Lecture 12: Salt tolerance

High permeability of cell membranes to water leads to problems for cells exposed to media, where the amount of solute (osmolarity) fluctuates.  Many animal tissues gain protection by secreting sulphated polysaccharide gels (e.g. mucus in the nose, slime on snails etc., agar) The polysaccharide gels are also found on some marine algae and my research group found these on salt-tolerant charophyte Lamprothamnium. However, the gel presence cushions the osmotic shock, but the cells still need to adjust internal ion concentrations to stop water entering (hypotonic shock) or leaving (hypertonic shock).

Hypotonic regulation

The medium becomes more dilute and water flows into the cells, which might lead to cell bursting. The acute response of most cells, whether animal, plant or fungal involves opening Cl^- and K⁺ channels, leading to membrane p.d. depolarization, efflux of Cl^- and K⁺, water loss and restoration of normal cell volume and turgor pressure. The details of channel activation are still being worked out.

In charophyte Lamprothamnium, young cells on top of the plant with small layer of polysaccharide gel, show great conductance increase over about one hour, which corresponds to outflow of Cl^- first, followed by K⁺ outflow through large conductance (maxi) K⁺ channels. We hypothesize that the cell is alerted to the osmotic shock by the membrane stretching and “stretch-activated” (SA) channels opening. These channels may not be very selective.

The Cl^- channels need high concentration of Ca²⁺ in the cytoplasm to open. The inflow of Ca⁺⁺ from the outside can be blocked by adding some La³⁺ in the outside medium. In steady state Ca²⁺ is very low in the cytoplasm and the cytoplasm streams around the cell. As Ca²⁺ concentration increases, the streaming is inhibited. The K⁺ channels can be blocked by TEA (tetra-ammonium).

The cells at the base of Lamprothamnium plant with thick gels, do not exhibit conductance increases, but adjust their K⁺ and Cl^- internal concentrations within about a day.

Hypertonic regulation

The medium becomes more concentrated and water flows out of the cells making them shrink. Cells need to be able to increase internal solute concentration. Again, K⁺ and Cl^- are employed for this purpose. This time ion flows are against the electrochemical gradient and energy is needed. In Lamprothamnium, inwardly rectifying K⁺ channels open at more positive membrane p.d.s. The action of the proton pump, which starts to work harder, makes the membrane p.d. more negative and K⁺ flows into the cell (coupling of flows). The Cl^- is thought to enter cells as symport with protons. Thus the proton pump, powered by ATP, is the primary mover of hypertonic regulation. If the pump is inhibited by metabolic inhibitors (DES – diethyl stilbestrol) hypertonic regulation does not happen.

Background notes on Lamprothamnium research

Lamprothamnium is a salt-tolerant charophyte surviving in media from freshwater to twice seawater. It is found in Tuggerah Lake system in central NSW. Cells about 2 cm long are mounted in three-compartment chamber, the compartments isolated by grease. Electrical current can the be passed across the compartments and the cell p.d. can be controlled. The cell p.d. is measured by inserting a glass microelectrode filled with KCl into the vacuole or cytoplasm. The membrane p.d. is “clamped” to a series of steps (the bipolar staircase), which allows the experimenter to obtain current-voltage (I/V) profile of the cell.

The total I/V curve can be modeled as an assembly of transporters. The conductance-voltage characteristics can be obtained from the data by differentiation. This approach is useful, as parallel conductances are additive.

Main transporters

K⁺ channels: inward (irc) and outward (orc) rectifiers only open at very negative and very positive p.d.s, respectively. They can be modeled by GHK equation multiplied by Boltzman distribution of open probability P_o:

   (12.1)

where z_g is number of gating charges (fixed to the protein transporter) and Dy₅₀ is the half activation p.d.

These types of K⁺ channels are ubiquitous in most cells preventing the cell p.d. to stray too far from steady state levels. If the K⁺ concentration in the medium rises over about 1 mM, large conductance K⁺ channels become the dominant conductance (K⁺ state) and the resting p.d. of the cell comes close to E_K. These channels close if the cell p.d. moves too far positive or negative, exhibiting a typical I/V profile, which can be modeled by GHK equation and two Boltzman distributions (as in eqns. 12.1).

If the proton pump is working hard, the I/V curve is again typical of this transporter and the resting p.d. is very negative (-200 to – 250 mV, pump state). The pump is modeled by the Hansen et al enzyme kinetics (eqn. 11.2). In some cells the pump does not seem to be working (or can be inhibited). These cells exhibit linear I/V characteristics with reversal p.d. (resting p.d.) of ~-100 mV (background state). This reversal p.d. does not correlate with  Nernst potential of any of the major ions involved. We hypothesize that at least part of the background current flows through stretch-activated channels, which may not be very ion selective (or they may be populations of specific SA channels, which open at the same time).