Osmolality tolerance and ion channels in protoplasts of entomophthoralean fungi

Lamb, Mary Patricia (1997) Osmolality tolerance and ion channels in protoplasts of entomophthoralean fungi. Masters thesis, Memorial University of Newfoundland.

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Abstract

This thesis documents the tolerance of E. maimaiga protoplasts to media with a broad range of pH levels and osmolalities. Growth curve data were statistically analyzed using a general linear model approach which utilizes the data from the entire growth curve. Although E. maimaiga grew in media ranging from pH 5.5 to 7.1, sensitivity to pH 5.5 was evident. Growth of E. maimaiga in media with osmolality levels of 250 to 400 mOsm did not show any significant differences. Further investigation of osmotic tolerance showed that this organism was capable of surviving osmolality treatments in solutions of 0 to 550 mOsm. However, a 350 to 550 mOsm range appeared optimal. The osmotolerance of E. aulicae was also determined and found to be similar to that exhibited by E. maimaiga. Further investigation is required to determine the mechanism used by these protoplasts for osmoregulation. Such a mechanism may include the activity of ion channels in the cell membrane. -- A protocol developed for patch clamping E. aulicae protoplasts is presented in this paper. An appropriate pipette solution (140 mM NaCl, 5mM KCl, 2 mM CaCl₂, 2.4 mM MgCl₂∙6H₂O, 10 mM MES, 3.8 mM glucose, 2.2 mM fructose, 29.8 mM sucrose, pH 6.2) and bath solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1.2 mM MgCl₂∙6H₂O, 10 mM MES, 3.8 mM glucose, 2.2 mM fructose, 36 mM sucrose, pH 6.2) were developed. Recording from cells 30 to 90 minutes after suspension in the bath solution using the cell-attached recording configuration and a pipette size of 20 MegaΩ resulted in low noise gigaseal recordings. -- Use of the developed patch clamping methodology resulted in the identification of outward rectifying, voltage-gated multichannel activity sensitive to membrane depolarization. Using the mean channel amplitude, the current-voltage relationship was identified as having a conductance value of 31 pS. Use of K⁺ channel blockers, TEA⁺ and Ba²⁺, caused reduced channel activity suggesting that the channels are involved in K⁺ transport. Further evidence of this classification is based on the reduced membrane conductance values obtained when elevated levels of K⁺ were present in the pipette solution. The conductance values were reduced to 10.6 pS and -20.1 pS with K⁺ concentrations of 60 and 140 mM respectively. This serves as further evidence that the voltage-gated channels in the protoplast membrane of E. aulicae are K⁺ channels.

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