Biology and Biomedical Sciences: Neurosciences
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
The SLO family channels are high conductance K+ channels that are gated both by voltage and intracellular ions. Structurally they resemble voltage gated channels but have additional large conserved intracellular domains appended on the C-terminal that allow them to be gated by different intracellular ions. Two members of this unique family of K+ channels are Slo1: BK) which is activated by Ca2+, and Slo2.2: Slack) which is activated by sodium. Both channels are widely expressed in the brain and other tissues in many species from C. elegans to humans.
The large conductance Ca2+- activated K+ channel: Slo1 or BK for Big conductance K+ channel) is widely distributed and controls many different physiological processes including cellular excitability, neurotransmitter release, muscle contraction, hair cell function, insulin release, and blood pressure. Defects in Slo1 channels have been associated with hypertension, autism and mental retardation, obesity, asthma, epilepsy, and cerebellar ataxia. Slo1 channels are activated by Ca2+, voltage, and Mg2+ through different allosteric pathways providing a model system to study allosteric coupling and pathways in channel gating and protein function. The structure of Slo1 has two functional domains, a "Core" consisting of seven transmembrane elements: S0-S6) which assemble to form a voltage sensing domain which allosterically confers voltage sensitivity to the pore gate domain, and a "Tail" that forms a large intracellular gating ring thought to confer Ca2+ and Mg2+ sensitivity through different transduction pathways from gating ring to Core. The large modular Slo1 channel is known to undergo many complex allosteric interactions during channel gating, some within subdomains of the Core itself, some within the massive Tail, and some between Tail and Core. Because of its great size and complexity it has not been possible to understand all these allosteric structural changes nor dissect the contributions of the different transduction pathways to channel gating. A new and valuable tool for answering these questions would be the ability to express the voltage-sensitive Core alone, free of the influence of the large and complex Tail. This would allow the determination of the baseline gating properties of the isolated Core, which would permit experiments such as adding the transduction pathways back one at a time and in combination, to reveal the functions of each. Unfortunately, it has not previously been possible to express the Core without the gating ring. However, we have been able to develop novel constructs of the Core without the gating ring that I have been able to express and analyze using the Xenopus oocyte heterologous expression system. I will show that currents from these constructs are from heterologously expressed gating ring-less channels and not from possible endogenously expressed channels. This allows determination for the first time, of the baseline properties of the Slo1 Core without passive or active allosteric input from the gating ring. The studies I performed show that the baseline properties of the isolated Core differ considerably from the properties of the intact Slo1 channel in the isolation of Ca2+. This shows that the gating ring imparts passive properties and interactions with the core, even in the absence of Ca2+. Thus, removing the gating ring reduces single-channel conductance ~30%, removes all Ca2+- and Mg2+-sensitivity, greatly reduces single channel mean open channel duration and burst duration; and right-shifts the G/V relation. Knowing these baseline properties of the Core then provides us with a novel tool and a guide for understanding the allosteric basis for gating in Slo1 channels. Such knowledge may facilitate the development of agents to restore normal function in genetic syndromes where Slo1 channels are involved. Also, this more complete understanding of how these complicated channels function could be important for understanding other channels that are activated by more than one factor: as TRP channels) or for other proteins which undergo complicated allosteric structural changes.
The goal of the second project was to reveal the physiological relevance of Slo2: Slack and Slick) Na+-dependent K+ channels. The discovery of high conductance Na+-dependent K+ channels in heart and brain presented a conundrum, the sodium concentrations needed to activate these channels in inside-out patches far exceeded the intracellular concentration of Na+ under normal physiological conditions. Thus, it was proposed that Na+-dependent K+ channels were an emergency conductance only activated under very special conditions such as during hypoxia or ischemia where the Na+ levels increase inside the cell. However, other reports indicated that these channels could be active under normal physiological conditions. Also, there is evidence of these channels being widely expressed all over the mammalian brain. I present data here showing that one of the largest components of the delayed outward current that is active under physiological conditions in many mammalian neurons, such as medium spiny neurons of the striatum and tufted-mitral cells of the olfactory bulb, is expressed by Na+-activated K+ channels and has previously gone unnoticed. Previous studies of K+ currents in mammalian neurons may have overlooked this large outward component because the sodium channel blocker tetrodotoxin: TTX) is typically used in studies of K+ channels. However, we found that TTX also eliminated this Na+-dependent delayed outward component in rat neurons as a secondary consequence. Unexpectedly, we found that the activity of persistent inward sodium current is highly effective at activating this large Na+-dependent: TTX sensitive) delayed outward current. Using siRNA techniques, I identified the Slo2.2 channel as a carrier of this delayed outward current. These findings have far reaching implications for many aspects of cellular and systems neuroscience, as well as clinical neurology and pharmacology.
The final part of this dissertation involves the study of the effect of divalent cations on Slo2.2 channels. The activating effect of virtually all divalent cations on Slo1 is well documented, but the effect of divalent cations on Slo2.2 channels is largely unstudied. In exploring this question, I was surprised to observe that all of the divalent ions that activate Slo1 channels have the opposite effect on Slo2.2 channels; they reduce channel activity. After making this observation I turned my attention towards understanding the mechanism of blocking. I considered two hypotheses: 1) Divalent ions blocked the pore of Slo2.2 channels, and 2) Divalent ions functioned at a site away from the pore and either competed with sodium ion binding or produced allosteric changes leading to channel inhibition. My results indicate that the effect of divalent ions on Slo2.2 is not by blocking the pore. I also showed that the blocking effect of divalent cations on Slo2.2 channels is conserved in the orthologous channel from Drosophila which has been cloned in our lab. In addition, I show that the Drosophila Slo2 channel is sodium dependent, unlike the Slo2 channel in another invertebrate, C. elegans, which lacks sodium sensitivity and is instead, activated by Ca2+. Finally, by site-directed mutagenesis, we have tentatively located the site of interaction of divalent cations with the Slo2.2 channel. In the conclusion to this section, I discuss the possible physiological relevance of my findings to a proposed mechanism of action of Slo2 channels.
Budelli, Gonzalo, "Novel Structural and Physiological Functions of High Conductance K+ Channels of the Slo Family" (2013). All Theses and Dissertations (ETDs). 1122.