Electro-mechanical measurements on membranes of Shaker-transfected cells.

摘要

Electro-mechanical (EM) and mechano-electrical (ME) transduction is a general, as well as a specialized process in biology. We have studied EM transduction at molecular resolution in membranes of live cells to examine membrane resident molecules in their near-native states. The experiments allow direct estimation of coupling energies. These studies involve AFM method development as well as biology experiments.; We measured the voltage-induced movements of membranes of cultured cells (HEK) transfected with voltage-gated potassium channels of the Shaker family (ShHEK) (1). We combined patch-clamp and the atomic force microscope (PC-AFM) as an electro-mechanical instrument capable of molecular resolution. The voltage-induced membrane movement (VDM) of ShHEK differed markedly from the VDM of wild-type cells (wtHEK). The VDM of wtHEK cells was roughly linear in the physiologic voltage range, whereas ShHEK showed a pronounced non-linearity in the voltage range of channel opening. While wt membranes move outward on depolarization, the movement increasing with voltage step amplitude, in ShHEK membranes the movement at short times ceased once the Shaker channels opened. We have ruled out mass flux dependence and voltage-clamp instabilities. This nonlinearity is time dependent, decreasing with time after the voltage transition. We present a set of potential models for the mechanism.; Driven by the demands for sensitivity in doing AFM of membranes, we have developed a set of novel cantilevers that resulted in order of magnitude increases in speed and mechanical resolution (2) and improved software. The sensitivity is now >1pN in a 1kHz bandwidth. The production process uses standard micro-fabrication techniques optimized to allow routine fabrication of micro-mechano-sensors with a variety of custom geometric and mechanical properties. We illustrate the improved noise by force spectroscopy of single GsmTx-4 molecules interacting wtHEK membranes.