Research in our lab focuses on both structural features and conformational changes which regulate K+ channel function. Mutations and abnormal processing of these channels lead to impaired cardiac, renal, and neuronal functions.
Our lab examines the roles of N-linked oligosaccharides on K+ channel: function, maturation and trafficking. Kv3 voltage gated K+ channels are critical components of neurons that fire repetitively at high frequency. All four Kv3 genes, including their splice variants, have two conserved N-glycosylation sites in their first extracellular loop (S1-S2). Using mutagenesis and biochemical assays, my laboratory has shown that both glycosylation sites of Kv3.1 are utilized in Spodoptera frugiperdae (Sf9) cells, an insect cell line. Additionally, whole cell current measurements demonstrated that Kv3.1 channels with N-glycans of simple type (Man3-GlcNac2-Asn) have different activation kinetics compared to those without N-linked oligosaccharides. Moreover, the activation kinetics of Kv3.1 expressed in Sf9 cells was much slower than that previously reported for Kv3.1 expressed in mammalian cells. Future studies will be to determine whether this dissimilarity in channel activation is caused by the type of oligosaccharide attached to Kv3.1 channels. ( See Figure 1 )
Secondly, we have shown that both glycosylation sites of Kv3.1, 3.2, and 3.3 channels are highly available in rat brain, and that at least one site is occupied by a complex oligosaccharide. Future studies will examine whether site occupancy and type of N-glycan are altered in the various regions of the brain. Additionally, it will be determined as to whether the type of N-linked oligosaccharide differs in the aging rat brain.
Glycosylation tagging studies from our lab have focused on two members of the inwardly rectifying potassium channel class, Kir1.1 and Kir2.1, which play critical roles in renal and cardiac physiology, respectively. (See Figure 2) Kir channel subunits cross the membrane two times, and a region between these transmembrane segments has been coined the putative pore-forming segment, P-loop. However, we have established that the putative pore-forming segment can be N-glycosylated for Kir1.1 and Kir2.1. These results lead us to propose that the M1-M2 linker was topologically extracellular. The focus of our present studies is to determine whether the extracellular topology of the putative pore-forming segment is conserved by other classes of K+ channels. Additionally, our lab is examining the protein insertion model for Kir channels. These protein structural and insertion studies should assist in drug design for heart and kidney disease.
Of recent, we have identified three conductance states, corresponding to the main open state and two subconductance states, of Kir2.1 channels. In addition, we have shown that Kir2.1 channels with mutations in the M1-M2 linker altered the transition, duration, and frequency of the defined populations of permeating and nonpermeating states. These findings led us to assign a novel functional role of the putative pore-forming segment in regulating transitions of the Kir2.1. The focus of our present studies is to identify additional pore conformations of the Kir2.1 channel, and to establish that the M1-M2 linker is a critical structural determinant in adjustments of pore conformations. These studies will assist in identifying subconductance states in Kir2.1 channels and their relevance in fine tuning the physiological response. (See Figure 3)
The multidisciplinary approach of our research provides outstanding training and later on career opportunities for all members of our lab. Each person has opportunities to gain experience in biochemistry, molecular biology, cell biology, glycobiology and electrophysiology. Additionally, lab members are expected to address significant questions thoroughly, to present their results at scientific conferences, and to publish in peer-reviewed journals. We seek talented and motivated undergraduate, graduate, and post-doctoral students to join our group and assist us in continuing to explore structural features and conformational changes that regulate K+ channel function. Please feel free to contact Ruth A. Schwalbe directly with inquiries at any time.
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Assistant Professor of Biochemistry & Molecular Biology
The Brody School of Medicine at East Carolina University
Greenville, NC 27834