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Homework #3 for Yue section of Systems Bioengineering I, Fall 2014, page 1 of 8

Homework 3: More gating and action-potential generation


(100 points in total)

1. (80 points total) ION-CHANNEL GATING. As a star engineer at Frankenstein Inc, you have
been asked to build the first artificial spiking cell. Inspired by your lectures on the Hodgkin-
Huxley model, you decided to use a voltage-gated Na channel (with 3 m activation gates and
1 h inactivation gate) in your cell. Your Na channel has a conductance (g
Na
) of 32 pS and
you inserted precisely 500 channels (hand-counted and in the correct orientation). The
channel was designed to have a steady-state inactivation curve (h(), black); and steady-
state activation (m
3
(), blue) as shown in Figure 1.



















Figure 1. Steady state probabilities of activation (m
3
()) and inactivation (h())

For your first experiment, you verified that your Na channel was functional by recording the
whole-cell currents after insertion of only the Na channels. The extracellular and intracellular
solutions you used were as follows:









Table 1. Intracellular and extracellular solutions

Extracellular Intracellular
150 mM NaCl
4 mM KCl
1 mM MgCl
2

0.5 mM CaCl
2

10 mM pH buffer
(pH adjusted to 7.4)
15 mM NaCl
140 mM KCl
1.5 mM MgATP
10 mM pH buffer

(pH adjusted to 7.4)
p
r
o
b
a
b
i
l
i
t
y

Homework #3 for Yue section of Systems Bioengineering I, Fall 2014, page 2 of 8

Your first recordings are shown below. In the following subsections, carefully deduce the
gating parameters at V
step
= -10 mV, o
m
, |
m
, o
h
, |
h
. Then, predict and plot the response of
these channels to an entirely new voltage protocol.



















Figure 2. Whole-cell Na current from your first artificial cell.

A. (5 pts) Derive the I* current-voltage relationship for the sodium current, assuming that all
channels are open and not inactivated (m = 1, h = 1) and given the experimental conditions
described above. For simplicity, you may assume a battery-resistor model of permeation.

V
reversal
= 25*ln(150/15) = 57.6 mV

I* = N
channel
*g
Na
*(V-V
reversal
)
= 500 * 0.032 pA/mV * (V 57.6)
= 16*(V 57.6)



-100 mV
-10
-100
Homework #3 for Yue section of Systems Bioengineering I, Fall 2014, page 3 of 8

1B. (15 pts) Using the relation from 1A, convert the trace into a time waveform for the
probability of being open. Plot the waveform carefully, presumably using computer software like
excel or MATLAB.














I* ( -10 mV) = -1081.6 pA; I*(-100 mV) = -2521.6 pA;

Obtained by dividing the whole-cell current (Figure 2) by I* (-10 mV) between 0 and 8 msec and
I* (-100 mV) for t < 0 msec and t > 8 msec

1C. (20 pts) Fit a time waveform proportional to the timecourse of h (probability that h gate is
enabled) for the time period between 0 and 8 msec. Reproduce the open probability trace from
1B and show the fit in red, give the parameters and form of the model below, and interpret these
parameters to deduce the values of o
h
and |
h
at V = -10 mV. For t < 0 ms, Assume that the
voltage was held at -100 mV for a long period (long enough to allow m and h gates to reach
steady-state)
The exact steady-state values can be obtained from excel spreadsheet.
h(t) = h(, -10mV) + (h(, -100mV) - h(, -10mV))*exp(-t/t) where t = 1/(o
h+
|
h
).
h(t) = 0.0074 + (1 - 0.0074)*exp(-t/t)

h(, -10mV) = o
h
/(o
h+
|
h
) ~ 0.0074.

Moreover, h(t) = 0.0074 + (1 - 0.0074)*exp(-t/1.1) is a good fit,
as shown in red

Thus o
h
= 0.0074/1.1 = 0.0067 ms
-1

and o
h
+ |
h
= 1/1.1 => |
h
~ 0.9 ms
-1
.


(Full points even if o
h
was deduced to be ~ 0 and o
h
was approximated to 0.9).




0
0.2
0.4
0.6
0.8
1
0 2 4 6 8
Homework #3 for Yue section of Systems Bioengineering I, Fall 2014, page 4 of 8

0
0.5
1
0 2 4 6 8
1D. (20 pts) Use the fit in part B to determine the time waveform for m (probability of m gates
being enabled) between 0 and 8 msec. Deduce the values for o
m
and |
m


Since the overall probability waveform (part 1B) is given by m
3
* h, the timecourse of m
3
could
be obtained by dividing through the overall probability waveform by the timecourse of h as
determined in part C. This procedure yields the blue line shown in figure. To get m, we take the
third root of the blue line, yielding the thick gray line.
m(t) = m(, -10mV) + (m(, -100mV) - m(, -10mV))*exp(-t/t
m
) where t
m
= 1/(o
m+
|
m
).
But from excel sheet, m(, -100mV) = 0.

m(, -10mV) = (.94)^(1/3) = 0.98 = o
m
/(o
m+
|
m
)
So we fit the gray waveform with the function
m(t) = 0.98*(1-exp(-t/0.265) (red fit)

Hence, o
m
+ |
m
= 1/0.265 = 3.76 ms
-1

o
m
/ (o
m
+ |
m
) = 0.98.

Solving two equations and two unknowns, o
m
= 0.98*3.76 = 3.7 ms
-1
and |
m
= 0.07 ms
-1
.

1E. (10 pts) Having deduced the parameters above in parts A through C, see if you can predict
the behavior of the same cell for a different voltage protocol. Here, assume the channel has
reached steady state with respect to a holding potential of +100 mV. Now plot the Na current
between 0 and 8 ms, when the voltage is returned to -10 mV.
If we hold at +100 mV for a long time the Na channels will inactivate (h(,+100mV) = 0. When
the voltage is returned to -10 mV, the Na channels will still be inactivated. So the whole-cell
current will be ~ 0 as shown in figure.

But if you wanted to be more precise,
m(t) = m(,-10mV) + (m(,+100mV) - m(,-10mV))*exp(-t/t
m
)
m(t) = 0.98 + (0.02)*exp(-t/0.265).
h(t) = h(,-10mV) + (h(,+100mV) - h(,-10mV))*exp(-t/t
h
)
h(t) = 0.0074 0.0074*exp(-t/1.1).
I(t) = m(t)^3*h(t)*g
Na
*N
channels
*(V-V
reversal
).

This would result in a very small current (steady-state ~ 8 pA).











Homework #3 for Yue section of Systems Bioengineering I, Fall 2014, page 5 of 8

1F. (10 pts) Though satisfied with the success of your initial experiments, you tried to repeat
them again as any good scientist would. However, this time around, your fellow engineers had
poor quality control resulting in 20% of your Na channels (i.e. 100/500) being defective. These
defective channels lacked an inactivation gate (h) but their activation gate (m) was normal. Plot
the whole cell currents in response to the voltage waveform shown below. Assume your
recording solutions are the same as before.



















I
Na
= 0.8*N
channels
*g
Na
*m
3
(t)*h(t) (V - V
reversal
) +0.2*N
channels
*g
Na
*m
3
(t)(V - V
reversal
)

where m(t) and h(t) were determined in 1C and 1D.

m(t) = m(,-10mV) + (m(,-100mV) - m(,-10mV))*exp(-t/t
m
)
m(t) = 0.98 * (1 exp(-t/0.265))

h(t) = h(,-10mV) + (h(,-100mV) - h(,-10mV))*exp(-t/t
h
)
h(t) = h(t) = 0.0074 + (1 - 0.0074)*exp(-t/1.1).

So then,
I
Na
= 12.8*m
3
(t)*h(t) (V 57.6) +3.2* m
3
(t)(V 57.6)







-100 mV
-10
-100
Homework #3 for Yue section of Systems Bioengineering I, Fall 2014, page 6 of 8

2. (20 points total) ACTION-POTENTIAL GENESIS

The defective Na channels in your artificial cell were a premonition, but it takes a lot more than a
mere 20% non-inactivating (persistent) Na channels to deter you from achieving your goal for
creating a spiking artificial cell. So you inserted 50 potassium (K) channels (always open) with a
conductance of 40 pS into your cell. The intracellular and extracellular solutions are as described
in Table 1 (page 1). You have an electrode in the cell to record and manipulate voltage.

2A. (2 pts) Precisely write the equation for the whole cell K current in your artificial cell. You
may assume a battery-resistor model for permeation.

I
K
= N
channel,K
*g
K
*(V-V
reversal,K
)
V
reversal,K
= 25mV*ln(4/140) = -88.9 mV
I
K
= 50*0.04 pA/mV*(V+88.9)

2B. (5 pts) On the graph below, carefully plot the quasi-instantaneous current-voltage relations
for I
K
, I
Na
and I
K
+ I
Na
before the Na channel inactivation sets in (h = 1) with quantitative
precision. Mark all fixed points of the system and label them as stable or unstable. What is the
voltage threshold for activation (V
th
)? If you depolarize the cell to -20 mV by a stimulus what
voltage will the system stabilize at?

You may assume that Na channel activation (m) gates equilibrate with changes in voltage faster
than the inactivation gate (h). Also remember that 20% of the Na channels lack an h gate but
have normal m gates.
I
Na
= 0.8*N
channel,Na*
g
Na
*m
3
(,V)*h(V)*(V-V
reversal
) + 0.2* N
channel,Na*
g
Na
*m
3
(,V)* (V-V
reversal
)
But h = 1. So, I
Na
= 16*m
3
(,V)*(V-57.6). Red = I
K
, black = I
Na
, blue = I
Na
+ I
K
.

V
th
~ -35 mV,
so if you depolarize to -20 mV, the voltage will stabilize near the stable fixed point at +40mV.

















I
Na

I
K

I
Na
+I
K

Stable fixed point
Unstable fixed point
Homework #3 for Yue section of Systems Bioengineering I, Fall 2014, page 7 of 8

2C. (5 pts) Now suppose you let the system evolve after the stimulus and Na channels with an
inactivation gate (h) are fully inactivated (h = 0). On the graph below, carefully plot the quasi-
instantaneous current-voltage relations for I
K
, I
Na
and I
K
+ I
Na
with quantitative precision. Mark
all fixed points of the system and label them as stable or unstable. What voltage does the system
stabilize at?

























I
Na
= 0.2* N
channel,Na*
g
Na
*m
3
(,V)* (V-V
reversal
)
= 3.2 * m
3
(,V)* (V-57.6)

Here the non-inactivating fraction of Na current is sufficiently large that there are still 3 fixed
points, so the system is not able to relax to the resting potential (stable point at -90 mV). Thus
the system would stabilize at ~ 0 mV.

I
Na

I
K

I
Na
+I
K

Stable fixed point
Unstable fixed point
Homework #3 for Yue section of Systems Bioengineering I, Fall 2014, page 8 of 8

2D. (4 pts) Following part 2C, you realized that your artificial cell is stuck midstream in a
perpetual action potential. Devise a strategy to allow the cell to repolarize, using methods from
which the company Medtronic would profit. You are not allowed to change the channel
composition in your cells or the bath solutions.

To repolarize the cell, you simply need withdraw current to bring the voltage to be just below the
unstable fixed point at ~ -25 mV. Now the system can repolarize back to the resting potential
(stable fixed point at -90 mV).












2E. (4 pts) Assuming 20% of your Na channels continue to be defective (lack h gate) as a
consequence of the manufacturing process, how would you redesign your system to allow it to
spike reliably.

One way would be to increase the number of K channels (in this case to 70 channels). Now
again, the K current is large enough to overwhelm the non-inactivating Na current resulting in a
single stable fixed point at -90 mV as shown below.















You could also decrease the number of Na channels to about 350, then again the system will
evolve to have only a single stable fixed point at -90 mV.