Optical measurement of voltage sensing by endogenous ion channels

Abstract

1A primary goal of molecular physiology is to understand how conformational changes of 2 proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging 3 method for measuring the conformational changes of a voltage-sensing protein within tissue. We 4 synthesized a fluorescent molecular probe, compatible with two-photon microscopy, that targets 5 a resting conformation of Kv2-type voltage gated K + channel proteins. Voltage-response 6 characteristics were used to calibrate a statistical thermodynamic model relating probe labeling 7 intensity to the conformations adopted by unlabeled Kv2 proteins. Two-photon imaging of rat 8 brain slices labeled with the probe revealed fluorescence consistent with conformation-selective 9 labeling of endogenous neuronal Kv2 proteins. In principle, this method of quantifying 10 endogenous protein conformational change from fluorescence images is generalizable to other 11 proteins labeled with conformation-selective probes. 12

The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

“…As GxTX-594 binding is determined by the stability of Kv2.1 in the earliest resting conformation, we converted the k∆F of GxTX-594 dissociation at +30 mV into a 1.9-fold change in equilibrium constant (Keq) for resting vs. activated voltage sensors (Thapa et al, 2021). To transform the experimentally measured k∆F into an approximate Keq, we used the k∆F vs. V vs. PActivated,Labeled relationship described in (Thapa et al, 2021). Briefly, experimentally measured k∆F values were interpolated onto the k∆F vs. V relation.…”

“…AMIGO1-YFP and Kv2.1 channel expression and GxTX-594 labeling was achieved following the same protocols as described in (Thapa et al, 2021) and were similar to those described in "GxTX-594 dose response time-lapse imaging". Briefly, instead of completely washing out GxTX-594 after incubation, the solution was diluted with 1 mL of a bovine serum albumin-deficient imaging solution for a working concentration of 9 nM GxTX-594 during experiments.…”

“…Immediately following the voltage stimulus, the fluorescence intensity decreased. The decay was fit with a monoexponential function as done in (Thapa et al, 2021). For presentation, fluorescence intensity traces were subtracted by the minimum intensity value and normalized to the maximum.…”

“…We also assessed AMIGO1-YFP and Kv2.1 colocalization using the Pearson's correlation coefficient (PCC) (Manders et al, 1992). Surface-expressing Kv2.1 on live cells was labeled with GxTX Ser13Cys(Alexa594) (referred to from here on as GxTX-594), the conjugate of the voltage sensor toxin guangxitoxin-1E derivative Ser13Cys with the maleimide of Alexa594 (Thapa et al, 2021). As auxiliary subunits can impede binding of toxins to voltagegated ion channels (Das et al, 2016;Gilchrist et al, 2013;Maffie et al, 2013;Wilson et al, 2011), we tested whether AMIGO1 impacted GxTX-594 binding to Kv2.1.…”

“…Alternately, if AMIGO1 acts directly on pore opening, the AMIGO1 impact on the pore opening equilibrium should be insensitive to voltage sensor modulation. To distinguish between these possibilities, we measured AMIGO1 modulation in the presence of GxTX-594, a GxTX derivative that acts by the same mechanism as GxTX (Thapa et al, 2021) and binds Kv2.1 similarly with or without AMIGO1 (Supp. Fig.…”