Diminishing neuronal acidification by channelrhodopsins with low proton conduction

Abstract

Many channelrhodopsins are permeable to protons. We found that in neurons, activation of a high-current channelrhodopsin, CheRiff, led to significant acidification, with faster acidification in the dendrites than in the soma. Experiments with patterned optogenetic stimulation in monolayers of HEK cells established that the acidification was due to proton transport through the opsin, rather than through other voltage-dependent channels. We identified and characterized two opsins which showed large photocurrents, but small proton permeability, PsCatCh2.0 and ChR2-3M. PsCatCh2.0 showed excellent response kinetics and was also spectrally compatible with simultaneous voltage imaging with QuasAr6a. Stimulation-evoked acidification is a possible source of disruptions to cell health in scientific and prospective therapeutic applications of optogenetics. Channelrhodopsins with low proton permeability are a promising strategy for avoiding these problems.

Optoelectronic control of cardiac rhythm: Toward shock‐free ambulatory cardioversion of atrial fibrillation

“…Most natural channelrhodopsins are nonspecific cation channels conducting a variety of different cations including protons. Due to their very high relative permeability for protons, they can acidify cells and thereby alter cellular behavior [57]. This problem can be tackled by using channelrhodopsins with low proton conductivity [58].…”

Behavioral control through the direct, focal silencing of neuronal activity

“…Photocaged STXs were then screened for potency against NaV1.2 stably expressed in CHO cells with uncaging elicited by 5 ms pulses of 130 mW, 355 nm light. For clarity of discussion, caged STXs have been divided into two categories: Generation 1, comprising the 'base model' STX MeNPOC, 2, as well as amide-modified photocaged derivatives of varying steric bulk and electronic substitution (3-7); and Generation 2, composed of carboxylic acid-modified (i.e., anionic) structures (11,12,13) and their uncharged, allyl ester counterparts (8, 9, 10) (Figure 1C, Extended Data Figure 1). Among Generation 2 photocaged STXs, we found carboxylate-substituted structures (11,12,13) to be fifteen-fold less potent than analogous allyl esters (8, 9, 10, respectively), thus establishing the importance of anionic charge incorporation to destabilize binding of the caged compound (Figure 1E, Extended Data Figure 2).…”

“…For clarity of discussion, caged STXs have been divided into two categories: Generation 1, comprising the 'base model' STX MeNPOC, 2, as well as amide-modified photocaged derivatives of varying steric bulk and electronic substitution (3-7); and Generation 2, composed of carboxylic acid-modified (i.e., anionic) structures (11,12,13) and their uncharged, allyl ester counterparts (8, 9, 10) (Figure 1C, Extended Data Figure 1). Among Generation 2 photocaged STXs, we found carboxylate-substituted structures (11,12,13) to be fifteen-fold less potent than analogous allyl esters (8, 9, 10, respectively), thus establishing the importance of anionic charge incorporation to destabilize binding of the caged compound (Figure 1E, Extended Data Figure 2). Steric bulk also decreased affinity of the caged inhibitor for NaVs, as bis-piperidine carboxylate 13 (STX-bpc) is approximately four-fold less potent than bis-carboxylate 12 (IC50: 13, 3.9 µM vs. 12, 1.0 µM; also compare compounds 10, 0.5 µM vs. 9, 0.07 µM).…”

“…Instead, methods like optogenetics 3,4 rely on the expression of exogenous ion channels that constructively (e.g, ChR2) 5 or destructively (e.g., WiChR) 6 interfere with endogenous currents to produce a net change in electrical output. While optogenetic methods have significantly improved our ability to control cellular activity and regulate neural circuits 7 , such technologies are often limited by toxicity, 8 delivery, 9 and expression 10 of the requisite ectopic proteins, as well as the altered capacitance, 11 electrochemical gradients, 12 and pH 13 of the target cells. 14 We have developed an alternative tool for manipulating APs both in vitro and in vivo that avoids genetic manipulation.…”