Bacterial lipopolysaccharide hyperpolarizes the membrane potential and is antagonized by the K2p channel blocker doxapram
Graphical abstract
Introduction
Understanding the regulation of resting membrane potential of cells is key to grasping how cells behave in relation to stimulation and inhibition of cellular responses as well as the transmission of signals to other cells. Many types of ion channels, pumps, and exchangers play a key role in maintaining a membrane potential. Pharmacological regulation of cell excitability and inhibition is a hallmark of many clinical treatments for cardiac and neural control. Additionally, understanding how to combat and treat pathogens which affect the cellular membrane potential is essential.
Interestingly, the larval Drosophila body wall muscle and skeletal muscle of crayfish results in rapid hyperpolarization followed by a return of the membrane potential to resting levels in the next few minutes when exposed to the endotoxin of gram-negative bacteria (i.e., lipopolysaccharides; LPS). The mechanism behind this rapid hyperpolarization has yet to be determined. The hyperpolarization of the muscle with LPS exposure still occurs in the presence of Cd2+ or Gd3+. The known blockers of voltage gated Ca2+ channel blockers, Cd2+ and Gd3+, used in previous studies confirmed LPS was not activating a Ca2+ influx in the muscle to activate calcium-activated potassium channels (KCa) (Cooper and Krall, 2022; Potter et al., 2021; Vacassenno et al., 2023). In addition, when no Ca2+ was added to the bathing saline, LPS still managed to hyperpolarize the membrane potential. Muscle contractions and depolarization of larval Drosophila body wall muscle occurs in part from Ca2+ entry from extracellular fluid across the plasma membrane and internal release of Ca2+ from sarcoplasmic reticulum (SER). Incubation with tetraethylammonium (TEA) (20 mM) for 20 min, which blocks KCa channels in Drosophila, did not dampen the LPS effect. However, the mechanism behind the larval body wall muscle still contracting when extracellular Ca2+ was substituted for Cd2+ is unclear in earlier studies, in which LPS was applied. This result led to the proposal that an internal release of Ca2+ from the SER occurs. Thus, if activating Ca2+ release from ryanodine-sensitive internal stores is possible then this could be an indirect means to stimulate KCa channels while the plasma membrane voltage-gated Ca2+ channels are blocked by Cd2+. 2-aminoethyl diphenylborinate (2-APB) was used in this study since it is known to block the sarco/endoplasmic reticulum Ca2+-ATPase pump (SERCA) and thus, reduce the release of Ca2+ via a ryanodine receptor (RyR) (Ma et al., 2000, Ma et al., 2002; Prakriya and Lewis, 2001;Goto et al., 2010).
Previous reports on adult Drosophila and moth muscles have indicated that the equilibrium potential for K+ may exceed −90 mV (Ikeda et al., 1976; Salkoff and Wyman, 1983). Therefore, the outflux of K+ could be key to hyperpolarization with LPS. The possibility of Cl− ion influx can be ruled out, as the chloride equilibrium potential is more depolarized above the resting membrane potential in body wall muscles of larval Drosophila (Rose et al., 2007; Stanley et al., 2019). Thoughts of acutely enhancing the action of the NaK ATPase pump were considered; however, no precedence for such a possibility exists, and the use of ouabain (10 mM) did not block the rapid onset of hyperpolarization by LPS (Potter et al., 2021). To explain the acute hyperpolarization of the muscle by LPS we proposed that two-pore domain potassium (K2p) channels are stimulated by LPS. The acute nature in the hyperpolarization may occur due to desensitization to account for the rapid hyperpolarization for 2 min followed by repolarization. With longer exposure times to LPS the cells depolarize until there is no electrical potential.
The K2p channels are responsible for maintaining resting membrane potential. These channels are generally referred to as leak channels because they are constitutively open and drive the membrane potential toward the potassium equilibrium potential. Various subtypes of K2p channels have different pharmacological profiles. The Drosophila genome has 11 known subtypes, and their distribution in expression within tissues is yet to be elucidated (Adams et al., 2000; Littleton and Ganetzky, 2000). The family of channels is divided into six subfamilies (tandem-pore weak inward rectifying K+ [TWIK], TREK, TASK, TALK, THIK, and TRESK) based on sequence similarity and functional resemblance (Plant and Goldstein, 2015). The nomenclature of the K2p subtypes is based on pharmacology and sensitivity to pH. Some of the classifications have been modified over the years (Duprat et al., 1997; Kamuene et al., 2021; Kim, 2005; Kim et al., 2000; Plant and Goldstein, 2015; Rajan et al., 2000).
The TWIK-1, TASK-1, and tandem-pore acid-sensing K+ (TASK-3) channels are sensitive to protonation, which is relevant to the larval Drosophila and crayfish muscle as the membranes of these tissues depolarize at lower pH levels (Badre et al., 2005; Bierbower and Cooper, 2010, Bierbower and Cooper, 2013). Interestingly, TASK-1 is activated only by halothane and isoflurane (Patel et al., 1999) and can lead to the hyperpolarization of cells. Moreover, the pharmacology of the K2p family of channels and their expression profiles in pathological conditions has clinical relevance (Lee et al., 2021).
Doxapram (stimulex or respiram) which is a blocker of some acid sensitive subtypes of K2p channels, and used in clinical applications, results in depolarization of larval Drosophila skeletal muscle (Vacassenno et al., 2023). Thus, in this study doxapram is used as a pharmacological approach to determine if the hyperpolarization induced by LPS can be blocked. The Drosophila model has served a vital role addressing the actions of the immune response induced by LPS which later led to a better understanding in the mechanisms of action in mammals and ultimately led to a few obtaining a Nobel Prize (i.e., Hoffmann and Beutler). The use of Drosophila continues to serve as a model, as a proof of concept, for many physiological studies related to other animals (Buckingham et al., 2005; Ugur et al., 2016; Yamaguchi and Yoshida, 2018).
K2p channels are known to function from yeast to humans with a wide range of subtypes in different organisms and their roles remain to be fully elucidated. The type of K2p channels expressed in skeletal muscle of Drosophila has yet to be identified. Even between rodents and humans, sensitivities to the enantiomers of isoflurane, an agonist of some K2p channels, are different. Additionally, regional differences exist in the central nervous system (CNS), including the spinal cord, of rodents and humans to the anesthetic bupivacaine and lidocaine, which are known to activate K2p channels (Keshavaprasad et al., 2005). Thus, examining further the function and pharmacology of K2p channels in various organisms will lead to an increase in the overall understanding of these unique channels (Buckingham et al., 2005).
Activation of K2p and/or KCa channels could result in hyperpolarization of the plasma membrane. 2-APB was used to further investigate the possibility of the Ca2+ release from SER, as loading of the SER should be blocked by this compound. Doxapram was used to examine if depolarization would occur upon exposure and if it could potentially block the hyperpolarization induced by LPS.
There are potential clinical reasons to determine the receptor subtypes for LPS that induce cytokine release, but also the potential for activation of K2p channels by LPS. However, whether LPS exerts an action on the K2p channels in mammals remains unknown. If so, recognizing that the receptors to LPS which could be blocked prior to a bolus of antibiotics to treat severe septicemia caused by gram-negative bacteria, would be advantageous in preventing a cytokine storm (Bone, 1991; Lin and Lowry, 1998). To date, blockers of the assumed LPS receptor complex, which is known as the CD14/TLR4/MD2 complex, remain unidentified in mammals (Du et al., 2000; Meng et al., 2010; Shimazu et al., 1999; Wright et al., 1990). Septicemia still causes high mortality worldwide, and in many cases, it is due to gram-negative bacterial strains and related endotoxins (CDC Statistics, 2022). Thus, understanding the potential action of LPS and developing models to examine the mechanisms of action are necessary.
Section snippets
Methods
Similar experimental conditions previously used to examine the possible mechanism of action of LPS in larval Drosophila muscles were used in this study (Bernard et al., 2020; Ballinger-Boone et al., 2020; Cooper et al., 2019; Istas et al., 2020; Potter et al., 2021; Vacassenno et al., 2023). The same procedures and Drosophila species as described by Vacassenno et al. (2023) were used in this study.
Effects of LPS and 2-APB
Exposure to LPS immediately resulted in hyperpolarization of the membrane potential. When the segmental motor nerve was electrically stimulated, the evoked EJPs dampened in amplitude quickly along with mEJPs (Fig. 1). Rapid hyperpolarization occurred every time after exposure to LPS (N = 6; P < 0.05 sign test). Considering that the reversal potential for the ionotropic glutamate receptors on the larval Drosophila muscle is close to −10 mV to 0 mV, the amplitude of the EJPs and mEJPs should have
Discussion
The mechanism of action by which LPS hyperpolarizes the membrane potential had not been resolved previously. A Cl− channel, or potentially KCa channels, as well as hyperactivation of the NaK ATPase pump, were considered to be responsible. However, these suggestions regarding the use of the larval Drosophila muscle as a model can now be put to rest. The Cl− equilibrium potential is more depolarized than the resting membrane potential of these cells (Rose et al., 2007; Stanley et al., 2019).
Funding
Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103436 (R.M.V.). Chellgren Endowed Funding (R.L.C.). University of Kentucky, College of Arts and Sciences summer fellowship (C.N.H.).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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