Electrophysiological characterization of sodium channel types in the HCN-1A human cortical cell line

Brain Research Bulletin, Vol. 30, pp. 577-583, Printed

in the USA. All rights

0361-9230/93

1993

$6.00 + .OO

CopyrightQ 1993Pergamon Press Ltd.

resewed.

Electrophysiological Characterization of Sodium Channel Types in the HCN-1A Human Cortical Cell Line ROBERT

E. SHERIDAN’

Neurotoxicology Branch, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425 Received

28 May 1992; Accepted

22 July 1992

SHERIDAN, R. E. Electrophysiological characterization ofsodium channel types in the HCN-IA human cortical cellline. BRAIN RES BULL 30(5/6) 577-583, 1993.-Electrically evoked sodium currents were recorded under whole-cell patch clamp from undifferentiated HCN- I A cells. Peak sodium currents had a half-maximal activation, V,,O.S, of -22.6 L 1.O mV with a voltage dependence, k,, of 7.28 ? 0.39 mV’. Steady-state inactivation indicated the presence of two types of sodium channel. One type inactivated with V,,,O.S= -93.8 ? 1.2 mV and kh = -6.8 + 0.4 mV_‘. The second type of sodium channel inactivated with V,,,O.S = -44.6 f 1.5 mV and k,, = -7.3 f 0.4 mV’. The occurrence of each channel type varied from cell to cell and ranged from 0 to 100% of the total sodium current. No variation in the rate of inactivation was seen when the holding potential was adjusted to eliminate the more negative of the two inactivation components. Application of tetrodotoxin (TTX) or saxitoxin (STX) revealed channel types with two different affinities for each toxin. TTX blocked peak sodium conductance with apparent ICSOsof 22 nM and 5.3 PM. STX was more potent, with apparent IC,,s of I .6 nM and I .2 WM.There was no statistical correlation between toxin sensitivity and steady-state inactivation voltage, suggesting that these properties varied independently among sodium channel types. HCN-IA

Human

Cortical

Neurons

Sodium channels

Saxitoxin

Tetrodotoxin

It is presently unknown whether cells in the central nervous system can express similar diversity in sodium channel physiology and pharmacology, although the potential from different cDNA sequences suggests this is possible. Recently, a new clonal cell line of human cortical origin was isolated from a patient with unilateral hemispheric megalencephaly (18). This cell line, designated HCN-1 A, has been biochemically identified as neuronal in character and could provide a useful model for studying human central neuronal sodium channels. Initial characterization of the sodium channels present in the HCN- 1A cell line in terms of classical channel kinetics and pharmacological sensitivity to sodium channel toxins are presented here. These studies have revealed a variety of channel types, some of which do not directly correspond with those seen in other CNS preparations or in peripheral tissues. Some of these data have been previously presented in abstract form (Biophys. J. 61;A107; 1992).

THE electrically excitable sodium channel plays a pivotal role in the function of neural systems, with differences in channel kinetics, pharmacology, and anatomical distribution having major impacts on signalling and information processing. Primary sequences of sodium channel cDNA have revealed at least three types of sodium channel in the rat brain (15,19,23) as well as distinct sodium channel types in mammalian skeletal muscle and heart (4,5,14). Some of these primary sequence differences are associated with differences in activation kinetics, inactivation kinetics, and toxin sensitivity of sodium currents from either native proteins or channels expressed in various cellular systems (2,3,7,11,12,2 I ,22,24-26). Studies performed with native sodium channels in cultured dorsal root ganglia or sympathetic ganglia have indicated the presence of at least two distinct types of sodium channel, often in the same cells. One type has a relatively negative inactivation potential, fast activation and inactivation kinetics, and a high sensitivity to tetrodotoxin (TTX) or saxitoxin (STX). The other channel type is much less sensitive to these toxins, has a more positive inactivation potential, and slower inactivation kinetics (1,9,10,16,17,20). Individual cells in cultured or explanted preparations have expressed either or both types of sodium current.

METHOD

Cell Culture Cells of the HCN- 1A line were obtained from American Type Culture Collection

under nonexclusive

’ Requests for reprints should be addressed to Commander, U.S.A.M.R.I.C.D., Attn: SGRD-UV-YN/Dc

577

license from Johns Hop-

R. E. Sheridan, AK?, MD 21010-5425.

578

kins University. The cells were maintained in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (Sigma Chem. Co.) at 37°C in an atmosphere of 10% COJ90% air and 295% RH. The cells were fed every 2-4 days with fresh medium and split 1:3 every 15 to 25 days using trypsin-EDTA in Dulbecco’s PBS. Cells from passages 18 to 20 were used for this study. For electrical recording, HCN- 1A cells were plated on either uncoated or polylysine-coated glass covershps (3 mm dia.) and cultured separately (Nunclon, 4-well multidishes). Electrical Recording Coverslips containing the HCN- I A cells were transferred to a Plexiglas recording chamber and held in place with a small drop of silicone grease (Dow Corning) under the coverslip. The solution bathing the cells was then replaced by continuous superfusion with a physiological saline solution consisting of (mM) 140 NaCl, 5 KCI, 1.8 CaQ, 1 MgC&, 10 tetraethylammonium bromide, 10 d-glucose, and 10 HEPES buffer adjusted to pH 7.4 with NaOH. Solutions of TTX (Sigma) and STX (Calbiochem) were prepared by adding toxin from I mM stock solutions to the physiological saline solution to give the concentrations indicated. The toxins were then applied by switching the superfusate from the control solution to the toxin-containing solution. Solution exchange was >90% complete within 1 min. Both the cells and the bathing solutions were maintained at room temperature (24-26°C). Patch-clamp recording of whole-cell currents followed standard techniques (6,2 I). Patch pipettes of 1-3 MB were fabricated of borosilicate glass (WPI, 1B 1SOF) and filled with a mock intracellular solution consisting of (mM) 140 CsCI, 5 NaCI, 1 MgC&, 5 EGTA, and 5 HEPES buffer adjusted to pH 7.4 with NaOH. A Dagan model 3900 patch clamp amplifier provided signals to a Zenith model 248 PC running a Labmaster AD/DA interface (Scientific Solutions) under control of pClamp (Axon Instruments) software. Membrane currents were typically lowpass filtered at 5 kHz and digitized at a rate of 30 kHz. Data Analysis Sodium currents following an applied step change in membrane potential were corrected for leakage current, and the initial capacitative transients were either subtracted or blanked from the resulting current trace. Peak sodium currents at each membrane potential were fitted using a modified Boltzmann equation:

where iNa is the peak sodium current in pA, V is the membrane voltage in mV, EN0 is the equilibrium sodium potential, g,,, is the maximum sodium conductance in nS, Vm,o.05is the half maximal voltage for activation (i.e., voltage at which 50% of the Hodgkin-Huxley m gates are shifted) and k, indicates the dependence of channel activation on membrane voltage. Doseresponse studies of toxin blockade of sodium current were based on fractional reduction of the maximum sodium conductance, g,,, measured above. For plots of fractional activation vs. membrane potential, the data were corrected for the driving force on the channel, g,,& V - E,,+J, and fitted as above. Steadystate inactivation was assessed with 100 ms prepulses to different membrane potentials from a holding potential of either - 140 or - 120 mV, followed by a brief test pulse to -10 mV to give near complete activation of the sodium channels that remained available for activation. The sodium current at the test potential

SHERIDAN

was measured as a fraction of the maximum current obtained at the test potential and fitted with the Boltzmann equation (above) to yield Vh,O.,and kh (which are the inactivation equivalents to I’m’,.O.s and km). When multiple components were seen in the inactivation curve(s), the data were fit with the weighted sum of two Boltzmann distributions. Time constant measurements for inaCtiVatiOn, Th, were obtained from fitting a single exponential to the falling phase of the sodium current from 80% of the peak current to baseline. All theoretical curves were fitted to the data using the Marquardt-Levenberg least squares minimization algorithm. All statistical estimates are mean f SEM (number of cells) unless specifically noted otherwise. RESULTS

Undifferentiated HCN- I A cells had a flat, polygonal structure on glass substrates. There was no substantial difference between the morphology or electrical properties of cells grown on polylysine-coated and uncoated glass coverslips, and the bulk of work was done on uncoated glass substrates. The apparent resting potential of cells was measured immediately after perforation of the cell attached patch and before perfusion of the intracellular contents with the pipette solution. Under these conditions, the average membrane potential was -31 + 3 mV (n = 33). This value declined within minutes, during perfusion ofthe cells with CsCl from the patch pipette. For cells with good gigaseals, the input resistance in the whole-cell mode was typically 3.5 f 2 GQ (n = 10). The total membrane capacitance of the cell was measured by integration of the currents resulting from application of a 20 mV peak-to-peak sawtooth waveform. Total membrane capacitance typically did not vary by more than 10% over the course of whole-cell recording from any given cell. Sodium currents were elicited by brief, lo-20 ms, depolarizing pulses from a holding potential of - 120 mV. These currents exhibited a typical sigmoidal rise to peak, and gradual decay due to channel inactivation. Figure I A shows a series of sodium currents obtained from a set of increasingly depolarized steps in membrane potential. Note that the currents did not completely inactivate by the end of the IO-ms depolarizations, as indicated by the small tail currents seen after repolarization to - 120 mV. Measurement of peak sodium current included both the fast and (much smaller) noninactivating component of this current. The overall sodium current density was low compared to the total cellular membrane area. The average ratio of maximum sodium conductance to membrane capacitance was 0.49 f 0.05 nS/pF (n = 13). Assuming a standard membrane capacitance of 1 pF/cm* of membrane, then the maximum sodium conductance density was 4.9 pS/pm*. Figure 1B shows the voltage dependence of activation (squares) and steady-state inactivation (circles) for two typical HCN-1A cells. In 12/35 cells tested there was one component in the steady-state inactivation curve, of which 8/12 displayed a negative characteristic voltage for inactivation like that shown in the open symbols. The majority ofcells tested, 23/35, showed mixed patterns of inactivation similar to that shown in the filled symbols. One component was identical to that seen in the cells with the negative steady-state inactivation component, type “ni.” The second, distinctly more positive steady-state inactivation component, type “pi,” constituted 8 to 93% of the total sodium current in cells where both inactivation processes were present and was the sole component in 4135 cells showing only one type of inactivation. The most negative steady-state inactivation process had an average vh$, of -93.8 +- 1.2 mV and kh = -6.8 f 0.4 mV_’ (n = 3 1). The more positive steady-state inactivation component had an average vh,O.sof -44.6 + 1.5 mV with kh

SODIUM CHANNELS

IN HCN-IA CELLS

579

A.

400 Pq 2 msec

“*“-I20

-100

-80

-60

Membrane

-40

-20

Potential,

0

20

40

mV

FIG. 1. Typical sodium currents in the HCN-IA cell line. (A) Whole-cell patch clamp currents. The cell was clamped at -120 mV and sequential pulses were applied to the potentials indicated. The resulting sodium currents (inward current down) were conected for voltage invariant leakage current and the initial capacitative transient was blanked. (B) Normalized sodium conduc~nce for the peak of the transient sodium current, corrected for the electrochemical potential for sodium at each potential and expressed as a fraction of the maximal sodium conductance seen. Data from two cells are shown and have been fitted with Boltzmann distributions as described in the Method section. The cells were both held at - 120 mV for 1-2 s between changes in membrane potential. (0) Indicates activation in cell I l’m,o.s = -18.7 mV and k, = 8.2/mV. (0) Indicates steady-state inactivation in cell 1 produced by 100 ms conditioning steps to the indicated membrane potentials followed by a IO ms test pulse to - 10 mV. A single inactivation component was seen in this cell with Vhs.5= -98.9 mV and kh = -7S/mV. (*f Was from a second cell that showed two inactivation components with 43% of the current inactivating with V h,0.5= -94.7 mV, kh = -6.l/mV and 57% of the current inactivating with V,,,05= -40.2 mV, k,, = -8.O/mV. (m) shows activation in cell 2 with V,,,,O.s= -18.4 mV and kh = 7.l/mV.

= -7.3r+-0.4mV-‘(n=

27).Althoughtwodistincttypesofsodium

channel inactivation could be discerned, only one type of voltage dependent channel activation was seen. There was no significant difference between the voltage-dependence of sodium channel activation in cells showing one inactivation component (open squares) and those showing two inactivation components Wed

The average values for half-maximal activation were V,,,,O.s= -22.6 +- I.0 mV with k, = 7.28 f 0.39 rnV_’ (n = 45). The kinetics of sodium channel inactivation did not appear to vary with the differences in voltage dependence of steadystate channel inactivation. Figure 2A shows sodium currents obtained from a cell with two different steady-state inactivation

squares).

580

SHERIDAN

channel type being the largest fraction. When the holding potential was changed to -70 mV. the more negative steady-state inactivation effectively removed all the type ni channels from the total sodium current. The peak sodium current at - 10 mV. trace 2, is then derived from essentially pure type pi steady-state inactivation and was reduced to about 40% of the maximum current in this cell. When scaled to the same peak value (scale bar = 400 PA), the sodium current traces are essentially identical. The major difference in the two traces does not appear to be in the rate of inactivation, rh, but a slightly slower rate of activation in 2. When the rate of sodium current inactivation was examined over the range of membrane voltages between -40 to +80 mV as shown in Fig. 2B, a similar lack of effect was seen when the dominant population of type ni inactivation sodium channels was removed by holding at -70 mV. When channels with the more positive steady-state inactivation voltage (A) are compared with sodium currents that contain a predominance of the more negative inactivation voltage (0) there was no consistent difference in the rate of inactivation at any voltage. The sodium currents showed their slowest rate ofinactivation at about -30 mV, and the rate increased exponentially with membrane depolarization and hyperpolarization from this value. The inactivation time constants, rh, were fitted with the reciprocal sum of two exponentials:

2 msec

l3. 2.0 c iii ii A-i 1.6 ii c, i! 8 1.2 -

71, =

2 .r( I- 0.8 k -l-l c, 5 0.4+-l c, :

0

C

r(

0.0’ . -60

.

’

-30

.

.

Membrane

’

0

*

.

’

’

30

Potential,

.

’

60

*

.

I

90

mV

FIG. 2. Effects of membrane holding potential on the kinetics of sodium channel inactivation. (A) A cell with mixed steady-state inactivation, 62% with Vh,05= -98.4 mV and 38% with Vh,0.5= -49.8 mV, the activation of the sodium current is shown for step changes in membrane potential from - 120 mV to -10 mV (trace 1) and from -70 mV to -10 mV (trace 2). The current scales have been adjusted to give the same peak current after elimination of the currents that inactivated at the more positive holding potential, -70 mV. (B) The falling phase of the sodium currents from the cell in A was fitted from 80% of the peak to baseline with a single exponential. The resulting time constants for the exponential fit are plotted as a function of the step change in membrane potential from a holding potential of (0) -120 mV or (A) -70 mV. The equation for the fitted curve is described in the text.

components, types ni and pi. The more negative, type ni, component comprised 62% of the total steady-state inactivation ( Vh,0.5 = -98.4 mV), and the more positive component, type pi, comprised 38% of the total ( Vh,O.s= -49.8 mV). Holding the membrane at - I20 mV and depolarizing to - 10 mV gave the current shown in 1 with the current scale at 1000 pA. This sodium current should be composed of channels with both types of steady-state inactivation, with the more negative inactivation

where Vis the membrane potential in mV and rh is the observed inactivation time constant in ms. The sodium currents in HCN- I A cells were not particularly sensitive to blockade by the site A sodium channel antagonists TTX or STX. Figure 3 shows the effect of extracellular application of a 1 PM solution of TTX on the whole-cell currents evoked from a holding potential of - 120 mV. The sodium currents were depressed from a g,, of 35 to 9.5 nS. but were still clearly present. Washing the cell with toxin-free solution resulted in a complete recovery of the sodium current with g,,,,, = 37 nS. Application of I PM TTX caused no significant changes in Vm,0.5r k, or ENa,as assessed by two-tailed t-tests (n = 6). TTX and STX sensitivities were examined in more detail by constructing dose-response curves for the effect of toxin on g,,. Evoked sodium currents were elicited with a series of depolarizing steps from holding potentials of - 120 mV so that currents from both high and low inactivation threshold type channels were represented. Figure 4A shows the suppression of sodium conductance by TTX. Each data point represents an average of three to six cells from several passages of HCN-I A cells. The data have been fitted with a two-component competition curve with KS0 values of 2 1.6 nM and 5.3 PM. The relative amplitudes of the high and low affinity components were 56% and 44%, respectively. No satisfactory theoretical fit to the data set could be obtained with only one component in the competition curve. Similarly, Fig. 4B shows the suppression of sodium conductance by STX. Again, two different STX affinities were needed to adequately describe the data, with ZC,, values of 1.6 nM and I .25 FM. In this case, only 29% of the total sodium current was blocked at the high STX affinity and 71% was blocked only at the low STX affinity. It should be noted that the STX and TTX data sets were obtained on different cells and the relative abundance of high and low affinity sites may be biased by the data sample. However, within each data set, the parameters of the fitted curves were estimated accurate to less than a t5% coefficient of variation.

SODIUM CHANNELS

IN HCN-1 A CELLS

581

sodium channels revealed by differences in the steady-state inactivation curves are superficially similar to those seen in dorsal ganglia root ganglia, nodose ganglia, and sympathetic [(9,10,16,17,20), but see also (l)]. These preparations all have multiple components in the steady-state inactivation curve with one characteristic voltage near -75 mV and another more positive component with Vh,0.5near -25 mV. What seems to be unique to the sodium currents in the HCN- 1A cell line is that there is little or no difference in the time constant of open channel inactivation that correlates with the significant differences in the steady-state inactivation. The sodium currents in

Membrane

Potential,

mV

FIG. 3. Current vs. voltage relation for peak sodium current with and without TTX. The peak sodium current was measured during a step from a holding potential of - 120 mV to the indicated membrane potential. Currents were measured in (0) control solution, (U) in I 1M TTX and (A) after washout of TTX and return to control solution. The smooth curves were fitted to the modified Boltzmann equation described in the text. The maximum sodium conductance, g,,, decreased from 35 nS to 9.5 nS in 1 PM TTX. EN0was 78 mV, V,,,.O.s was -26 mV and k, was 9.75 mV-’ throughout.

Possible correlations between toxin sensitivity and channel inactivation voltages were explored in cells showing mixed steady-state inactivation components, ni and pi. Figure 5 shows the relative reduction in each component following addition of either 0.1 PM or 1.O PM STX. At these toxin concentrations, more than 98% of the sensitive component of the total sodium conductance should have been blocked. There was no significant difference in the average sensitivity of the ni and pi currents to the block by STX (p > 0.7, two-tailed f-test). The results, however, were variable with individual instances where either the ni or pi component disappeared in toxin with little reduction in the other component.

0

t

0.01 lo-‘0

,”

1.0 -

ITe:rOoklto:O;n;. :o-s

5

$ z 0.8 os EB i u ’ .6 :: *I J 5 0.4 m P o-. Z 5

0.2 -

z )I

DISCUSSION

Undifferentiated HCN- 1A cells exhibit a low sodium channel density. The calculated density of sodium channel conductance was 4.9 pS/pm’. If one assumes a single sodium channel conductance of 11 pS (2 I), then the estimated density of functional sodium channels in the HCN-1 A membrane becomes 0.44 channels/rm’ (or 1 channel/f.2 pm*). This compares with channel densities of 78 channels/pm* in N 18 neuroblastoma cells to 2000 channels/pm2 at mammalian nodes of Ranvier (8). This low channel density may also be responsible for the relatively high input impedance of the HCN- 1A cells and the relatively depolarized resting membrane potential, assuming that leakage currents are similarly sparse. Sodium currents in undifferentiated HCN- 1A cells indicate the presence of a variety of channel types. The differences in

10-s

10-s

Y I=

0 .o

10-10

10-a

‘\ .“_-

10-s

..“‘A

10-6

10-7

[Ssxltoxinl,

\ “‘..A

10-s

M

FIG. 4. Dose vs. response curves for block of sodium conductance by TTX and STX. The maximum sodium conductance, g,, was measured as described in the text. The value ofg,, in the indicated concentration of toxin was compared to the value in control solutions before and after application of toxin (see Fig. 3) and expressed as a fraction of the control conductance. These values were then averaged for each toxin dose (n = 3 to 6 cells each) and expressed as a mean f SEM. The smooth lines were least squares fits of two component competition curves. (A) Suppression of sodium conductance by TTX. In this population of cells, 56% of the conductance was inhibited with an I&, value of 21.6 nM and 44% of the conductance was inhibited with an 1C,,, of 5.3 pM. (B) Suppression of sodium conductance by STX. In a different population of cells, 29% of the conductance was inhibited with an IC,,, of 1.57nM and 7 1% was inhibited with an KY,,,of I .25 FM.

SHERIDAN

0.1

FM

STX

1.0

/LM STX

FIG. S. Correlation

between steady-state inactivation voltage and SIX sensitivity. Cells showing mixed steady-state inactivation voltages were exposed to either 0. I or I .O PM STX and the relative reduction in each inactivation component was calculated from the fitted Boltzmann curve (as in Fig. 1B). There was no significant difference in the suppression of sodium current by either concentration of toxin (p > 0.7. two-tailed, paired /-test).

HCN-IA cells are. thus, distinct from sodium currents in ganglionic preparations, where the channels with more positive inactivation voltages are often described as slow because the associated inactivation kinetics are three- to fivefold slower than the fast currents that have the more negative steady-state inactivation potential (9,lO. 16,17,20). This kind of kinetic/inactivation voltage correlation was not seen in the HCN- IA sodium currents which exhibit only the fast type of inactivation. In terms of TTX and STX sensitivity, there is also an indication of multiple sodium channel types in the HCN- 1A cell line. Both TTX and STX inhibit sodium conductance with high and low affinities. The higher affinities for both TTX and STX. IC,, = 2 1.6 nM and 1.57 nM respectively, are consistent with values obtained from human and rat brain channels reconstituted into bilayers (2,3,5,7) as well as with measurements on rat hippocampal neurons (11) and human, rat, and chicken brain RNA expressed in Xenopus oocytes or cultured mammalian cell lines (12,15,22-26). It should be noted that bilayer studies indicate a voltage dependence in the dissociation constants for TTX and STX that were generally not studied in other systems. The affinities reported for the HCN- 1A cells are based primarily on the changes in maximum sodium conductance and are, thus, determined primarily by membrane potentials where the conductance was maximally activated, i.e., between 0 and +50 mV. The observed affinities in HCN-IA cells were also consistent with direct binding measurements on rat brain or muscle (13,14). The lower affinity for TTX and STX inhibition seen in HCN-IA cells is more characteristic of cardiac sodium channels (4,5) or the slowly inactivating, toxin resistant sodium currents in dorsal root ganglia and spinal ganglia neurons (10,17). In the HCN- I A cell line, these low-affinity

channels represent a significant fraction (44 to 71%>) of the total channels seen in the cell population. Unlike the situation in ganglionic sodium channels of the slow or TTX-resistant type, there seemed to be no correlation in HCN-IA cells between the sensitivity to TTX or STX and the time course of the sodium currents or in their steady-state inactivation voltages. In comparison with the subtypes of sodium channel that have been isolated from rat brain (types I, II, and III), the sodium channels in HCN-IA cells do not clearly match the characteristics of the rat brain type II. IIA, and III cDNAs expressed in oocytes. The toxin sensitivity is uniformly high in the rat and is a mixed population in the HCN-IA cells. The steady-state inactivation voltage for the rat type II, IIA. and III channels (about ~60 mV) lies between the two voltages seen in the HCN- I A channels (15,19,23,25,26). However, the time constant of open channel inactivation, fast, and the activation voltages. I’,,,“,5, seem consistent between the HCN- 1A cells and human or rat brain cDNAs. It is possible that expression of the cy-subunit of the brain sodium channels in isolation contributes to the differences in properties compared to the HCN-1.4 channels. Expression of the brain sodium channel message in oocytes along with lower molecular weight message shifts sodium channel steady-state inactivation to more negative voltages and increases the rate of inactivation ( 12). Expression of sodium channels in mammalian cell lines has a similar effect (19.25.26). Thus, the ni component of the HCN-1A currents are within the voltage range that might represent CNS sodium channel types. However, toxin sensitivities raise doubt that the channels expressed in undifferentiated HCN- 1A cells of human cortical origin actually represent the characteristics of mature human CNS neurons. Brain sodium channels, expressed from nucleic acids, reconstituted into bilayers or in isolated tissues, exhibit a uniformly high TTX/STX sensitivity (2,3,7,1 l15.19,23-26). This is clearly not the case with the HCN-IA sodium currents. Neither do the HCN- I A sodium channels clearly resemble the several subtypes of sodium channels that have been described in ganglia preparations, because toxin sensitivity does not correlate with steady-state inactivation voltage. The type ni sodium channel in HCN-IA cells seems to correlate with the fast sodium channel type in ganglia and spinal cord. However, there was no evidence that this channel type constituted the high-affinity TTX/STX-sensitive component in total HCN-1A sodium current. The type pi sodium channel, by contrast, does not match the characteristics of the ganglionic slow sodium channel type in terms of channel kinetics and did not constitute the toxin-resistant fraction of the sodium current. The independent expression of ni and pi currents as well as high and low affinities to TTX and STX suggest four sodium channel types in the HCN- I A human cortical cell line. Some of the combined properties of these channel subtypes have not been described in other neuronal preparations. At this time, it is unclear whether these different sodium channels derive from differences in the sequence of message coding for the sodium channels. differences in the posttranslational processing, differences in phosphorylation, or some combination of these factors. ACKNOWLEDGEMENTS

Thanks are extended to Mr. Bryce Doxzon for his technical assistance. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the U.S. Army or the Department of Defense.

SODIUM

CHANNELS

583

IN HCN-1 A CELLS REFERENTS

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