Regulation of K+ channel current magnitude by mechanisms occurring in the outer vestibule

Voltage-gated potassium channels are potassium selective ion pores that open in response to changes in membrane potential. The molecular mechanisms behind channel modulation of gating and permeation are currently not well understood. One region that has been shown to be involved in several functional channel properties is the channel outer vestibule. Here we focus on two potassium channels, Kv1.5 and Kv2.1, which are known to display outer vestibule dependent current modulation. For Kv 1.5, currents are uniquely sensitive to changes in external pH in the physiological range. We found that a shift in the voltage-dependence of activation, via modification of sites contributing to a local surface potential, was entirely responsible for this effect. Importantly, we found that a specific amino acid residue in the outer vestibule is, at least in part, responsible for shifting Kv1.5 sensitivity into the physiological pH range. For Kv2.1, current magnitude is modulated by changes in external [K+] in the physiological range. In order to examine this observation we required the ability to study current through single potassium channels, which typical methods of heterologous channel expression, in mammalian cells, precludes. Through the use of a tetracycline inducible expression system, we were able to lower channel expression to a membrane density where we could routinely study single channel currents. By combining macroscopic and single-channel studies with hidden Markov modeling we addressed the Kv2.1 outer vestibule mechanism. Our results support a model whereby an outer vestibule lysine selectively interferes with K+ flux through Kv2.1. Potassium dependent reorientation of this lysine, into one of two functionally defined conformations, results in just two single channel conductance states. Moreover, the results demonstrate a mechanism where macroscopic current magnitude is determined by the distribution of channels open in one of the two conductance states, which is determined by potassium concentration. Together, data in this dissertation describe how potassium channels modulate currents through mechanisms occurring in their outer vestibules.