Abstract
Domains in macromolecular complexes are often considered structurally and functionally conserved while energetically coupled to each other. In the modular voltage-gated ion channels the central ion-conducting pore is surrounded by four voltage sensing domains (VSDs). Here, the energetic coupling is mediated by interactions between the S4-S5 linker, covalently linking the domains, and the proximal C-terminus. In order to characterize the intrinsic gating of the voltage sensing domain in the absence of the pore domain, the Shaker Kv channel was truncated after the fourth transmembrane helix S4 (Shaker-iVSD). Shaker-iVSD showed significantly altered gating kinetics and formed a cation-selective ion channel with a strong preference for protons. Ion conduction in Shaker-iVSD developed despite identical primary sequence, indicating an allosteric influence of the pore domain. Shaker-iVSD also displays pronounced 'relaxation'. Closing of the pore correlates with entry into relaxation suggesting that the two processes are energetically related.
Highlights
Voltage-gated ion channels play a fundamental role in a wide range of physiological processes, including the generation and propagation of electrical signals in excitable cells and muscle tissues, neurotransmitter release in pre-synaptic nerve endings, and maintaining cell homeostasis (Hille, 1978)
Shaker-iVSD was site-directed fluorescently labeled by attaching an extrinsic fluorescent probe to a cysteine introduced at position A359C in the S3–S4 linker just N-terminal to S4
Our initial objective was to obtain the gating chargevoltage (QV) relation of Shaker-iVSD. Unlike those observed in Shaker-W434F (Figure 1b bottom), the gating currents of Shaker-iVSD could not be estimated reliably because of substantial ionic currents in particular when holding the potential at À90 mV
Summary
Voltage-gated ion channels play a fundamental role in a wide range of physiological processes, including the generation and propagation of electrical signals in excitable cells and muscle tissues, neurotransmitter release in pre-synaptic nerve endings, and maintaining cell homeostasis (Hille, 1978). They comprise four subunits of each six transmembrane segments (S1–S6), which are assembled into two functionally distinct parts: a central ion-selective pore formed by the combined S5 and S6 segments of four domains, surrounded by four voltage-sensing domains (VSDs) formed by the four helices S1–S4. In addition to the coupling in this region, a direct structural and functional interaction between the VSD and pore has been demonstrated (Lee et al, 2009a; LiSmerin et al, 2000; Petitjean et al, 2015)
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