Developing instrumentation for multi-parametric investigation of mechanisms of mechanosensitivity in ion channels.

摘要

Mechanosensitive (MS) channels are implicated in pathologies of the renal and pulmonary systems. Abnormal activity in MS channel reduces cell viability causing a variety of pathologies. MS channels are also responsible for sensation of pain and hearing. Despite the vital importance of MS channels, very little is known about the gating mechanisms of these channels. Attempts to study the mechanisms are severely limited by the lack of suitable instrumentation. A better understanding of the structure-function interaction of MS channels is necessary to find pharmacological leads for the pathologies. Activation data based on indirect activation of MS channels using hypo- or hyper-osmotic solutions or viscous drag is confounded by factors like membrane stretch and cytoskeletal stress. Traditional patch clamp does not allow direct access to the cell by other probes. While a planar patch clamp chip may allow for such access, most of the existing planar patch clamp chips are focused on high throughput screening for pharmaceutical targets and have designs that limit multi-parametric studies.;We present here instrumentation that combines atomic force microscopy with cellular electrophysiology based on planar patch clamp approach. The instrumentation allows multi-parametric studies on single cells and provides unique insights into mechanisms of activation of not just MS channels, but ion channels in general by combining cellular electrophysiology, optical microscopy and atomic force microscopy. Using HaCaT cells as our model system we have obtained functional maps of distribution MS channels across cell surface. The maps reveal that the distribution of MS channels on HaCaT cells is highly non-uniform and that the channels are present in small clusters instead of dispersed as single entities. Our results using direct mechanical stimulation of single cells reveal that threshold stress level is required in order to activate MS channels and that the stress has a limited spatial range. Investigation of kinetics of the electrical response to direct mechanical stimulation reveals that the MS channels respond to the mechanical signal after a small time lag, which we attribute to the conformational changes necessary while the channel is being gated.;We hope that the insights gained from studying the mechanosensitive channels of HaCaT cells will also advance the understanding of MS channels in general. Apart from opening new avenues in MS channel research, the instrumentation can also be useful in studying the dynamics and gating of ligand gated channels by appropriately tagging the AFM cantilever. With further improvements in the speed of AFM imaging, it will also be possible to observe the gating of channels in real time at molecular scale by imaging the channel on the cell while the channel is being gated.