Ion Transport from Physics to Physiology: the Missing Rungs in the Ladder
Molecular rearrangements underlying BK channel functionTeresa Giraldez
In neurons, sites of Ca2+ influx and Ca2+ sensors are located within 20-50 nm, in subcellular Ca2+ nanodomains. Such tight coupling is crucial for the functional properties of synapses and neuronal excitability. Two key players act together in nanodomains, coupling Ca2+ signal to membrane potential: the voltage-dependent Ca2+ channels (Cav) and the large conductance Ca2+ and voltage-gated K+ channels (BK, hslo or KCa1.1). BK channels are characterized by synergistic activation by Ca2+ and membrane depolarization, but the complex molecular mechanism underlying channel function is not adequately understood. Information about the pore region, voltage sensing domain or isolated intracellular domains has been obtained separately using electrophysiology, biochemistry and crystallography. Nevertheless, the specialized behavior of this channel must be studied in the whole protein complex at the membrane in order to determine the complete range of structures and movements critical to its in vivo function. In our laboratory we use a combination of genetics, biochemistry, electrophysiology and spectroscopy, which we correlate with protein structural analysis, to investigate the real time structural dynamics underlying the molecular coupling of Ca2+, voltage and activation of BK channels in the membrane environment, its regulation by accessory subunits and channel effectors (Miranda et al., 2013; Miranda et al., 2016). BK subcellular localization and role in Ca2+ neuronal nanodomains make these channels perfect candidates as reporters of local changes in [Ca2+] restricted to specific subcellular regions close to the neuronal membrane. We have created fluorescent variants of the channel that report BK activity induced by Ca2+ binding, or Ca2+ binding and voltage (Giraldez et al., 2005). We aim to optimize and deploy these novel optoelectrical reporters to study physiologically relevant Ca2+-induced processes both in cellular and animal models. Overall, optically-active BK channels with spectrally-separate photoactivation and FRET modules offer many possibilities for the study of activation in mammalian cells.