1. Modeling the current-voltage characteristics of Ca 2+ - 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 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 Method modelingResults Conclusions 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 linear background current i background 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 background 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 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.