The fundamental processes that underlie ion channel function are permeation/selectivity and gating. In an effort to understand ion channel gating, we have used an approach that combines reporter-group spectroscopic techniques (spin labeling/EPR) and electrophysiological methods with classical biochemical and molecular biological procedures. Through site-directed spin labeling, cysteine chemistry was used to introduce nitroxide radicals into specific sites within these channels with high reactivity and specificity. EPR spectroscopy analysis of the spin labeled mutants yields two types of structural information: 1) mobility and solvent accessibility of the attached nitroxide through collisional relaxation methods and 2) distances between pairs of nitroxides through dipole-dipole interactions. The crystal structures of the bacterial voltage-dependent K+ channel (KvAP) and its isolated voltage-sensor domain have raised several interesting questions about the relative orientation of the membrane domains and the loop regions within the voltage-sensor. Here, we will focus on the structure and dynamics of the voltage-dependent K channel KvAP in the context of the lipid bilayer. We show in reconstituted full-length KvAP that the S4 segment lies at the protein-lipid interface, with most of the gating charges protected from the lipid environment. Additionally, the segment is highly flexible, consisting of two helices separated by a short linker. Accessibility and dynamics data position the S1 segment at the contact interface between the voltage sensing and pore domains. Analysis of our EPR measurements of the isolated voltage-sensor domain correlates well with the helical nature of several TM segments as predicted by its crystal structure. The probe dynamics and accessibility data show that the S4 segment is highly flexible and is exposed to lipid environment, as in the full-length channel. However, in sharp contrast to the full-length channel, the S1 segment shows significantly higher mobility and oxygen accessibility, which appears to be due to lack of the pore domain which is in close proximity to S1. Our results confirm that S1 in the full-length channel is protected by a protein environment while S4 is exposed to lipids. These results establish the general principles of voltage-dependent channel structure under physiological conditions and thus limit the types of structural models underlying voltage-dependent gating.