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articles
Calcium channel gating and
modulation by transmitters depend
on cellular compartmentalization
Patrick Delmas, Fe C. Abogadie, Noel J. Buckley and David A. Brown
Wellcome Laboratory for Molecular Pharmacology, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK
© 2000 Nature America Inc. • http://neurosci.nature.com
Correspondence should be addressed to P.D. (ucklpds@ucl.ac.uk)
Voltage-gated Ca2+ channels participate in dendritic integration, yet functional properties of Ca2+
channels and mechanisms of their modulation by neurotransmitters in dendrites are unknown. Here
we report how pharmacologically identified Ca2+ channels behave in different neural compartments.
Whole-cell and cell-attached patch-clamp recordings were made on both cell bodies and electrically
isolated dendrites of sympathetic neurons. We found not only that Ca2+ channel populations differentially contribute to somatic and dendritic currents but also that families of Ca2+ channels display
gating properties and neurotransmitter modulation that depend on channel compartmentalization.
By comparison with their somatic counterparts, dendritic N-type Ca2+ currents were hypersensitive to
neurotransmitters and G proteins. Single-channel analysis showed that dendrites express a unique Ntype channel that has enhanced interaction with Gβγ. Thus Ca2+ channels in dendrites seem to be specialized elements with unique regulatory mechanisms.
Integration of synaptic inputs in the dendritic tree depends
largely on active cable properties. Neuronal dendritic arbors
express a large variety of voltage-gated ion channels that
enable action potentials and synaptic inputs to propagate
throughout the dendrites1,2. Such active responses in dendrites
are capable of activating Ca2+ channels, which in turn increase
intradendritic free Ca2+ and regulate the integration and propagation of information3–6.
Multiple subtypes of voltage-gated Ca2+ channels, designated as T, L, N, P/Q and R7, may coexist in a single neuron. These
Ca2+ channel subtypes have different functional characteristics
and are differently distributed in dendrites, nerve terminals and
cell bodies, suggesting that they serve highly specialized subcellular roles. Several other factors add to the functional diversity
of Ca2+ channels, such as post-transcriptional modification8–10,
differential association with auxiliary subunits11 and multiple
modulation by receptors and G proteins12. Electrophysiological
and molecular studies show that multiple types of Ca2+ channels
are also present in dendrites1,2,13–17 and can be modulated by
transmitters17. However, there is limited information about the
contributions of individual types of Ca2+ channels to dendritic
Ca2+ currents, or their functional properties and regulation.
In the present experiments, we compared Ca2+ channel properties and their modulation in identified dendrites and cell bodies of sympathetic neurons. We used both single-channel
recording and macroscopic dendritic current recording, using a
method for acute electrical uncoupling of the dendrites from the
ganglion cell soma that allows direct comparison of dendritic
and somatic currents. We found that dendritic Ca2+ current is
dominated by the discrete localization of an unusual N-type Ca2+
channel that is sparsely represented in the soma, and that is more
sensitive to inhibition by receptors and G proteins than the predominant somatic channels. The characteristics of this channel
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seem suited for providing a mechanism that limits excessive Ca2+
influx in synaptically active dendrites.
RESULTS
Voltage clamp of electrically isolated dendrites
We first determined how to obtain patch-clamp recordings of
macroscopic Ca2+ currents in large neurites of sympathetic neurons (Fig. 1). Electrical isolation of a given neurite from the cell
body was achieved by compressing the neurite’s proximal part
with a bent glass micropipet (Fig.1a and Methods). This formed
a tight junction of high access resistance between the soma and
the neurite of interest. The loss of electrical coupling was tested
by double current-clamp recordings in the soma and neurite of
the same neuron (Fig. 1b). Under control conditions, action
potentials and electrotonic responses evoked in the cell body were
propagated to the neurite, although they were attenuated. By
contrast, in the presence of the isolating pipet, propagation of
somatic action potentials was severely impaired, though the neuritic compartment remained excitable. In additional experiments,
we tested whether the exchange of cytosolic elements between
somatic and neuritic compartments was also prevented. Although
M-type K+ currents recorded in neurites could be blocked by
injection of Cs+ in the cell body, the same currents were not
appreciably altered when neurites were uncoupled using the isolating pipet (Fig. 1c). Although the percentage of successful
experiments was relatively low, the above data indicate that the
isolating pipet was able to electrically and chemically isolate the
neuritic segments from the cell body.
Using this method, we were able to record Ca2+ channel activation in visually identified processes up to 90 µm long (Fig. 1a).
In each experiment, we used the tail current deactivation and
activation rate of ICa to assess the quality of voltage control. As
expected from an experiment with good clamp, ICa activated
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gradually, and tail current deactivation was less than 1 ms.
Recordings with inadequate spatial and temporal controls were
discarded. Calcium current density in large neurites (16–36
pA/pF) was about the same as in the somata for a mean capacitance ∼4-fold less (13 ± 4 pF).
For technical reasons, most recordings were made on large
processes similar to that shown in Fig. 1a. To identify these structures further, we double labeled neurons for tau, an axonal marker, and microtubule-associated protein 2 (MAP2), a dendritic
marker18. On days one to three of culture, MAP2 and tau were
colocalized in cell bodies and in most processes. On day four and
later, occasional thin and long axon-like processes exhibited segregation of tau, whereas MAP2 labeling was restricted to thick
neurites of the type we selected for recordings (Fig. 1d). These
data indicate that the large neurites used in the present study are
related to dendritic structures. Other well-known somatodendritic currents, including Ih, IK(A) and various Ca2+-activated K+
and Cl– currents (Methods), were also detected in these processes. Accordingly, we refer to these processes as ‘dendrites’ or ‘dendrite-like structures’.
Differential expression of dendritic Ca2+ currents
The quantitative contribution of the different populations of dendritic Ca2+ channels was estimated in 2 mM extracellular calcium ([Ca2+]o), using various blockers of Ca2+ channels. Calcium
currents could be divided into N currents (70%) sensitive to
ω-conotoxin (ω-CgTx) GVIA and P/Q currents (14%) sensitive
to ω-agatoxin (ω-Aga) IVA (Fig. 2a–c). After blockade of N and
P/Q Ca2+ channels, dendritic recordings showed a residual current
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Fig. 1. Calcium current recordings in processes of sympathetic neurons. (a) Whole-cell patch-clamp recording of a
large neurite (17 pF, 75 µm long) of a sympathetic neuron.
Voltage protocol and isolated Ca2+ currents are shown
below. The patch electrode (P) was sealed ∼25 µm away
from the isolating pipet (arrow). (b) Simultaneous currentclamp recordings in soma and neurites (85–95 µm away) in
the presence or absence of the isolating pipet. Electrotonic
responses and action potentials evoked in the soma were
not propagated to the neurite in the presence of the isolating pipet (bottom). Inset, responses to current injections
into the neurite. Rate of success, 55% (n = 9).
(c) Intracytoplasmic injection of Cs+ in the soma (top trace)
results in blockade of M-type K+ current recorded in a neurite 85 µm away. Bottom trace, same experiment (different
neuron) but in the presence of the isolating pipet.
(d) Double staining for tau and MAP2 proteins. Top, MAP2
immunoreactivity of a large neurite selected for recording.
The isolating and recording pipets are indicated. The Ca2+
current recorded in this neurite is shown in Fig. 2b. Bottom,
representative example of a large neurite (white arrows)
labeled for the dendritic marker MAP2 but not for tau.
(∼16%) that activated at –45 mV and partly inactivated with τdecay over 20 ms at 0 mV. This current was
blocked by low concentrations of Ni2+ (Fig. 2a). It was
tentatively identified as R-type because it appeared to
deactivate rapidly (τ < 0.5 ms), a property associated with R-type rather than T-type Ca2+ currents19. No
L-type Ca2+ currents could be observed in dendrites
(Fig. 2a and b). For comparison, somatic Ca2+ currents recorded on cell bodies without identifiable
processes indicated the presence of N (84%), R (9%)
and L (7%) currents but not P/Q currents (Fig. 2a
and b), consistent with previous reports20,21. Thus, these data
indicate that P/Q channels (the mRNA for which is detected in
rat sympathetic neurons22) may contribute to the global Ca2+ current in dendrites, and that R and L channels provide different
fractions of dendritic and somatic currents. Further exploration
of the dendritic N-type Ca2+ current also showed that it differs
from that expressed in the soma, in that the dendritic N current
had a more negative activation curve (Fig. 2d) and a 20-fold higher half maximal block by ω-CgTx (Fig. 2e and f).
G-protein modulation of dendritic Ca2+ currents
The dendritic current was more susceptible to inhibition by
receptors coupled to members of the Go/Gi class of G protein than
its somatic counterpart. Thus, inhibition following maximal stimulation of α2 adrenoceptors and somastostatin receptors (both
of which act through Go/i23,24) significantly increased from 50%
and 43%, respectively, in the soma to 75% and 64% in the dendrites (Fig. 3). By contrast, voltage-independent M1 muscarinic
inhibition, which is mediated via the Gαq subunit25, was not
increased in the dendrites (Fig. 3b). Voltage-dependent inhibition of both N and P/Q Ca2+ currents is mediated by G-protein βγ
dimers26,27. To test for the involvement of Gβγ in noradrenergic
and somastostatin inhibition of dendritic Ca2+ currents, we overexpressed retinal Gα transducin, which acts as a Gβγ sequestering agent in SCG neurons24. Overexpression of Gα transducin
was equally effective in blocking neurotransmitter responses in
the two compartments (Fig. 3b), indicating that Gβγ mediates
inhibition in both dendrites and soma. Enhanced modulation
by noradrenaline in dendrites was associated with an alteration of
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Fig. 2. Components of the Ca2+ current in dendrites. (a) Macroscopic Ca2+ currents recorded in the soma (47 pF) and dendrite (12 pF) of sympathetic neurons in control (ctr) conditions and after sequential application of ω-CgTx GVIA (500 nM, for 10 min), ω-Aga IVA (500 nM, for 10 min),
nimodipine (10 µM) and Ni2+ (50 µM), in an additive manner. Traces are scaled for comparison. (b) Components of dendritic (14 cells) and somatic
(11 cells) Ca2+ currents as determined in (a). Inset traces are difference currents, isolating the different components of global ICa in a single dendrite.
The sizes of R and P/Q currents have been magnified. (c) I–V relationships of Ca2+ currents in a dendrite (14 pF) in control (thick line) and in the presence of ω-Aga IVA, ω-CgTx GVIA and 500 µM Cd2+. Difference currents are shown below. (d) Tail-current activation curves for ω-CgTx GVIA-sensitive currents (difference currents) determined in soma and dendrites. The continuous curves represent single Boltzmann fits to the data. Tail
currents were evoked on repolarization to –90 mV after 5 ms depolarization. The mean shift for midpoint values between dendrite and soma was
–7.5 ± 3 mV (n = 4–6). Inset, example of ω-CgTx GVIA-sensitive tail currents in a dendrite (reversed for convenience). (e, f) Dose dependence of
ω-CgTx GVIA block in soma (e) and dendrite (f). Solid lines are the best least-square fits to single (soma) and double (dendrite) binding site equations. Calculated Kd values are indicated. Kd for the soma was consistent with ref. 32. n = 4–7 cells per point.
the voltage dependence of inhibition. Noradrenergic inhibition of
somatic Ca2+ currents can be partly reversed by applying strong
positive voltages, resulting in a phenomenon termed facilitation28.
We found that although modulated somatic and dendritic Ca2+
currents were equally facilitated (facilitation,
1.5–2.1), depolarization was only about half as
effective in reversing inhibition in the dendrites as a
in the soma, as assessed from the inhibition ratio24
(1.4 ± 0.2, n = 13 and 2.7 ± 0.4, n = 11, respectively).
Taken together, these data indicate that,
although somatic and dendritic Ca2+ currents are
modulated via Gβγ dimers, they display different
Fig. 3. N-type Ca2+ current in dendrites is hypersensitive to neurotransmitters. (a) Inhibition of Ca2+ currents
by noradrenaline (NA, 10 µM) in soma and dendrite.
Currents were evoked using the protocol illustrated in
inset and recorded in presence of ω-Aga IVA and nifedipine. Note the moderate facilitation of the tail current in
dendrite compared with soma (arrows). Outward currents were truncated for clarity. (b) Summary of inhibition (mean ± s.e.m.) by noradrenaline, somatostatin (Sst,
500 nM) and oxotremorine-M (Oxo-M, 1 µM) of N-type
Ca2+ currents, in control cells and in cells expressing
retinal Gα transducin (200 µg per ml cDNA). Oxo-M
(M1 receptor) inhibition was recorded in cells pretreated with pertussis toxin (1 µg per ml for 24 h).
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sensitivities to neurotransmitters and G-protein subunits. To
investigate this further, we evaluated the time course of re-inhibition following facilitatory depolarization, which would reflect
the rate of reblock of Ca2+ currents by Gβγ dimers. When we did
b
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Fig. 4. Time course of re-inhibition and relief from inhibition
are different in dendrite and soma. (a, b) Re-inhibition of
somatic (a) and dendritic (b) Ca2+ currents in the presence of
either noradrenaline (top) or Gβ1γ2 (bottom). Ca2+ currents
were evoked by using the three-voltage-step protocol illustrated above (∆T = 5 ms). (c, d) Re-inhibition kinetics for NA
(c) and Gβ1γ2 (d) as a function of interpulse duration at
–70 mV in the soma (open symbols) or dendrite (filled symbols). Time constants of re-inhibition from individual experiments were as follows: for NA, 42, 36, 33, 29 and 53 ms in the
soma and 18, 7, 10, 8 and 16 ms in dendrite; for Gβ1γ2, 43, 24
and 30 ms in the soma and 10, 12 and 14 ms in dendrite.
(e) Representative examples of relief of G-protein block in
dendrite and soma. Tail currents were evoked on repolarization from a step to +90 mV (∆T = 2.5 ms). (f) Relative tail
Ca2+ currents plotted as a function of step duration at
+90 mV. The time course of removal of inhibition was best fitted by one exponential in the soma (τ = 6.7 ms) and by two
exponentials in the dendrite (τ1 = 4.5 ms and τ2 = 18 ms). All
recordings made in presence of ω-Aga IVA and nifedipine.
this on pharmacologically isolated N-type Ca2+ currents, we found that re-inhibition was two- to threefold faster in the dendritic compartment than in the
somatic one (Fig. 4a–d). This could not be ascribed to
either a higher amount of Gβγ activated by neurotransmitters29 or to the involvement of different Gβγ
dimers 24,30 , as overexpression of a single specific
βγ dimer (β 1 γ 2 ) led to the same results
(Fig. 4b and d). Consistently, the time course of relief
from inhibition (which reflects the rate of dissociation
of Gβγ from the Ca2+ channel) was slower in dendrites.
Thus, the time constants for maximal facilitation were
6 ± 1 ms at +90 mV for somatic Ca 2+ currents and
20 ± 2 ms for dendritic currents (Fig. 4e and f).
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A low-conductance Ca2+ channel in dendrites
The above data favor the view that the differential
effects of neurotransmitters and Gβγ dimers on dendritic Ca2+
currents arise from a difference in affinity of the Gβγ dimer for
the dendritic Ca2+ channels. This seems to result from the intrinsic characteristics of dendritic N Ca2+ channels rather than the
involvement of different signaling pathways. To test this further,
we used cell-attached patch recordings from different membrane
a
b
loci to examine the properties and distribution of Ca2+ channels.
These recordings revealed a high density of inward (Ba2+) channel currents on both somatic and dendritic membranes, with two
main channel conductances, high (16–24 pS) and low (8–13 pS).
The most frequently encountered high- and low-conductance
channels (Fig. 5a and b) were detected independently of each
c
d
Fig. 5. Two groups of Ca2+ channel conductances in dendrite and soma. (a, b) Representative single-channel activities recorded from the soma (a) or
dendrite (b) of sympathetic neurons in response to depolarization. Note the difference in unitary current in these two patches. (c) I–V relationships
for the small Ca2+ channel observed in dendrite (n = 12, open symbols) and the large Ca2+ channel observed in both soma and dendrite (n = 9, filled
symbols). Solid lines are linear regression fits giving slope conductances of 10.5 and 19 pS for low- and high-conductance channels, respectively.
Dashed lines, 95% confidence limits. (d) Open probability (Po) plotted against voltage for individual patches containing high (filled symbols) or low
(open symbols) conductance channels. Recordings were made in presence of Bay K 8644 to identify L channels and exclude them from the analysis.
Solid lines are Boltzmann fits (unconstrained) to all points.
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Fig. 6. High- and low-conductance Ca2+ channels are
blocked by ω-CgTx GVIA.
(a–c) Single-channel currents
and ensemble-averaged currents for two time points (3
and 21–24 min) after seal formation. The pipet was tipfilled with toxin-free solution
(90 mM Ba2+) for 2 min and
then back-filled with the
same solution containing
50 µM ω-CgTx GVIA.
Patches were from the soma
in (a) and (b) and from the
dendrite in (c). They contain
a high-conductance channel,
two L-type Ca2+ channels and
a low-conductance Ca2+
channel, respectively. Sweep
currents were evoked every
10 s. Bay K 8644 (1 µM) was
present in all recordings.
(d–f) Ensemble currents from
high-conductance channels
(d), L-type channels (e) and
low-conductance channels (f)
as a function of time in the
presence of ω-CgTx GVIA or
ω-Aga IVA (5 µM). Each point
represents the mean ± s.e.m.
of 3–5 experiments. The
amplitude of the averaged
currents was measured as the
mean current value over the
20–45 ms test pulse.
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other, although their relative frequency depended on patch location. Thus, the number of active patches containing low-conductance Ca2+ channels increased from less than 8% (3/41) on
the cell body to ∼61% (23/38) on dendrites. In striking contrast,
70% (39/56) of the patches with high-conductance Ca2+ channels (excluding L channels) were from the somata or the proximal
part of the dendrites. When we corrected for charge screening in
90 mM Ba2+ pipet solution (estimated shift of ∼30 mV; Methods), most of the high- and low-conductance channels had characteristics of high-voltage-activated channels, with an apparent
activation threshold of –4 ± 2 mV (∼–35 mV) and –11 ± 3 mV
(∼–38 mV), respectively. Consistently, both types of channels
could still be activated when stepped from a holding potential of
–40 mV (n = 5–9), which should inactivate R-type channels31.
The open probabilities (po) for both high- and low-conductance
channels (Fig. 5d) were well fitted using Boltzmann functions,
although variability occurred from patch to patch. The fits from
all patches yielded a V1/2 of +26 mV and a slope of 8.7 mV for
the large channel and a V1/2 of +17 mV and a slope of 7.3 mV for
the small channel. In the presence of Bay K 8644 in the bath, we
could also resolve (in ∼5% of somatic and perisomatic patches)
long openings of large-conductance channels (22–24 pS; Fig. 6),
very similar to those reported for L-type Ca2+ channels31.
Both Ca2+ channel types are blocked by ω-CgTx GVIA
The findings that the low-conductance channel is located predominantly on dendrites and that the N-type Ca2+ current is the
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main component of global dendritic Ca2+ current suggested that
this channel(s) might be N-type. To test this, we examined the
pharmacology of individual channels using intrapipet diffusion
of ω-conotoxin GVIA31, applied at a high concentration to ensure
block in high Ba2+ solution32. Figure 6 shows the effects of the
toxin on ensemble-averaged currents resulting from the activity
of a high-conductance channel (21 pS) recorded from the soma
(Fig. 6a) and a low-conductance channel (12 pS) recorded
∼65 µm away (Fig. 6c). In most patches thus recorded, the diffusion of ω-conotoxin GVIA progressively blocked the activity
of large as well as small channels within ∼20 minutes (Fig. 6d and
f). In three patches containing L channels (identified by their
high conductance and long openings), activity was not altered
by ω-conotoxin GVIA after up to 25 minutes (Fig. 6b and e).
Activities of high- and low-conductance (ω-conotoxin GVIAsensitive) channels were not blocked in patches tested with ωagatoxin IVA (Fig. 6d and f). In an additional set of experiments,
ω-conotoxin GVIA (1 µM) was pre-incubated in Ca2+-free media
for 6–8 min. No putative N channels could be observed in patches made after such pre-incubation. However, in few dendritic
patches (3/14), relatively large channels (14–18 pS) with very
rapid gating that prevented accurate conductance measurement
were observed at potentials positive to –5 mV (data not shown).
Taken together, these results indicate that the most frequently
encountered high- and low-conductance channels belong to the
N-type Ca2+ channel family, consistent with the predominance
of this current in both somatic and dendritic compartments.
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Gβγ modulation of dendritic Ca2+ channels
Because the low-conductance N channel is the predominant one
in dendrites, it is likely to be the channel responsible for conferring the unique G-protein regulatory features described above
on the N-type dendritic current. This prompted us to compare
single-channel behavior of high- and low-conductance N channels during their modulation by noradrenaline or by Gβγ overexpression, at +20 mV where channel activity is high and best
resolved (Fig. 7). In control conditions, neither the large nor the
small N channel seemed to be significantly modulated. Thus
depolarizing prepulses had no clear effects on channel activation
delay or kinetics of ensemble-averaged currents, one of the characteristic features of voltage-dependent modulation of N channels 33,34 . This is consistent with the lack of facilitation of
macroscopic currents in control conditions (Figs. 3a and 4a).
However, the percentage of large N-channel patches with delayed
channel activation and slowly activating averaged currents
markedly increased when the patch was made with a pipet containing noradrenaline (57% of patches versus 2%) or from cells
expressing Gβ1γ2 (79% of patches). In these patches, the inhibitory effect of noradrenaline and Gβ1γ2 dimers was first noticed as a
significant decrease in open probability of channels (+20 mV,
po control 0.42, po NA 0.23, po βγ 0.17, n = 5–8 patches). This change in
po was mainly accounted for by an increase in first latencies of
channel opening that gave ensemble-averaged currents the slower rising phase characteristic of modulated currents (Fig. 7a).
Delayed activation of large N channels was removed by short
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Fig. 7. Prepulse facilitation of large but not small
ω-CgTx GVIA-sensitive channels. (a, b) Cellattached patch recordings of high (a, soma) or
low (b, dendrite) conductance Ca2+ channels with
50 µM noradrenaline in the patch pipet. Shown
are two representative current traces with
resolved openings and corresponding ensembleaveraged currents. The patches were stepped to
+20 mV from a holding potential of –70 mV after
(lower traces) or before (upper traces) a 20-ms
conditioning depolarization to + 90 mV. Test and
conditioning pulses were separated by a 4-ms
repolarization to –70 mV. Ensemble-averaged currents with (dashed lines) or without (solid lines)
conditioning prepulse (+PP) are shown superimposed. Traces are averages over 18–25 sweeps.
Smooth curves are best fits from ensemble-averaged currents. (c, d) Prepulse-induced change in
the rising phase of ensemble-averaged currents
from large (c, soma) or small (d, dendrite) Ca2+
channels. Currents were recorded either with NA
in the patch pipet or after expression of Gβ1γ2.
The activation kinetics of the ensemble currents
(thin broken lines) were obtained by averaging
data from 3–4 patches (17–35 single-channel
sweeps per patch). Data are shown with and without conditioning prepulses. The solid and dashed
lines represent curve fittings of the activation
phase using exponential functions. Calculated
time constants are indicated.
facilitatory prepulses (Fig. 7a). In patches
containing small N channels, there was also
a clear difference in single-channel activity in
the presence of noradrenaline or after Gβ1γ2
expression. Mean po at +20 mV fell from a
control value of 0.48 to 0.21 or 0.14 with NA
or Gβ1γ2, respectively (n = 7–11 patches). However, channel inhibition was not associated with prominent slowing of ensembleaveraged currents and was only weakly reversed by a depolarizing
prepulse (Fig. 7b). Comparing the kinetics of modulated and
facilitated ensemble-averaged N currents in the soma (Fig. 7c)
and dendrites (Fig. 7d) shows that, in contrast to the high-conductance N Ca2+ channel, the low-conductance channel shows
little sign of kinetic slowing or prepulse-induced facilitation, suggesting that Gβγ inhibition of this particular N channel is largely resistant to modulation by voltage.
DISCUSSION
Our parallel investigation of Ca2+ currents in cell bodies and dendrites of sympathetic neurons provides evidence that Ca2+ current
properties and regulation by neurotransmitters strongly depend
on cell compartmentalization. The different Ca2+ signaling capabilities found in soma and dendrites arise from, first, the different
subcellular distribution of the multiple Ca2+ channel subtypes, second, the presence in dendrites of a distinct class of N-type Ca2+
channel and, third, a differential sensitivity of dendritic N-type
Ca2+ channels to neurotransmitters and G-protein βγ subunits.
Pharmacological analysis revealed three main components of
high-voltage-activated calcium current in the dendrites: N, R and
P/Q channels. The predominant current (70%) seems to be Ntype, as it was blocked by low concentrations of ω-conotoxin
GVIA. The second largest component (16%) in the dendrites displays the characteristic profile for R-type Ca2+ currents19. This
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current showed moderate inactivation and sensitivity to low concentrations of Ni2+ but was resistant to both ω-conotoxin GVIA
and ω-agatoxin IVA. RT-PCR analysis shows that abundant
amounts of α1E mRNA are present in rat SCG22. However we
failed to identify the R-type current at the single-channel level.
More surprising was the detection in dendrites of a small component of Ca2+ current (14%) that was sensitive to ω-agatoxin
IVA. Although α1A mRNA is found in rat SCG22, ω-agatoxin IVAsensitive currents have never been observed in cell bodies of these
neurons21 (in contrast to mouse SCG35). Although infrequent
channel recordings precluded a definite identification, we occasionally detected channels in dendrites with unitary properties
(∼16 pS) very similar to those previously reported for α1A channels36. Hence, a small population of P/Q-type channels may be
present in the dendrites of sympathetic neurons. However, it
should be pointed out that we detected overlapping blocking
effects of the toxins (see Methods) within a concentration range
typically considered selective. In addition, the P/Q-type component detected here displays low sensitivity to ω-Aga IVA and noninactivating properties (Methods), characteristics related to but
distinct from either P- or Q-type channels. It is therefore possible
that these channels might be splice-variant products of the α1A
gene10 or composed of different ancillary β subunits37.
Overall, our findings suggest that the multiple Ca2+ channel
subtypes are differentially distributed on the soma and dendrites
of sympathetic neurons. This accords with previous studies on
brain neurons showing differential subcellular localization of
Ca2+ channels2,13–15. Whether the distribution we observed in cultured neurons also occurs in vivo remains to be tested.
Although the N-type Ca2+ current is the main current in both
dendrites and cell bodies, there were significant differences in its
properties in these two compartments. First, block by ω-CgTx
GVIA was less potent in dendrites and was best fitted with a double binding site equation, indicating the presence of a second
binding site of lower affinity (∼40 nM versus 2 nM in somata).
Second, the dendritic N-type current had an I–V relationship
that was consistently (although variably) shifted leftward by 2–14
mV. From the single-channel recording presented here, it seems
that these two different profiles result from the existence of more
than one type of N channel. Using the property of CgTx GVIA
sensitivity as the predominant identifying characteristic, we identified two main types of N-type Ca2+ channels: one with high
conductance (19 pS) and distributed in both somatic and dendritic membranes, and one with lower conductance (10.5 pS)
expressed predominently in dendrites. Single-channel recording
and ensemble-averaged currents from both types of channels
showed the appropriate biophysical characteristics of high-voltage-activated Ca2+ currents, although the activation of the lowconductance channel appeared steeper and at slightly more
negative potentials. The latter finding is consistent with the properties of the macroscopic N-type Ca2+ currents in dendrites.
N-type Ca2+ channels show diverse properties in native systems. With CgTx GVIA responsiveness as pharmacological criterion, N-channel conductance of 11 pS is found in cerebellar
granule cells38 whereas channels of 18–20 pS are described in
bullfrog sympathetic neurons31. Also, putative N channels of 12
pS39, 14 pS40, 17 pS16 or 18–20 pS41,42 have been described in various tissues, although some of these may have been misidentified31. In addition, exogenously expressed α1B channels yield
unitary conductances of 6 as well as 19 pS43.
What N-channel molecules may account for the presence of
different N-channel currents in recordings from dendrites and cell
bodies? As the major pharmacological and functional determi676
nants of the N-type Ca2+ current are contained in the pore-forming α1B subunit, it may be hypothetized that both high- and lowconductance channels are composed of the same α1B subunit,
interacting with different auxiliary subunits/tiers molecules (for
example, synapsin). In COS7 cells, expression of α1B subunits,
without β or α2-δ auxiliary subunits, can form low-conductance
channels (∼6 pS)43. Thus, one possibility is that the low-conductance channel encountered in our study is formed by the α1B subunit alone—the ‘naked’ channel. This view is supported by its
enhanced sensitivity to Gβγ modulation43,44. Alternatively, different unitary currents might arise from the expression of structurally
and functionally different channel isoforms. Splice variants of α1B
have been identified in rat SCG and give functionally different N
currents when heterologously expressed9. The variants are distinguishable on the basis of short and localized regions, including
divergence in the intracellular linker from IS6–IIS1 and in the
extracellular loops from IIIS3–IIIS4 and IVS3–IVS4. Interestingly,
these discrete sites are crucial for the interaction of Gβγ (the I–II
linker45,46), the voltage dependence of activation (S3 segments and
flanking regions47) and potentially, for ω-CgTx GVIA binding48,
three of the most notable phenotypic divergences between large
and small channels. Very similar alternative splicing sites in the
α1A gene produce phenotype variants with altered sensitivity to G
proteins, voltage and ω-Aga IVA10.
Functionally, the predominant expression of the low-conductance N-type Ca2+ channel in dendrites may have important implications for dendritic integration. As in most dendritic structures
of central neurons, dendrites of sympathetic neurons are the principal sites for synaptic input. Calcium channels in sympathetic dendrites open during fast excitatory synaptic inputs and during
backpropagating action potentials (unpublished data). The preferential localization of the low-conductance N channels in dendrites would then confer two special properties on the dendritic
Ca2+ currents that differentiate them from the somatic current.
First, their greater sensitivity to G-protein βγ dimers would confer
an increased amount of inhibition by stimulating G-protein-coupled receptors—for example, the stimulation of M4 muscarinic
receptors by synaptically released acetylcholine. Thus, the ability
of these channels to trigger Ca2+ influx would be minimized in
synaptically active zones. Second, the reduced voltage sensitivity of
the G-protein inhibition of these channels would mitigate against
the ability of action potentials (or synaptic potentials) to relieve Gprotein inhibition, as has been noted in somatic recordings49. Hence,
the overall characteristics of these channels make them well suited
for countering unrestricted Ca2+ influx in dendrites.
In conclusion, our study provide evidence that the distribution, properties and modulation of voltage-gated Ca2+ channels
depend on cell compartmentalization. This strengthens the concept that neuronal dendrites form units of integration relatively
independent from the cell body, which are capable of supporting specialized functions.
METHODS
Whole-cell voltage-clamp recording. Rat superior cervical ganglion neurons were dissociated and cultured on glass coverslips as described24.
Neurons were recorded after 3–6 days in vitro using the whole-cell configuration of the patch-clamp technique. Pipets were coated with Sylgard and had resistance of 1.5–2.5 MΩ (soma) and 2.5–5 MΩ (dendrite)
when filled with an internal solution consisting of 120 mM NMDG, 14
mM CsCl, 10 mM HEPES, 5–11 mM EGTA, 0.5–1 mM CaCl2, 10 mM
phosphocreatine, 4 mM MgATP and 0.2 mM Na2GTP (pH 7.34). The
extracellular solution consisted of 130 mM NaCl, 3 mM KCl, 1 mM
MgCl2, 10 mM HEPES, 0.5 µM TTX, 2 mM CaCl2, 1 mM CsCl and 11
mM glucose (pH 7.3). Currents were measured with an Axopatch 200A
nature neuroscience • volume 3 no 7 • july 2000
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articles
© 2000 Nature America Inc. • http://neurosci.nature.com
amplifier (Axon Instruments, Foster City, California), filtered at 1–5 kHz
and leak-subtracted using P/6 protocol. Data were sampled every 50–100
µs. Recording of somatic currents were made on small cells (30–50 pF)
lacking identifiable processes. Series resistances were 3.4 ± 1 MΩ and 8.6
± 2 MΩ before compensation (75–85%) in soma and dendritic segments,
respectively. All potentials were corrected for a junction potential of –10
mV. Double current-clamp recordings were made using an Axoclamp2B amplifier for somatic recording and an Axopatch 200A amplifier for
dendritic recording. In these recordings, a KCl-based internal solution
was used, and TTX was omitted. All experiments were done at 30–32°C.
Isolation of neurites. ‘Isolating’ pipets used to disrupt the connection
between neurites and cell bodies were pulled in one stage using a Flaming-Brown horizontal puller. They typically had long, thin necks and
resistances of 50 MΩ (filled with external solution during experiment).
The micropipet was positioned just above a process of interest (Fig. 1a)
with a Sutter micromanipulator and then moved down with an angle of
incidence of less than ∼35°. The pipet bent (or eventually broke as in
Fig. 1a) when contact was made with the bottom of the recording chamber, which isolated the process from the cell body by compression.
Although this procedure did not appear to damage the integrity of the
cell, at least 10 min was allowed before conducting electrophysiological
recording on the dendrite. To improve the clamp, we used only dendritic segments with a low axial resistance for recording macroscopic currents. In these experiments, the patch pipet was typically sealed onto the
middle of the dendritic segment (about 40 µm away from the position
of the isolating pipet). TTX was used to block the fast-inactivating voltage-gated Na+ current. Although we did not investigate it extensively, the
Na+ current in the dendrites is small compared with that in the cell body.
Sodium currents are commonly observed in dendritic structures16,17.
Large Ca2+-activated chloride, Ih-like inward rectifier and A-type K+ currents were also encountered in dendrites. The use of intracellular NMDG
greatly minimized the impact of the chloride current, and external Cs+
was used to block inwardly rectifying currents.
Cell-attached patch recording. Sylgard-coated electrodes had resistances of
13–25 MΩ when filled with 90 mM BaCl2, 10 mM HEPES, 10 mM TEACl, 3 mM 4-aminopyridine and 0.5 µM TTX (pH 7.3 with TEAOH). Cell
membrane potential was zeroed (–0.7 ± 1 mV, n = 6) using 120 mM KCl, 10
mM HEPES, 5 mM EGTA, 3 mM MgCl2 and 11 mM glucose (pH 7.3).
Where noted, the zeroing solution contained 1 µM Bay K 8644. Voltages
are given in the normal convention and were not corrected for junction
potentials. Uncompensated capacitive and voltage-independent leak currents were corrected offline by subtracting null current sweeps or scaling
traces of smaller amplitude if no nulls. This was done within a single data set,
typically 50 sweeps (4.14–8.3 min recording). Single-channel activities were
elicited by 50-ms steps to minimize inactivation. All experiments were done
at 29°C to facilitate comparison with whole-cell data and also because
recordings at room temperature significantly increased the percentage of
null sweeps (at +20 mV, 5.4% at 29–30°C versus 19% at 20–22°C). Singlechannel current levels were measured by all-points histograms, or by lowvariance analysis if necessary31. Open probability (po) was calculated from
20–30 consecutive sweeps (including nulls) in patches containing only one
channel. During voltage steps to +20, the po of active sweeps was relatively
high (0.4–0.6) for most high- and low-conductance channels. Occasionally, we detected sweeps in which the high-conductance N channel gated with
low po (0.12–0.18 at +20 mV). This gating mode was easily discriminated
from the one produced by G proteins in that it was largely insensitive to
depolarizing prepulses. The frequency of this mode of activity was low
(∼8%) and reminiscent in some respect to the ‘LLP’ (large conductance–low
po) activities previously described42. Patches containing large N channels
with such low po gating were excluded from analysis.
In most patches, we could not resolve single-channel activity at +45
or +50 mV. The Boltzmann fits in Fig. 5d should therefore be considered as tentative; even so the properties of the large channel are consistent
with available information on N channels50. Voltage-dependent shift of
Ca2+ channel activation with Ba2+ pipet solutions was estimated in
macropatch recordings from the shift of the I–V relationships. Shift from
5 mM Ca2+ to 90 mM Ba2+ gave a 31 ± 3 mV and 27 ± 4 mV rightward
shift in soma and dendrites, respectively (n = 3–5).
nature neuroscience • volume 3 no 7 • july 2000
Peptide toxins and pharmacology. The peptide toxins ω-CgTx GVIA
and ω-Aga IVA were dissolved in distilled water and kept in stock aliquots
at –20°C. Single-channel data recorded during intrapipet diffusion of ωCgTx GVIA was achieved using a described method31. For dose–response
curves (Fig. 2e and f), ω-CgTx GVIA was applied for the time necessary
to approach equilibrium binding in 0.1 mM Ca2+ (5–8 min). Calcium
(2 mM, in presence of the toxin) was returned for 2 min to record block.
Somatic and dendritic currents were corrected for a 15% and 22% rundown per 30 min, respectively. Calcium currents identified in this study
as P/Q were not sensitive to ω-Aga IVA at concentrations less than 80
nM. In addition, the ω-Aga IVA-sensitive component was still observed
but reduced by ∼30% when ω-Aga IVA was applied after ω-CgTx GVIA.
ω-Aga IVA block of Ca2+ currents in dendrites was not reversed by the
20-ms prepulse to +90 mV used in Fig. 3a. All drugs were from Sigma
(St. Louis, Missouri), except nifedipine and nimodipine (Calbiochem,
San Diego, California).
Nuclear microinjection of cDNA. Plasmids were diluted into Ca2+-free
KCl-based solution containing 0.5 % FITC-dextran to a final concentration of 200 µg per ml and injected into the nucleus of sympathetic neurons as described24,25. The cDNAs for bovine β1 and γ2 G-protein subunits
and retinal α transducin were provided by G. Milligan (Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK). These
cDNAs were subcloned using standard procedures into the pcDNA3
expression vector (Invitrogen, San Diego, California). Recordings were
made 24 h after cDNA delivery.
Immunocytochemistry. Immunodetection was done essentially as
described25. Briefly, cells were fixed for 20 min and then incubated for 1 h
at room temperature with mouse monoclonal anti-MAP2 antibody (1:500)
or rabbit anti-tau antiserum (1:100; Sigma). Bound antibodies were then
detected using FITC- or TRITC-conjugated secondary antibodies.
ACKNOWLEDGEMENTS
We thank M. Dayrell for technical assistance and G. Milligan for the gift of
Gα-transducin, Gβ1 and Gγ2 cDNAs. This work was supported by The
Wellcome Trust.
RECEIVED 3 MARCH; ACCEPTED 16 MAY 2000
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