First published online December 2, 2005
Journal of Experimental Biology 208, 4747-4756 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01967
Biophysical properties of voltage-gated Na+ channels in frog parathyroid cells and their modulation by cannabinoids
Yukio Okada1,*,
Kotapola G. Imendra4,
Toshihiro Miyazaki2,
Hitoshi Hotokezaka3,
Rie Fujiyama1,
Jorge L. Zeredo1,
Takenori Miyamoto5 and
Kazuo Toda1
1 Integrative Sensory Physiology, Graduate School of Biomedical Sciences,
Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
2 Oral Cytology and Cell Biology, Graduate School of Biomedical Sciences,
Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
3 Orthodontics and Biomedical Engineering, Graduate School of Biomedical
Sciences, Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
4 Department of Physiology, Faculty of Medicine, University of Rhuna, Galle,
Sri Lanka
5 Department of Chemical and Biological Sciences, Faculty of Science, Japan
Women's University, Bunkyo-ku, Tokyo 112-8681, Japan
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Fig. 1. Freshly isolated frog parathyroid cells are white and oval-shaped in this
phase-contrast image. Two large cells in the center of the image are the
erythrocytes. Bar, 50 µm.
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Fig. 2. A representative example of perforated whole-cell current of a frog
parathyroid cell in normal saline solution. (A) Transient inward currents were
elicited in response to 15 ms voltage steps between –74 to +56 mV in 10
mV increments from a holding potential of –84 mV. The leak currents were
not subtracted from the current traces. (B) Pooled current–voltage
(I–V) relationships for the inward currents (N=17)
elicited by the voltage steps. Values are means ±
S.E.M.
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Fig. 3. Effects of the elimination of external Na+ (A) and the addition
of 3 µmol l–1 TTX to external normal saline solution (B)
on the transient inward currents. The currents were elicited by 15 ms step
pulses from a holding potential of –84 mV to a test potential of
–24 mV.
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Fig. 4. Voltage dependence of activation of the Na+ currents, determined
by I–V relationships for the Na+ currents elicited
by the voltage steps. Smooth curves are simple Boltzmann functions. (A)
V1/2=–45.7 mV, k=–6.4 mV for
perforated recording (filled circles), V1/2=–46.1
mV, k=–5.2 mV for conventional recording (open circles). (B)
V1/2=–35.5 mV, k=–7.6 mV for 0.5 mmol
l–1 GTP S (filled triangles),
V1/2=–45.1 mV, k=–4.4 mV for 1 mmol
l–1 GDPßS (open triangles). For comparison, the broken
line for conventional recording is plotted in B. The values are means ±
S.E.M. obtained from 6–18 cells.
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Fig. 5. Voltage dependence of steady-state inactivation of the Na+
currents. (A) The dependence was determined by measuring peak current elicited
by a single depolarization to –34 mV from a range of 200 ms conditioning
voltages. (B) V1/2=–79.8 mV, k=8.2 mV for
perforated recording (filled circles),
V1/2=–86.3 mV, k=9.4 mV
for conventional recording (open circles). (C)
V1//2=–98.4 mV, k=9.6 mV for 0.5 mmol
l-1 GTPS (filled triangles),
V1/2=–86.1 mV, k=10.0 mV for 1 mmol
l–1 GDPßS (open triangles). For comparison, the broken
line for conventional recording is plotted in B. The values are means ±
S.E.M. obtained from 7–14 cells.
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Fig. 6. Time course of recovery from inactivation. (A) Inward currents recorded
using a double pulse protocol in which a 20 ms control pulse from –84 to
–24 mV was followed by a second identical voltage pulse. (B) The plots
show the recovery time course of the Na+ currents. The curves
represent the fits of a single exponential function, giving values of time
constants of 9.6 ±1.2 ms (N=5) for perforated mode (filled
circles), 13.1±0.7 ms (N=5) in conventional mode (open
circles) and 16.5±2.4 ms (N=5) for the condition containing
0.5 mmol l–1 GTPS in the internal solution (open
triangles).
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Fig. 7. Effects of WIN 55,212-2 and 2-AG ether on the Na+ currents. (A)
Reversible inhibition of the Na+ current by 10 µmol
l–1 WIN 55,212-2. The currents were elicited by a 20 ms pulse
from a holding potential of –84 mV to a test potential of –24 mV.
The current traces labelled a–c were obtained at the times indicated by
the same letter on the time course. (B) Representative example of time course
of the current signal. (C) Na+ current magnitudes before and after
superfusion with each drug. Values are means ±
S.E.M. Numerals within parentheses are number
of the cells sampled.
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Fig. 8. Effects of WIN 55,212-2 and 2-AG ether on activation and inactivation of
the Na+ currents. (A) Voltage dependence of activation before and
after superfusion with 10 µmol l–1 WIN 55,212-2.
V1/2=–48.6 mV, k=–5.1 mV for control
(open squares), V1/2=–37.0 mV, k=–5.0
mV for WIN 55,212-2 (filled circles). (B) Voltage dependence of inactivation
before and after superfusion with 10 µmol l–1 WIN 55,212-2
(filled circles). V1/2=–76.9 mV, k=8.1 mV
for control (open squares), V1/2=–94.4 mV,
k=9.0 mV for WIN 55,212-2. (C) V1/2 of activation
before (white bars) and after superfusion with each drug (hatched bars). (D)
V1/2 of inactivation before (white bars) and after
superfusion with each drug (hatched bars). Values are means ±
S.E.M. Numerals within parentheses are number
of the cells sampled.
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Fig. 9. Effects of PDBu and forskolin on the Na+ currents. (A)
Representative example of time course of the current signal after superfusion
with 10 µmol l–1 PDBu. (B) Representative example of time
course of the current signal after superfusion with 10 µmol
l–1 forskolin. (C) Na+ current magnitude before
(white bars) and after superfusion with each drug (hatched bars). The currents
were elicited by 20 ms pulse from a holding potential of –84 mV to a
test potential of –24 mV. Values are means ±
S.E.M. Numerals within parentheses are number
of the cells sampled.
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Fig. 10. Effect of 10 µmol l–1 PDBu on activation and
inactivation of the Na+ currents. (A) Voltage dependence of
activation. V1/2=–47.7 mV, k=–5.3 mV
for control (open squares), V1/2=–44.3 mV,
k=–6.0 mV for PDBu (filled circles). (B) Voltage dependence of
inactivation. V1/2=–81.8 mV, k=8.1 for
control (open squares), V1/2=–93.7 mV,
k=9.0 mV for PDBu (filled circles). Values are mean ±
S.E.M. obtained from 5–8 cells.
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Fig. 11. Effect of WIN 55,212-2 on the Na+ currents in the presence of an
inhibitor of PKC. (A) Representative example of time course of the current
signal. The currents were elicited by a pulse from a holding potential of
–84 mV to a test potential of –24 mV. (B) Mean values of
Na+ current magnitudes before (white bar) and after superfusion
with 10 µmol l–1 chelerythrine and 10 µmol
l–1 WIN 55,212-2 (hatched bar). Numerals within parentheses
are number of the cells sampled. (C) Voltage dependence of activation.
V1/2=–42.0 mV, k=–6.1 mV for control
(open squares), V1/2=–30.7 mV, k=–8.0
mV for chelerythrine plus WIN 55,212-2 (filled circles). (D) Voltage
dependence of inactivation. V1/2=–83.5 mV,
k=8.4 mV for control (open squares),
V1/2=–103.8 mV, k=7.6 mV for chelerythrine
plus WIN 55,212-2 (filled circles). The values are mean ±
S.E.M. obtained from three cells.
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© The Company of Biologists Ltd 2005
