| Summary |
Voltage-gated K⁺ (Kv) channels, such as Kv3.1, play a central role in cellular excitability, specifically regulating firing patterns and the duration of action potentials in neurons. Here the aim was to identify regions of the channel that are topologically extracellular. Techniques of patch clamping, A/-glycosylation tagging, and membrane fractionation were used to characterize native and introduced glycosylation sites of Kv3.1. Immunoband shifts in the presence and absence of tunicamycin, an A/-glycosylation inhibitor, showed that the two native glycosylation sites, N220 and N229, in the S1-S2 linker were occupied by N-linked oligosaccharides and also that targeting of Kv3.1 to the plasma membrane did not require A/-glycosylation. Moreover, the absence of A/-glycosylation did not significantly affect whole cell or single channel current amplitudes. To simplify interpretations of the topology of the S5-S6 linker, N220Q/N229Q was used as template for construction of the A/-glycosylation mutant, M398N. Position 398, the central and deepest residue of the putative pore-forming segment, was demonstrated to be glycosylated and targeted to the plasma membrane. Whole cell current patterns, although reduced, and single channel current amplitudes, although of low opening probability, were similar between M398N, wild type Kv3.1, and N220Q/N229Q. Collectively, this data reveals that both the S1-S2 and the S5-S6 linkers of Kv3.1 are exposed to an extracellular aqueous environment. |
