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4 m A. Membrane current. 0 current. Membrane PD. 200 mV. 0 PD. amplifier. 1sec. Voltage commands. Modeling the current-voltage characteristics of Ca 2+ - activated Cl - channels of salt-tolerant charophyte Lamprothamnium. Mary J. Beilby and Virginia A. Shepherd

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slide1

4 mA

Membrane current

0 current

Membrane PD

200 mV

0 PD

amplifier

1sec

Voltage

commands

Modeling the current-voltage characteristics of Ca2+- activated

Cl- channels of salt-tolerant charophyte Lamprothamnium

Mary J. Beilby and Virginia A. Shepherd

Biophysics Department, School of Physics, the University of NSW, NSW 2052

Fit parameters

Introduction

modeling

Results

Ca2+-activated Cl- channels play an important role in depolarizing phase of charophyte action potential and in the hypotonic regulation in salt-tolerant charophytes. At the time of hypotonic challenge the Cl- channels in young Lamprothamnium cells with thin polysaccharide mucilage stay open for sufficient time to apply current-voltage (I/V) analysis (1). The modelling of these I/V profiles provides insights into characteristics of the Cl- channels and the cytoplasmic/vacuolar Cl- concentrations.

Fitting the Cl- current

Large conductance K+ channels, which contribute to the hypotonic regulation, were blocked by 20 mM TEA (2).

The total current was modeled as sum of up to four currents (3):

Ca2+- activated Cl- current iCl

inward K+ rectifier iirc

outward K+ rectifier iorc

linear background current ibackground

The rectifiers did not contribute to total current in this set of data.

GHK only

GHK with Boltzmann distribution of open channels, V50+ = -40 mV

V50- = -255 mV

Thick gray line: GHK with V50+ = - 40mV only

Method

Apparatus to gather I/V data

ibackground

The colour coding here refers to the time after hypotonic exposure. The steady state I/V profile contains a H+ pump component. The pump began to contribute to the total current again at later times, not shown here.

The cells were acclimated in 1/3 seawater and the hypotonic medium was 1/6 seawater (2). The thick cell wall makes internal electrode position uncertain (2).

Boltzmann probability distribution

simulates PD-dependent channel closure

iCl with and without Boltzmann distribution

Conclusions

  • To model the low reversal PD at the time of Cl- flow, [Cl-]i has to be set to 160 -180 mM, close to vacuolar concentration (1). We assume that cytoplasmic Cl- has already increased from vacuole inflow in the initial 9 min.
  • The GHK PD - dependence is too weak to model iCl. PD - dependent channel closure needs to be invoked to fit the data.
  • The Ca++ concentration, necessary for the Cl- channels to open, is less than that which inhibits cytoplasmic streaming. The time of hypotonic streaming inhibition is similar to that seen in L. succinctum (4).
  • The NClPCl parameter declines with time, presumably as [Ca++]cyt drops.
  • The background current, which is thought to flow through mechanosensitive channels (5), exhibits transient depolarization of Vbackground and increase of gbackground, as observed before at the time of hypotonic regulation (5).

V membrane PD

R gas constant

T temperature in K

F Faraday’s constant

z ion valency

zggating charge

V50 half activation PD

NP number of channels, channel permeability, treated as single parameter

[X]i

[X]oconcentration of ion X inside or outside the cell

gbackground background conductance

Vbackground reversal PD of background current

gbackground is constant, so

total conductance reflects gCl

with and without Boltzmann distribution

The I/V profiles at the time of hypotonic effect

: the bipolar staircase

34 min

19 min

15 min 9 min

11 min

References

ibackground = gbackground (V – Vbackground)

(1) Beilby MJ, Cherry CA and Shepherd VA, 1999, Dual turgor regulation response to hypotonic stress in Lamprothamnium papulosum. Plant, Cell and Environment22, 347 - 359

(2) Beilby MJ and Shepherd VA, 1996, Turgor regulation in Lamprothamnium papulosum. I. I/V analysis and phamacological dissection of the hypotonic effect. Plant, Cell and Environment19, 837 - 847

(3) Beilby MJ and Shepherd VA, 2001, Modeling the current-voltage characteristics of charophyte membranes: II. The effect of salinity on membranes of Lamprothamnium papulosum. J. Membrane Biol.181, 77 - 89

(4) Okazaki Y, Ishigami M and Iwasaki N, 2002, Temporal relationship between cytosolic free Ca2+ and membrane potential during hypotonic turgor regulation in a brackish water charophyte Lamprothamnium succinctum. Plant Cell Physiol.43, 1027-35

(5) Shepherd VA, Beilby MJ and Shimmen T, 2002, Mechanosensory ion channels in charophyte cells: the response to touch and salinity stress. Eur. Biophys J. 31, 341 - 355

Example of data fit:

The data points in (a) come from a cell exposed to 1/6 seawater for 19 min. The black line is the total fitted current, the coloured lines are from the separate transporters as described above. (b) the G/V curve calculated the I/V data by differentiation.

The PD was falling too rapidly to do I/V scan in first 9 min of hypotonic exposure. Cytoplasmic streaming slowed after 3 min of hypotonic challenge, stopped totally after 7 min, large shards of cytoplasm started moving after 13 min, almost back to normal after 18 min.

Each pulse contains 50 to 70 data points. The last 10 data points for each current and PD pulses are averaged to yield one point on the I/V profile.