Single-channel physiology in small compartments

Abstract

Cellular membranes are replete with integral transport proteins which regulate key processes from biochemical and electrical signaling to osmotic homeostasis. Bulk transport measurements obscure kinetic details due to ensemble averaging. Fluorescent reporters of ions and small molecules provide sensitive readouts of solute concentrations but rapid diffusion and clearance of solute fluxes in the bulk limits the scope of transporters which can be measured. In this dissertation, we use experiment and theory to explore how small membrane-derived compartments can be leveraged for highly sensitive in cellulo transport measurements and distinct electrophysiology of native nanoscale structures. Native densities of membrane transport proteins (e.g. ion channels, transporters, pumps) in a neuron vary by ~four orders of magnitude. Transport rates through integral proteins (e.g. solute molecules s-1) vary by ~nine orders of magnitude. It follows that there exist membrane area and lumen volume regimes where transporter numbers are small and single-unit flux alters lumenal solute concentrations. In fact, biology is replete with nanoscopic structures including thin filopodia (area ~10 µm2, volume ~1.5 fL), dendritic spines (~1 µm2, ~0.1 fL), the primary cilium (~2.5 µm2, ~0.1 fL), and synaptic vesicles (~5×10^(-3) µm2, ~30 zL). Membrane compartments of similar scales can be created in living cells on-demand using physical (e.g. micromanipulation) and optogenetic tools. The electrophysiology of native and membrane-derived compartments is accessible using advanced optogenetic techniques to measure electrolyte and voltage dynamics. In Chapter Two we leverage membrane tethers – tubes of membrane extracted from cells – for optical recordings of Ca2+ flux through single voltage-gated Ca2+ channels. Tethers are a facile means of isolating individual channels from the bulk membrane. Tether radii are subdiffraction-sized such that Ca2+ transported into the tether lumen remains within the microscope focus. Ca2+ transported into the lumen is isolated from bulk membrane transport because the quasi-1D tether geometry restricts diffusion of transported solute. Despite low channel conductance ( pS) and estimates of high lumenal Ca2+ buffering, membrane tethers enabled the detection of single-channel Ca2+ transport via a genetically encoded Ca2+ indicator. Comparison of event statistics to stochastic single-channel simulations suggest that tether Ca2+ indicators are sensitive to as little as ~0.4 fC or ~1250 transported Ca2+ ions. This corresponds to as few as 6-13 free Ca2+ ions under typical buffering conditions. In Chapter Four we discuss how tether-based transport measurements might generalize to other integral proteins and solutes. In membrane tethers, single-channel gating measurably perturbs lumenal ionic concentrations but voltage remains well clamped by the cell body. However, many nanoscopic structures (e.g. synaptic vesicles, dendritic spines, endocytic vesicles, and microbes) are electrically and biochemically isolated. In Chapter Three we use stochastic single-channel and ensemble simulations in model membranes to identify compartment size and channel density regimes in which membrane noise becomes a dominant driver of membrane voltage and lumenal electrolyte fluctuations. Within these regimes, we explore the qualitatively distinct dynamics which emerge when voltage and electrolyte fluctuations feedback onto voltage-dependent channel gating. We identify regimes which alter effective voltage-gated channel kinetics and produce patterns of single-channel and ensemble channel activity which cannot be described using deterministic conductance-based models. We provide several predictions for nanoscale electrophysiology which are experimentally testable using the tools of optical electrophysiology. Nanoscale structures support patterns of electrical activity which differ substantially from the electrophysiology of large cells. The experimental and computational work described herein provides a glimpse into how nanoscale electrophysiology can be leveraged for sensitive studies of membrane transport and interpreted in native contexts.