Auxiliary Hyperkinetic β Subunit Of K～+ Channels Modulates Properties Of K～+ Currents In Adult Drosophila Neurons

Object: The availability of genetic, molecular and biophysical techniques makes Drosophila an ideal system for the study of ion channel function. Since several culture systems were developed, more and more electrophysiological recordings have been obtained from Drosophila neurons. Dissociated neuronal culture systems have been used for patch-clamp analysis of embryonic, larval and pupal neurons. However, earlier attempts to culture neurons from Drosophila adult have attained little success. In this study we focused on searching for the whole-cell voltage-gated K+ currents and the effect of auxiliary Hyperkinetic (Hk) p subunit on K+ currents in adult Drosophila neurons. Methods: We obtained wild type and Hk mutant adult neurons with good activity using the mechanically vibrating method and studied the electrophysiological properties of adult Drosophila neurons with patch clamp technique. Results: Two types of lA-like currents, mainly including fast-inactivated component, and two types of It-like currents, which inactivated very slowly, were defined in both wild type (WT) and p subunit mutant Drosophila. A Shaw-like K+ current with the properties of distinct voltage-dependence activation, large conductance and different rise rime at different pulse contributes a subpopulation of adult neurons. Drosophila mutants alters the numbers of IA-like currents, which were fitted with two-exponential, inactivating rapidly or less rapidly. In addition, the HkP subunit modulates other kinetic properties of the lA-like currents, including their steady-state inactivation and recovery. Differing from the expression system and cultured "giant" neurons and larval muscles, the adult P subunit mutant Hk1 accelerated the inactivation of K+ currents. Further experiments on the allele mutant Hk4 show the same tendency, but the changement was not so obvious as in Hk1. P subunit mutant Hk1 only affected parts of the neurons in the Drosophila brain. The less sensitivity to K+ channel blockers in Hk1 mutant revealed that p subunit modulated both the intra- and extracellular regions of the channel pore by interacting with some a subunits. Conclusion: Our results indicate that Hk stabilizes the structure of adult K+ channel. The morphology of neurons changed obviously from larval to adult. The K+ channels may be modulated during the development of Drosophila and the altered kinetic and pharmacological properties of U-like currents may underlie the high excitability of neurons and leg-shaking behavioral in Drosophila Hk mutant. The mechanical dissociated method, which is convenient and economical, does not need to use the expensive enzymes and the complicated equipment needed by culture method. The cells isolated with the method keep the physiological properties in vivo better than those with enzyme treatment. At the same time, the mechanical method avoids damaging the protein such as receptors and ion channels on membrane by enzymes.Xu, Taixiang (Pharmacology) Directed by Prof. Jin, Yi; Prof. Xu, Tianle...