Modeling the current-voltage characteristics of Ca 2+ - activated
1
Modeling the current-voltage characteristics of Ca Modeling the current-voltage characteristics of Ca 2+ 2+ - - activated activated Cl Cl - - channels of salt-tolerant charophyte channels of salt-tolerant charophyte Lamprothamnium Lamprothamnium Mary J. Beilby and Virginia A. Shepherd Biophysics Department, School of Physics, the University of NSW, NSW 2052 Ca 2+ -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 Fit parameters (1) Beilby MJ, Cherry CA and Shepherd VA, 1999, Dual turgor regulation response to hypotonic stress in Lamprothamnium papulosum. Plant, Cell and Environment 22, 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 Environment 19, 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 Ca 2+ 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 Introduction Introduction Method Method modeling modeling Results Results Conclusions Conclusions References References amplifier Membrane PD Voltage commands Membrane current 1sec 0 PD 200 mV 0 current 4 A Apparatus to gather I/V data : the bipolar staircase 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. 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). 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): Ca 2+ - activated Cl - current i Cl inward K + rectifier i irc outward K + rectifier i orc e 1 1 1 P ) e 1 ( RT ) e ] K [ ] K ([ V ) zF ( P N P RT ) 50 V V ( F g z o RT zFV RT zFV o i 2 K K irc , o irc i RT ) 50 V V ( F g z o RT zFV RT zFV o i 2 K K orc , o orc e 1 1 P ) e 1 ( RT ) e ] K [ ] K ([ V ) zF ( P N P i e 1 1 P e 1 1 1 P model GHK ) e 1 ( RT ) e ] Cl [ ] Cl ([ V ) zF ( P N P P RT ) 50 V V ( F g z o RT ) 50 V V ( F g z o RT zFV RT zFV o i 2 Cl Cl o o Cl i i background = g background (V – V background ) 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. GHK only GHK with Boltzmann distribution of open channels, V 50+ = -40 mV V 50- = -255 mV i backgrou nd i Cl with and without Boltzmann distribution Boltzmann probability distribution simulates PD-dependent channel closure V membrane PD R gas constant T temperature in K F Faraday’s constant z ion valency z g gating charge V 50 half activation PD NP number of channels, channel permeability, treated as single parameter [X] i [X] o concentration of ion X inside or outside the cell g background background conductance V background reversal PD of background current g background is constant, so total conductance reflects g Cl with and without Boltzmann distribution Thick gray line: GHK with V 50+ = - 40mV only The rectifiers did not contribute to total current in this set of data. The I/V profiles at the time of hypotonic effect [Cl - ] o time Cl - current i irc i orc [Cl - ] i g back ground V back ground E Cl E R N Cl P Cl V 50+ V 50- z g- z g+ N KP K V 50 z g N KP K V 50 z g mM/ min x 10 -7 m 3 .s -1 m V m V x 10 -7 m 3 .s -1 m V x 10 -7 m 3 .s -1 mV mM S.m -2 mV mV mV 184 / steady state 4.9 -275 1 60 115 2 4.2 -100 -118 92 / 9 m in 0.425 +35 -235 1 0.5 165.0 6.2 -25 +14.7 +2.1 92 / 11 min 0.34 -40 -260 1 0.5 2.0 210 2 180.0 7.9 -20 +16.9 -4.5 92 / 15 min 0.285 -65 -310 1 0.5 2.0 200 2 180.0 6.7 -30 +16.9 -12.9 92 / 19 min 0.17 -50 -300 1 0.5 8.0 -255 1 2.0 190 2 80.0 6.2 -50 -3.5 -37.1 92 / 34 min 8.0 -267 1 0.52 88 2 4.7 -80 -78.8 34 min 19 min 15 min 9 min 11 min • 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 i Cl . 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 N Cl P Cl 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 V background and increase of g background , as observed before at the time of hypotonic regulation (5). 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. 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.
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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 - PowerPoint PPT Presentation
Transcript of Modeling the current-voltage characteristics of Ca 2+ - activated
Modeling the current-voltage characteristics of CaModeling the current-voltage characteristics of Ca2+2+- activated- activatedClCl-- channels of salt-tolerant charophyte channels of salt-tolerant charophyte LamprothamniumLamprothamnium
Mary J. Beilby and Virginia A. ShepherdBiophysics Department, School of Physics, the University of NSW, NSW 2052
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
Fit parameters
(1) Beilby MJ, Cherry CA and Shepherd VA, 1999, Dual turgor regulation response to hypotonic stress in Lamprothamnium papulosum. Plant, Cell and Environment 22, 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 Environment 19, 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
IntroductionIntroduction
MethodMethod
modelingmodeling ResultsResults
ConclusionsConclusions
ReferencesReferences
amplifier
Membrane PD
Voltagecommands
Membrane current
1sec
0 PD
200 mV
0 current
4 A
Apparatus to gather I/V data
: the bipolar staircase
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.
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).
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 iircoutward K+ rectifier iorc
linear background current ibackground
e1
11P
)e1(RT
)e]K[]K([V)zF(PNP
RT
)50VV(Fgzo
RTzFV
RTzFV
oi2
KKirc,oirc
i
RT
)50VV(Fgzo
RTzFV
RTzFV
oi2
KKorc,oorc
e1
1P
)e1(RT
)e]K[]K([V)zF(PNP
i
e1
1P
e1
11P
modelGHK
)e1(RT
)e]Cl[]Cl([V)zF(PNPP
RT
)50VV(Fgzo
RT
)50VV(Fgzo
RTzFV
RTzFV
oi2
ClClooCl
i
ibackground = gbackground (V – Vbackground)
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.
GHK only
GHK with Boltzmann distribution of open channels, V50+ = -40 mVV50- = -255 mV
ibackground
iCl with and without Boltzmann distributionBoltzmann probability distributionsimulates PD-dependent channel closure
V membrane PD R gas constantT temperature in KF Faraday’s constantz ion valencyzg gating chargeV50 half activation PD
NP number of channels, channel permeability, treated as single parameter
[X]i [X]o concentration 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
Thick gray line: GHK with V50+ = - 40mV only
The rectifiers did not contribute to total current in this set of data.
The I/V profiles at the time of hypotonic effect
[Cl-]o
time Cl- current i irc iorc [Cl-]i
gbackground
Vbackground
ECl ER
NClPCl V50+ V50- zg- zg+ NKPK V50 zg NKPK V50 zg
mM/ min
x 10-7
m3.s-1mV mV x 10-7
m3.s-1mV x 10-7
m3.s-1mV mM S.m-2 mV mV mV
184 / steady state
4.9 -275 1 60 115 2 4.2 -100 -118
92 / 9 min 0.425 +35 -235 1 0.5 165.0 6.2 -25 +14.7 +2.1
92 / 11 min 0.34 -40 -260 1 0.5 2.0 210 2 180.0 7.9 -20 +16.9 -4.5
92 / 15 min 0.285 -65 -310 1 0.5 2.0 200 2 180.0 6.7 -30 +16.9 -12.9
92 / 19 min 0.17 -50 -300 1 0.5 8.0 -255 1 2.0 190 2 80.0 6.2 -50 -3.5 -37.1
92 / 34 min 8.0 -267 1 0.52 88 2 4.7 -80 -78.8
34 min
19 min
15 min 9 min
11 min
• 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).
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.
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.
