane potentials SA channels were inhibited by Cs+, Ba2+ or Na+. The relationship between average mechanosensory current and pressure was biphasic for SA channels and monophasic for CS channels. Combinations of SA and CS channels could be observed in the same patch. We propose that CS channels are non-specific cation channels which sense membrane tension only when the patch membrane is in a specific, permissive curvature. SA channels appear to be K(+)-selective channels that sense membrane tension independent of the direction of curvature.     

Chlorine channels activated by suberyldicholine in mollusk neurons]

Patch-clamp technique in cell-attached configuration was applied to investigate ion permeability induced by suberyldicholine in the neurons of freshwater mollusc Planorbarius corneus. The inward currents through single channels were registered at patch potentials 50-100 mV more negative than resting potential of the cell. When the patch pipette contained suberyldicholine (5 mol/l), single channel currents were recorded which grouped in bursts and bursts of openings could themselves be grouped together in clusters of bursts. The desensitisation was not too pronounced: usually we were able to observe single-channel currents for 20-30 minutes. The unit conductance gamma was found to be about 10 pS.     

Ionic channels in mouse astrocytes in culture

We observed Na, K, and Cl voltage-dependent currents in a patch-clamp study of mouse brain astrocytes. In whole-cell recordings, depolarizations activated inward currents that were identified as Na currents since they were blocked by TTX, although complete block required high concentrations (greater than 1 microM). The corresponding single-channel Na currents were observed in outside-out patches. The channels were opened by a depolarizing pulse applied from a holding potential identical to the resting potential (-70 to -80 mV). Therefore, they may be considered functional Na channels. After addition of veratridine and an alpha-scorpion toxin, the decay of Na currents in whole-cell recordings was slower than observed under control conditions. At the single-channel level, the channels appeared to open in bursts. Depolarization did not increase the duration of the bursts, but inside each burst, increased the time spent in the open state. The K currents observed in the whole-cell recording mode were separated into inactivating and noninactivating currents. The inactivating current resembled the A current in its kinetics, its insensitivity to tetraethylammonium, and its sensitivity to 4-aminopyridine. At the single-channel level, at least 3 classes of K channels were observed at steady depolarized potentials. They resembled the K channels found in chromaffin cells by Marty and Neher (1985). Large conductance channels (385 pS) activated around 0 mV were identified as Cl channels.     

Characterization ofClostridial botulinum neurotoxin channels in neuroblastoma cells

The channel and chaperone activities of Clostridial botulinum neurotoxin (BoNT) A were investigated in Neuro 2a neuroblastoma cells under conditions that closely emulate those prevalent at the endosome. Channel activity occurs in bursts interspersed between periods of little or no activity. The channels are voltage dependent, opening only at negative voltages. Within bursts, the channel resides preferentially in the open state. The channels open to a main conductance of 105 +/- 5 pS or 65 +/- 4 pS in 200 mM CsCl or NaCl, respectively. The BoNT channels display a conspicuous subconductance of 10 +/- 2 pS. The neuroblastoma cell line appears, therefore, to be a suitable system to characterize the BoNT channel and to pursue evaluation of plausible strategies for targeted drug delivery thereby minimizing the requirement for in vivo animal testing.     

Ion channels activated by quinolinic acid in cultured rat hippocampal neurons

The membrane responses to quinolinic acid, an excitotoxic brain metabolite, were studied in cultured rat hippocampal neurons with the patch-clamp technique. In the whole-cell recording mode, pressure applications of quinolinic acid elicited inwardly directed membrane currents over a membrane potential range of −60 to −5 mV. The current response reversed at about 0 mV. The current-voltage (I–V) relation of the response had a negative slope conductance at membrane potentials more negative than −40 mV. On removal of Mg²⁺ from the extracellular solution, the current response showed no region of negative slope conductance at potentials more positive than −60 mV. In Mg²⁺-free solution applications of quinolinic acid elicited discrete pulse-like current flows through the outside-out membrane patch. The single channel conductance was 40–46 pS over a membrane potential range of −40 to −80 mV, and 50–55 pS at membrane potentials more positive than +30 mV, showing an outward rectification. These values of the single channel conductance were similar to those of the main conducting state of the channels activated by (NMDA). The responses to quinolinic acid were completely suppressed by the NMDA receptor antagonist (±)-2-amino-5-phosphonovaleric acid. The results indicate that quinolinic acid selectively activates NMDA receptors in the cultured rat hippocampal neurons.     

Single potassium channels in neuropile glial cells of the leech central nervous system

We performed patch-clamp experiments to identify distinct K⁺ channels underlying the high K⁺ conductance and K⁺ uptake mechanism of the neuropile glial cell membrane on the single-channel level. In the soma membrane four different types of K⁺ channels were characterized, which were found to be distributed in clusters. Since no other types of K⁺ channels were observed, these appear to be the complete repertoire of K⁺ channels expressed in the soma region of this cell type. The outward rectifying 42 pS K⁺ channel could markedly contribute to the high K⁺ conductance and the maintenance of the membrane potential, since it shows the highest open probability of all channels. The channel gating occurred in bursts and patch excision decreased the open probability. The outward rectifying 74 pS K⁺ channel was rarely active in the cell-attached configuration; however, patch excision enhanced its open probability considerably. This type of channel may be involved in neuron-glial crosstalk, since it is activated by both depolarizations and increases in the intracellular Ca²⁺ concentration, which are known to be induced by neurotransmitter release following the activation of neurons. The 40 pS and 83 pS K⁺ channels showed inward rectifying properties, suggesting their involvement in the regulation of the extracellular K⁺ content. The 40 pS K⁺ channel could only be observed in the inside-out configuration. The 83 pS channel was activated following patch excision. At membrane potentials more negative than −60 mV, flickering events indicated voltage-dependent gating.     

Characterization of large conductance Ca2+-activated K+ channels in cerebellar Purkinje neurons

We investigated the role of large conductance, calcium-activated potassium channels (BK channels) in regulation of the excitability of cerebellar Purkinje neurons. Block of BK channels by iberiotoxin reduced the afterhyperpolarization of spontaneous action potentials in Purkinje neurons in acutely prepared cerebellar slices. To establish the conditions required for activation of BK channels in Purkinje neurons, the dependence of BK channel open probability on calcium concentration and membrane voltage were investigated in excised patches from soma of acutely prepared Purkinje cells. Single channel currents were studied under conditions designed to select for potassium currents and in which voltage-activated currents were largely inactivated. Micromolar calcium concentrations activated channels with a mean single channel conductance of 266 pS. BK channels were activated by both calcium and membrane depolarization, and showed no sign of inactivation. At a given calcium concentration, depolarization over a 60-mV range increased the mean open probability (P(O)) from < 0.1 to > 0.8. Increasing the calcium concentration shifted the voltage required for half maximal activation to more hyperpolarized potentials. The apparent affinity of the channels for calcium increased with depolarization. At -60 mV the apparent affinity was approximately 35 micro m decreasing to approximately 3 micro M at +40 mV. These results suggest that BK channels are unlikely to be activated at resting membrane potentials and calcium concentrations. We tested the hypothesis that Purkinje cell BK channels may be activated by calcium entry during individual action potentials. Significant BK channel activation could be detected when brief action potential-like depolarizations were applied to patches under conditions in which the sole source of calcium was flux across the plasma membrane via the endogenous voltage-gated calcium channels. It is proposed that BK channels regulate the excitability of Purkinje cells by contributing to afterhyperpolarizations and perhaps by shaping individual action potentials.     

Voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671

A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay. The rates of early activation and subsequent inactivation accelerate and approach a maximum in response to test potentials, V, of greater depolarization. The magnitude of peak sodium current increased from negligible values below V = -50 mV and reached a maximum at V = -3.6 mV +/- 2.7 mV (mean +/- S.E.M., n = 12). Sodium currents reversed at V = + 70 mV, near the predicted Nernst equilibrium potential for a Na+ selective channel. The peak sodium conductance, gpeak increased with depolarizing voltages to a maximum at V = approximately 0 mV, exhibiting half-activation voltage at V approximately equal to -36.8 mV and an e-fold change in gpeak/9.5 mV. The Hodgkin-Huxley inactivation parameter h infinity indicates that at V = -73.6 mV half of the sodium currents were inactivated. Single channel current recordings demonstrated the occurrence of discrete events: the latency for first opening was shorter as the depolarizing pulse became more positive. The single-channel current amplitude was ohmic with a slope conductance, gamma = 17.13 pS +/- 0.66 pS. Sodium channel currents were reversibly blocked by tetrodotoxin (TTX).(ABSTRACT TRUNCATED AT 250 WORDS)     

Conductance properties of GABA-activated chloride currents recorded from cultured hippocampal neurons

The conductance characteristics of gamma-aminobutyric acid-activated single channel currents from cultured hippocampal neurons were examined using patch clamp techniques. GABA-activated currents had amplitudes which were linearly correlated to the membrane potentials over a range of -80 to +70 mV and an open time and burst time of 2.2 and 4.3 ms, respectively. The conductance of the gamma-aminobutyric acid-activated channels was 19 pS. These data demonstrate that cultured hippocampal neurons have channel conductances which have characteristics different from those of adult neurons.     

Involvement of non-selective cationic channels in the generation of pacemaker depolarizations and firing behaviour in cultured frog melanotrophs

The firing patterns of cultured frog melanotrophs were studied using the patch-clamp technique. In the cell-attached mode, unitary currents were frequently observed as well as biphasic waveforms which were attributed to action potentials ‘leaking’ through the patch membrane An inwardly rectifying single-unit current was observed with pipette solutions containing either 100 mM K⁺ or 100 mM Na⁺. Under both conditions, these channels displayed an identical I/V relationship, yielding a unitary conductance of 110 pS. The channel opening time was extremely long (50–3000 ms) and single-channel currents showed typical relaxations, which triggered bursts of action currents. In the whole-cell configuration large (2–12 mV) fluctuations in the membrane voltage of current-clamped cells frequently occured. The deflections appeared to result from single-channel currents. Depolarizing ‘events’ often led to the discharge of action potentials. Taken together, our data provide evidence for the existence of high-conductance cationic channels in frog pars intermedia cells. These channels may, at least in some cases, be responsible for the generation of pacemaker depolarizations, thereby regulating firing behaviour. It is concluded, that the current traversing a single channel can seriously affect the membrane potential and excitability of frog melanotrophs.     

Activation and inactivation of batrachotoxin-modified sodium channels of nerve fiber membranes in the frog

Currents through batrachotoxin (BTX)-modified sodium channels in frog myelinated nerve were measured under voltage-clamp conditions. Nonlinearity of "instantaneous" current-voltage relations was taken into account when determining steady-state parameters of channel activation. BTX induces the shift of voltage dependence of channel activation towards more negative potentials by 67 mV, without changes in its steepness. Current kinetics and effect of preceding depolarization on current size suggest that BTX-modified channels are capable for partial inactivation. High level of steady-state conductance of BTX-modified channels can be explained by suggestion that open state of the channel is energetically more profitable than inactivated one. It is concluded that effect of BTX on inactivation is different in principle from that of pronase and protein reagents.     

Characteristics of acetylcholine-activated channels of innervated and chronically denervated skeletal muscles

Characteristics of the ACh-activated channels before and after denervation of the frog interosseal muscle were studied using the patch clamp technique. Acetylcholine sensitivity was increased on extrajunctional portions of the muscle 7, 42, and 73 days after sectioning of the sciatic nerve. Nonjunctional regions of the innervated muscle appeared to contain one type of ACh channels having a conductance of 28 pS and a mean channel lifetime of 3.8 ms at -90 mV. The denervated muscles contained two classes of channels with conductance of 18 and 28 pS which were present as early as 7 days postdenervation and remained for 93 days. The channel open times of the innervated muscles increased with membrane hyperolarization. The open times of the channels present at 42 days postdenervation showed longer lifetimes than those of innervated muscles and were 10.8 ms and 9.6 ms at -90 mV. These channels also showed less voltage dependence than the control fibers.     

Characterisation of the L- and N-type calcium channels in differentiated SH-SY5Y neuroblastoma cells: calcium imaging and single channel recording.

We have used single cell imaging of [Ca2+]i and single channel cell-attached patch clamp recording to characterise the Ca2+ channels present on the plasma membrane of retinoic acid-differentiated human neuroblastoma (SH-SY5Y) cells. Exposure to raised K+ (45 or 60 mM) for 1 min resulted in a transient rise in [Ca2+]i which was abolished by cadmium (100 microM). The amplitude of the evoked rise varied from cell to cell. Both omega-Conus toxin (500 nM) and nifedipine (10 microM) reduced, but did not abolish, the rise in [Ca2+]i whereas Bay K 8644 (3 microM) potentiated it. In single channel records both L- and N-type Ca2+ channel openings were observed during membrane depolarisations from a holding potential of -90 mV. L-type channel openings (unitary conductance 22.5 pS) were prolonged by S(+)-PN 202-791 (500 nM) and could still be evoked from a depolarised holding potential (-40 mV). N-type channel openings (unitary conductance 12.5 pS) were unaffected by the dihydropyridine agonist but were inactivated at a holding potential of -40 mV. These results indicate that, in contrast to previous observations using whole cell recording, retinoic acid-differentiated SH-SY5Y cells express both L- and N-type Ca2+ channels.     

Three conductance classes of nicotinic acetylcholine receptors are expressed in developing amphibian skeletal muscle

Two previously described classes of nicotinic AChRs in vertebrate skeletal muscle have conductances of 40 and 60 pS. In addition, a third conductance class of AChR channels is present in developing Xenopus muscle. This class appears to represent an independent channel type, rather than a subconductance state of the larger conductance channels. The channel has a slope conductance of 25 pS and a reversal potential of about 0 mV membrane potential. Its kinetic properties resemble those of the 40 pS channels present in early embryonic myotomal muscle. The channel has a mean open time of about 6 msec (at 40 mV applied potential). The open time is dependent on membrane potential and increases e-fold for every 60 mV of hyperpolarization. Consecutive openings were often separated by brief closures of about 0.4 msec in duration. The identity of the channel as a nicotinic AChR was established by blocking the channel openings with alpha-BTX and by demonstrating bursting and desensitization in the presence of high agonist concentrations. In some muscles (e.g., extraocular), this channel may be a predominant form at early developmental stages and could therefore be important to the function of developing synapses in those muscles.     

Single inward rectifier channels in horizontal cells

The ion channels responsible for inward rectification in horizontal cells were studied using the patch clamp technique applied to isolated cells from goldfish retina. Inward currents recorded from these cells were identified as due to the opening of inward rectifier channels based on their ion selectivity, channel gating behavior, and the effects of external blocking ions. The single channel conductance was 20 pS in 125 mM external K+. The null current potential shifted with changes in the K+ concentration as expected for a channel permeable to K+, and the channel appeared to have little permeability to Na+. The probability of a channel being in an open state increased as the membrane was hyperpolarized from the K+ equilibrium potential (0 to -10 mV) over potentials ranging to -80 mV, in the presence of external Na+. The closing rate was insensitive to membrane potential in the presence of external Na+. The opening rate of the channel increased as the membrane was hyperpolarized. The increase in the probability of a channel being open at negative potentials was therefore caused by the voltage sensitivity of the rate of channel opening.