A patch-clamp analysis and a characterisation of the cyclic nucleotide-gated channels involved in mammalian olfactory signal transduction
[thesis]
Sarada Balasubramanian
1997
In olfactory signal transduction cationic current through cyclic nucleotide-gated (CNG) channels is largely responsible for the generation of the receptor potential. In order to better understand mechanisms underlying peripheral odorant transduction a patch-clamp analysis and characterisation of CNG channels from enzymatically dissociated olfactory receptor neurons from rats was undertaken as the theme of this thesis. Permeation of large monovalent organic cations through olfactory CNG channels
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... (activated by cAMP), revealed that these channels poorly select among monovalent cations and that they have minimum pore dimensions of at least 6.5 x 6.5 A since they allow TEA and Tris to permeate readily. The permeability sequence, PNH4 > PNa > Pdma > Ptma > Pchoiine > Ptea> was indicative of high field strength binding sites within the channel. Calcium-dependent modulation of the affinity of these CNG channels for cAMP revealed that the addition of [Ca~ ], caused a reversible affinity shift from 3 to 30 jliM. This affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by exposure to Mg +EGTA and was not restored by the application of calmodulin. Calmodulin, when applied along with Ca" , shifted cAMP affinity further to about 200 juM and this affinity shift, which was reversed by mastoparan, was not affected by Mg" +EGTA exposure. The Ca -only induced affinity shift was maintained in the presence of 8-bromo-cAMP and microcystin-LR ruling out the possible roles of phosphodiesterases or phosphatases. Thus, the results indicated that these channels could be modulated by calmodulin as well as by an as yet unidentified factor, distinct from calmodulin. NaCl concentration dependence of permeation of sodium ions in these essentially cation selective channels revealed ion binding sites within the channels with a Km of about 60-150 mM. The results also indicated that the channels are essentially symmetrical and display conductances dependent on both solution concentrations irrespective of voltage. Preliminary ratetheory modelling indicated that the ion-binding sites were close to the centre of the channel. Information provided in this thesis should aid in our understanding of ion permeation and modulation of these olfactory transduction channels. Methods 95 Cell preparation Solutions and perfusion system Electrophysiological recording Results Decrease in the cAMP sensitivity by Ca~+ does not wash out with time Irreversible loss of affinity shift caused by the exposure of the patch to 3 mM Mg" and 2 mM EGTA Exogenous calmodulin (CAM) decreases the affinity of the CNG channel for cAMP Effect of mastoparan on Ca" +CAM and Ca" -only affinity shifts Ca" -only and Ca"+CAM effect are still present hydrolysis-resistant analogue 8-bromo-cAMP Ca" -only effect is present in the presence of microcystin-LR, phosphatase inhibitor Discussion Comparison with other studies Physiological role Chapter 6 Concentration dependence of sodium permeation and sodium ion interactions in mammalian olfactory cyclic nucleotide-gated (CNG) channels vi VII Methods Cell preparation Electrophysiological recording Solutions and perfusion system Determination of current-voltage relations and reversal potentials Results The dependence of CNG channel current on internal Na+ concentration Currents through the olfactory CNG channel deviate from the independence principle Current through the olfactory CNG channel: relative permeabilities to Na+and Cl" Discussion Summary of results Comparison with previous studies Conclusion Chapter 7 Conclusion Membrane Biology
doi:10.26190/unsworks/6474
fatcat:y6u2g2ygdzfgtnil5rmfp7d4ve