NITRIC OXIDE (NO*) DONORS INCREASE THE OPENING OF NATIVE INTESTINAL AND CLONED ALPHA SUBUNITS OF HUMAN BRAIN BK_Ca CHANNELS

Emily L. Mulholland¹, Billie Hunne², Craig B. Neylon², Richard J. Lang¹, 1 Department of Physiology, Monash University, 2 Department of Anatomy & Cell Biology University of Melbourne, Vic.

Although endogenously-released or exogenously-applied nitric oxide (NO*) is thought have its major inhibitory role in smooth muscle via the activation of cytosolic guanylate cyclases (GC), membrane hyperpolarization and muscle relaxation can be evoked in human myometrium, rat blood vessels and guinea-pig intestinal preparations after GC blockade. It is thought that NO* binds to thiols on cysteine residues (Cys) of large conductance Ca²⁺-activated K⁺ (BK_Ca) channels to form S-nitrosothiols that influence the opening kinetics of these channels. We have recently established that the NO* donor, S-nitroso-L-cysteine (NOCys)-evoked increases of guinea-pig intestinal BK_Ca channel activity can be prevented by the negatively-charged methanethiosulfonate (MTS) reagent (MTSES C₃H₇O₅S₃Na), but not by positively-charged MTS reagents. As MTS reagents irreversibly convert Cys thiols to non-reactive disulphides, NOCys must be binding to single or viscinal water-facing Cys within basic regions of the BK_Ca channel protein(s) that allow MTSES to approach but exclude positively-charged MTS reagents.

As there is >90% homology in the sequences of the alpha subunit of BK_Ca channels (alpha-slo) isolated from the mouse and rat brain, dog intestine and human aorta and myometrium we have subcloned alpha-slo from human brain (alpha-hbr1) into the mammalian expression vectors pcDNA3 and pEGFP-C3 and transfected these constructs into HEK293 cells plated onto lysine-coated coverslips. Cells were then incubated at 37 ^OC for a minimum of 24 hours before recordings commenced. Single channel and whole cell membrane currents attributable to the opening of BK_ca channels were recorded at potentials positive to -20 mV in cells transfected with the alpha-hbr1 constructs. As with native intestinal BK_ca channels, the disulfide reducing agent, dithiothreitol (DTT), and the NO* donor, S-nitroso-N-acetylpenicillamine (SNAP), both increased whole-cell alpha-hbr1 construct currents in the presence of the GC blocker, ODQ (10 µM). Site directed mutagenesis techniques will be employed to establish which Cys are being bound by NO*.