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.     

Analysis of single cyclic nucleotide-gated channels in olfactory receptor cells.

In the accompanying article (Firestein et al., 1991b), we have demonstrated that odor- and cyclic nucleotide-sensitive channels exist at a low density in the dendritic membranes of isolated salamander olfactory receptor neurons. Here, we analyze the cyclic nucleotide sensitivity of these channels using the inside-out patch recording technique. Both cAMP and cGMP, at micromolar concentrations, are capable of inducing channel openings. The biophysical parameters of channel activity are nearly the same in response to either ligand. The unitary conductance is about 45 pS, the reversal potential of single-channel currents is +5 mV, and the I/V relation is linear over the range -80 to +80 mV. The channel activity shows no obvious voltage dependence in divalent cation-free symmetrical solutions. The channel shows no desensitization, even to agonist exposures lasting 15 sec. Mean open time is about 1.5 msec; the closed time distribution is best fit by two exponentials with a fast time constant in the submillisecond range (ca. 0.15 msec) and a slower time constant in the millisecond range (ca. 1.5 msec). The only clear difference in the activity of the two ligands is in their affinity constants. The K1/2 for cAMP is 20 microM; that for cGMP is 4 microM. In both cases, the Hill coefficient is greater than 2, suggesting that channel opening requires the cooperative action of three ligand molecules.     

Biophysical properties of scorpion alpha-toxin-sensitive background sodium channel contributing to the pacemaker activity in insect neurosecretory cells (DUM neurons)

A scorpion alpha-toxin-sensitive background sodium channel was characterized in short-term cultured adult cockroach dorsal unpaired median (DUM) neurons using the cell-attached patch-clamp configuration. Under control conditions, spontaneous sodium currents were recorded at different steady-state holding potentials, including the range of normal resting membrane potential. At -50 mV, the sodium current was observed as unclustered, single openings. For potentials more negative than -70 mV, investigated patches contained large unitary current steps appearing generally in bursts. These background channels were blocked by tetrodotoxin (TTX, 100 nm), and replacing sodium with TMA-Cl led to a complete loss of channel activity. The current-voltage relationship has a slope conductance of 36 pS. At -50 mV, the mean open time constant was 0.22 +/- 0.05 ms (n = 5). The curve of the open probability versus holding potentials was bell-shaped, with its maximum (0.008 +/- 0.004; n = 5) at -50 mV. LqhalphaIT (10-8 m) altered the background channel activity in a time-dependent manner. At -50 mV, the channel activity appeared in bursts. The linear current-voltage relationship of the LqhalphaIT-modified sodium current determined for the first three well-resolved open states gave three conductance levels: 34, 69 and 104 pS, and reversed at the same extrapolated reversal potential (+52 mV). LqhalphaIT increased the open probability but did not affect either the bell-shaped voltage dependence or the open time constant. Mammal toxin AaHII induced very similar effects on background sodium channels but at a concentration 100 x higher than LqhalphaIT. At 10-7 m, LqhalphaIT produced longer silence periods interrupted by bursts of increased channel activity. Whole-cell experiments suggested that background sodium channels can provide the depolarizing drive for DUM neurons essential to maintain beating pacemaker activity, and revealed that 10-7 m LqhalphaIT transformed a beating pacemaker activity into a rhythmic bursting.     

Sodium channel currents in maturing acutely isolated rat hippocampal CA1 neurones.

Sodium channel currents were recorded in excised inside-out patches from immature (P(4-10)) and older (P(20-46)) rat CA1 neurones. Channel conductance was 16.6+/-0.013 pS (P(20-46)) and 19.0+/-0.031 pS (P(4-10)). Opening patterns varied with step voltage and with age. In some patches bursting was apparent at voltages positive to -30 mV. Non-bursting behaviour was more dominant in patches from younger animals. In older animals mean open time (m.o.t.) was best described by two exponentials especially in the older cells; in the immature, there were fewer cases with two exponentials. The time constant of inactivation (tau(h)) estimated in ensemble averages was best described by two exponentials (tau(hf) and tau(hs)) in most patches from older cells. tau(hf) decreased with depolarization; tau(hs) increased in the range -30 to 0 mV. The voltage dependence of tau(hf) in the older cells is identical to that of the single tau(h) found in the younger; the results indicate a dominance of tau(hf) in the younger. Patches from younger cells more often showed one apparent active channel; in such cases, m.o.t. was described by a single exponential. However, in two cases, channels showed bursting behaviour with one of these channels showing a shift between bursting and non-bursting modes. Our findings are consistent with a heterogeneous channel population and with changes in the population in the course of maturation.     

Single-channel recordings of three K+-selective currents in cultured chick ciliary ganglion neurons

Multiple distinct K+-selective channels may contribute to action potential repolarization and afterpotential generation in chick ciliary neurons. The channel types are difficult to distinguish by traditional voltage-clamp methods, primarily because of coactivation during depolarization. I have used the extracellular patch-clamp technique to resolve single-channel K+ currents in cultured chick ciliary ganglion (CG) neurons. Three unit currents selective for K+ ions were observed. The channels varied with respect to unit conductance, sensitivity to Ca2+ ions and voltage, and steady-state gating parameters. The first channel, GK1, was characterized by a unit conductance of 14 pico-Siemens (pS) under physiological recording conditions, gating that was relatively independent of membrane potential and intracellular Ca2+ ions, and single-component open-time distributions with time constants of approximately 9 msec. The second channel, GK2, was characterized by a unit conductance of 64 pS under physiological recording conditions and gating that was affected by membrane potential but was not dependent on the activity of intracellular Ca2+ ions. Open-time distributions indicated 2 open states, with open-time constants of 0.09 (61%) and 0.35 (39%) msec, at +40 mV membrane potential. The third channel, GKCa2+, was identified in isolated patch recordings in which the concentration of internal Ca2+ was 10(-7) M or greater, which was an absolute prerequisite for channel opening. GKCa2+ was characterized by a unit conductance of 193 pS in symmetrical 0.15 M KCl solutions, an open-state probability that was a function not only of [Ca2+]i, but also of membrane potential, and single-component open-time distribution with a time constant of 1.11 msec at -10 mV patch potential. These results suggest the presence of at least 3 distinct K+ channel populations in the membrane of cultured chick CG neurons.     

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.     

BK channels in human glioma cells have enhanced calcium sensitivity

We have previously demonstrated the expression of large-conductance, calcium-activated potassium (BK) channels in human glioma cells. In the present study, we characterized the calcium sensitivity of glioma BK channels in excised membrane patches. Channels in inside-out patches were activated at -60 mV by 2.1 x 10(-6) M cytosolic Ca(2+), were highly K(+)-selective, and had a slope conductance of approximately iqual 210 pS. We characterized the Ca(2+) sensitivity of these channels in detail by isolating BK currents in outside-out patches with different free [Ca(2+)](i). The half-maximal voltage for channel activation, V(0.5), of glioma BK currents in outside-out patches was +138 mV with 0 Ca(2+)/10 EGTA. V(0.5) was shifted to +81 mV and -14 mV with free [Ca(2+)](i) of 1.5 x 10(-7) M and 2.1 x 10(-6) M, respectively. These results suggest that glioma BK channels have a higher Ca(2+) sensitivity than that described in many other human preparations. Data obtained from a cloned BK channel (hbr5) expressed in HEK cells support the conclusion that glioma BK channels have an unusually high sensitivity to calcium. In addition, the sensitivity of glioma BK channels to the BK inhibitor tetrandrine suggests the expression of BK channel auxiliary beta-subunits by glioma cells. Expression of the auxiliary beta-subunit of BK channels by glioma cells may relate to the high Ca(2+) sensitivity of glioma BK 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.     

Characterisation of large-conductance calcium-activated potassium channels (BK(Ca)) in human NT2-N cells

Large-conductance calcium-activated potassium (BK(Ca)) channels were studied in inside-out patches of human NTERA2 neuronal cells (NT2-N). In symmetrical (140 mM) K(+) the channel mean conductance was 265 pS, the current reversing at approximately 0 mV. It was selective (P(K)/P(Na)=20:1) and blocked by internal paxilline and TEA. The open probability-voltage relationship for BK(Ca) was fitted with a Boltzmann function, the V((1/2)) being 76.3 mV, 33.6 mV and -14.1 mV at 0.1 muM, 3.3 muM and 10 muM [Ca(2+)](i), respectively. The relationship between open probability and [Ca(2+)](i) was fitted by the Hill equation (Hill coefficient 2.7, half maximal activation at 2.0 muM [Ca(2+)](i)). Open and closed dwell time histograms were fitted by the sum of two and three voltage-dependent exponentials, respectively. Increasing [Ca(2+)](i) produced both an increase in the longer open time constant and a decrease in the longest closed time constant, so increasing mean open time. "Intracellular" ATP evoked a concentration-dependent increase in NT2-N BK(Ca) activity. At +40 mV half-maximum activation occurred at an [ATP](i) of 3 mM (30 nM [Ca(2+)](i)). ADP and GTP were less potent, and AMP-PNP was inactive. This is the first characterisation of a potassium channel in NT2-N cells showing that it is similar to the BK(Ca) channel of other preparations.     

Capsaicin differentially modulates voltage-activated calcium channel currents in dorsal root ganglion neurones of rats

It is discussed whether capsaicin, an agonist of the pain mediating TRPV1 receptor, decreases or increases voltage-activated calcium channel (VACC) currents (I(Ca(V))). I(Ca(V)) were isolated in cultured dorsal root ganglion (DRG) neurones of rats using the whole cell patch clamp method and Ba2+ as charge carrier. In large diameter neurones (>35 micorm), a concentration of 50 microM was needed to reduce I(Ca(V)) (activated by depolarizations to 0 mV) by 80%, while in small diameter neurones (< or =30 microm), the IC50 was 0.36 microM. This effect was concentration dependent with a threshold below 0.025 microM and maximal blockade (>80%) at 5 microM. The current-voltage relation was shifted to the hyperpolarized direction with an increase of the current between -40 and -10 mV and a decrease between 0 and +50 mV. Isolation of L-, N-, and T-type calcium channels resulted in differential effects when 0.1 microM capsaicin was applied. While T-type channel currents were equally reduced over the voltage range, L-type channel currents were additionally shifted to the hyperpolarized direction by 10 to 20 mV. N-type channel currents expressed either a shift (3 cells) or a reduction of the current (4 cells) or both (3 cells). Thus, capsaicin increases I(Ca(V)) at negative and decreases I(Ca(V)) at positive voltages by differentially affecting L-, N-, and T-type calcium channels. These effects of capsaicin on different VACCs in small DRG neurones, which most likely express the TRPV1 receptor, may represent another mechanism of action of the pungent substance capsaicin in addition to opening of TRPV1.     

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)     

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.     

Acidic regulation of junction channels between glomus cells in the rat carotid body. Possible role of [Ca(2+)](i)

The purpose of this work was to characterize the gap junctions between cultured glomus cells of the rat carotid body and to assess the effects of acidity and accompanying changes in [Ca(2+)](i) on electric coupling. Dual voltage clamping of coupled glomus cells showed a mean macrojunctional conductance (G(j)) of 1.16 nS+/-0.6 (S.E.), range 0.15-4.86 nS. At normal pH(o) (7.43), a steady transjunctional voltage (DeltaV(j)=100.1+/-10.9 mV) showed multiple junction channel activity with a mean microconductance (g(j)) of 93.98+/-0.6 pS, range 0.3-324.5 pS. Single-channel conductances, calculated as variance/mean g(j), gave a mean value of 16.7+/-0.2 pS, range 5.13-39.38 pS. Manual measurements of single-channel activity showed a mean g(j) of 22.03+/-0.2 pS, range 1.3-160 pS. Computer analysis of the noise spectral density distribution gave a channel mean open time of 12.7+/-1.5 ms, range 6.37-23.42 ms. The number of junction channels, estimated in each experiment from G(j)/single-channel g(j), showed a range of 7 to 258 channels (mean, 107.2). Optical measurements of [Ca(2+)](i) gave a mean value of 80.2+/-4.27 nM at pH(o) of 7.43. Acidification of the medium with lactic acid (1 mM, pH 6.3) induced: 1) Variable changes in G(j) (decreases and increases); 2) A significant decrease in mean g(j) (to 80.36+/-0.34 pS) and in single-channel conductance (g(j)=12.8+/-0.2 pS in computer analyses and 17.23+/-0.2 pS when measured by hand); 3) Variable changes in open times, resulting in a similar mean (12.8+/-1.5 ms) and 4) No change in the number of junction channels. When pH(o) was lowered to 6.3 [Ca(2+)](i) did not change significantly (there were increases and decreases). However, when pH(o) was lowered to 4.4, [Ca(2+)](i) increased significantly to 157.1+/-8.1 nM. It is concluded that saline acidification to pH 6.3 depresses the conductance of junction channels and this effect may be either a direct effect on channel proteins or synergistically enhanced by increases in [Ca(2+)](i). However, there are no studies correlating changes of [Ca(2+)](i) and intercellular coupling in glomus cells. Stronger acidification (pH(o) 4.4), producing much larger changes in [Ca(2+)](i), may enhance this synergism. But, again, there are no studies correlating these effects.     

Ionic currents of cultured olfactory receptor neurons from antennae of male Manduca sexta.

Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the ionic currents of insect olfactory receptor neurons (ORNs) in vitro. The cells were isolated from the antennae of male Manduca sexta pupae at stages 3-5 of adult development and maintained in primary cell culture. After 2-3 weeks in vitro, the presumptive ORNs had resting potentials of -62 +/- 12 mV (n = 18) and expressed at least 1 type of Na+ channel and at least 3 types of K+ channels. Na+ currents, recorded in the whole-cell mode, were reversibly blocked by 0.1 microM tetrodotoxin. The predominant type of K+ channel observed was a voltage-activated K+ channel (gamma = 30 pS) with characteristics similar to those of the delayed rectifier. The activity of the 30-pS K+ channel could be inhibited by the application of nucleotides to the cytoplasmic face of inside-out patches of membrane. The nucleotides had relative potencies as follows: ATP greater than cGMP greater than cAMP, with an inhibition constant for ATP of Ki = 0.18 mM. Raising the intracellular Ca2+ concentration from 0.1 to 5 microM induced the opening of a Ca2(+)-activated K+ channel (gamma = 66 pS at 0 mV) that had a low voltage sensitivity. A third, transient type of K+ channel (gamma = 12-18 pS) could be activated by depolarizing voltage steps from very negative resting potentials. Properties of this channel were similar to those of the "A-channel." These results support the conclusion that M. sexta ORNs differentiate in vitro and provide the basis for studying primary mechanisms of olfactory transduction.     

Voltage-gated potassium channels in larval CNS neurons of Drosophila

The availability of genetic, molecular, and biophysical techniques makes Drosophila an ideal system for the study of ion channel function. We have used the patch-clamp technique to characterize voltage-gated K+ channels in cultured larval Drosophila CNS neurons. Whole-cell currents from different cells vary in current kinetics and magnitude. Most of the cells contain a transient A-type 4-AP-sensitive current. In addition, many cells also have a more slowly inactivating TEA-sensitive component and/or a sustained component. No clear correlation between cell morphology and whole-cell current kinetics was observed. Single-channel analysis in cell-free patches revealed that 3 types of channels, named A2, KD, and K1 can account for the whole-cell currents. None of these channels requires elevated intracellular calcium concentration for activation. The A2 channels have a conductance of 6-8 pS and underlie the whole-cell A current. They turn on rapidly, inactivate in response to depolarizing voltage steps, and are completely inactivated by prepulses to -50 mV. The KD (delayed) channels have a conductance of 10-16 pS and can account, in part, for the more slowly inactivating component of whole-cell current. They have longer open times and activate and inactivate more slowly than the A2 channels. The K1 channels have a slope conductance, measured between 0 and +40 mV, of 20-40 pS. These channels do not inactivate during 500 msec voltage steps and thus can contribute to the sustained component of current. They exhibit complex gating behavior with increased probability of being open at higher voltages. Although the K1 channels are sufficient to account for the noninactivating component of whole-cell current, we have observed several other channel types that have a similar voltage dependence and average kinetics.     

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.     

Mislocalization of Kir channels in malignant glia

Inwardly rectifying potassium (K(ir)) channels are a prominent feature of mature, postmitotic astrocytes. These channels are believed to set the resting membrane potential near the potassium equilibrium potential (E(K)) and are implicated in potassium buffering. A number of previous studies suggest that K(ir) channel expression is indicative of cell differentiation. We therefore set out to examine K(ir) channel expression in malignant glia, which are incapable of differentiation. We used two established and widely used glioma cell lines, D54MG (a WHO grade 4 glioma) and STTG-1 (a WHO grade 3 glioma), and compared them to immature and differentiated astrocytes. Both glioma cell lines were characterized by large outward K(+) currents, depolarized resting membrane potentials (V(m)) (-38.5 +/- 4.2 mV, D54 and -28.1 +/- 3.5 mV, STTG1), and relatively high input resistances (R(m)) (260.6 +/- 64.7 MOmega, D54 and 687.2 +/- 160.3 MOmega, STTG1). These features were reminiscent of immature astrocytes, which also displayed large outward K(+) currents, had a mean V(m) of -51.1 +/- 3.7 and a mean R(m) value of 627.5 +/- 164 MOmega. In contrast, mature astrocytes had a significantly more negative resting membrane potential (-75.2 +/- 0.56 mV), and a mean R(m) of 25.4 +/- 7.4 MOmega. Barium (Ba(2+)) sensitive K(ir) currents were >20-fold larger in mature astrocytes (4.06 +/- 1.1 nS/pF) than in glioma cells (0.169 +/- 0.033 nS/pF D54, 0.244 +/- 0.04 nS/pF STTG1), which had current densities closer to those of dividing, immature astrocytes (0.474 +/- 0.12 nS/pF). Surprisingly, Western blot analysis shows expression of several K(ir) channel subunits in glioma cells (K(ir)2.3, 3.1, and 4.1). However, while in astrocytes these channels localize diffusely throughout the cell, in glioma cells they are found almost exclusively in either the cell nucleus (K(ir)2.3 and 4.1) or ER/Golgi (3.1). These data suggest that mislocalization of K(ir) channel proteins to intracellular compartments is responsible for a lack of appreciable K(ir) currents in glioma cells.     

Potassium channels in crustacean glial cells.

Unitary currents through single ion channels in the glial cells, which ensheath the abdominal stretch receptor neurons of the crayfish, were characterized with respect to their basic kinetic properties. In cell-attached and excised patches two types of Ca(++)-independent K+ channels were observed with slope conductances of 57 pS and 96 pS in symmetrical K+ solution. The 57 pS K+ channel was weakly voltage-dependent with a slope of the Po vs. membrane potential relationship of +95 mV for an e-fold change in Po. In addition to the main conductance level, the channel displayed conductance levels of 80 and 109 pS. In excised patches, channel activity of this "subconductance" K+ channel showed "rundown" that could be prevented with 2 mM ATP-Mg on the cytoplasmic side of the membrane. The 96 pS K+ channel was strongly voltage-dependent with a slope of +12 mV for an e-fold change in Po. Averaged single-channel currents elicited by voltage jumps proved the channel to be of the delayed rectifying type. Channel activity persisted in excised patches with minimal salt solution and in virtually Ca(++)-free saline. Because of its dependence on intracellular ATP-Mg, the subconductance K+ channel is discussed as a target of modulation by transmitters or peptides via phosphorylation of the channel.     

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.