UC Berkeley

Membrane proteins respond to both chemical and electrical stimuli. This work explores the molecular mechanisms by which membrane voltage controls voltage-gated proteins and describes the development of tools to modulate voltage-gated protein function.

Voltage-gated potassium (Kv) channels are tetrameric transmembrane proteins that translate changes in the membrane electric field into the controlled permeation of potassium across the plasma membrane. Kv channels mediate the initiation and regulation of action potentials, muscle contraction, hormone secretion, and information processing, rendering them important drug targets. We employed organic synthesis, molecular dynamics and electrophysiology techniques to demonstrate that calix[4]arenes with free phenolic OH groups at the lower rim and positively-charged groups at the upper rim constitute a versatile class of reversible ligands for homotetrameric Kv1.x channels. Synthesis of a panel of calix[4]arenes with variable upper and lower rim substituents enabled the systematic development of Kv1.x channel-compatible ligands. We used molecular modeling to predict calix[4]arene binding to the pore domain, and through electrophysiology experiments, we demonstrated that the calix[4]arene ligands function as reversible blockers of Kv1.x channels. We probed the mechanism of calix[4]arene-channel interactions using voltage clamp fluorometry and found these ligands modify the voltage-dependent motions of the Shaker Kv channel in addition to inhibiting ion current. These calix[4]arene ligands provide a new set of tools to control cell excitability by specifically targeting Kv channels.

Until recently, ion channels were the only proteins known to sense changes in membrane potential. This changed with the discovery of Ciona intestinalis voltage-sensor containing phosphatase (Ci-VSP) which has a voltage sensing domain like voltage-gated ion channels and a cytosolic phosphatase domain resembling the phosphoinositide phosphatase PTEN. Ci-VSP is the first member of the voltage dependent family of proteins that is not an ion channel. Instead, Ci-VSP takes an electrical signal in the form of membrane voltage and converts it to a chemical signal through its phosphatase activity. To study the mechanism of voltage-sensing in Ci-VSP, we combined electrophysiology and fluorescence methods in living cells to determine the oligomerization state of Ci-VSP and monitor the functional transitions that result in Ci-VSP mediated changes in phosphoinositide pools. We find that Ci-VSP is a functional monomer which undergoes complex voltage-dependent conformational changes to control a cytosolic phosphoinositide phosphatase domain. As Ci-VSP catalyzes several reactions, we also developed fluorescent-based methods to study Ci-VSP substrate specificity and monitor Ci-VSP-mediated changes in multiple phosphoinositide pools in a single cell. Finally, we find that basic residues in the interdomain linker connecting the voltage sensing domain and phosphatase domains in Ci-VSP are essential for coupling the two domains. Our results indicate that a single voltage sensing domain can function in the membrane on its own and suggests that voltage sensing domains are modular units that can impart voltage sensitivity to a variety of effector domains.