Biology subjects

Peters, C. H.

Publications and source records attributed to Peters, C. H..

6 recordsLinked to original sources

bioRxiv2026

The proximal N-terminus of IRAG is required for potentiation of HCN4 channels

The inositol triphosphate-associated, ER transmembrane proteins IRAG and LRMP are isoform specific regulators of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, while IRAG mimics the effect of cAMP on the channel. We previously showed that regulation by LRMP requires both the N-terminus of HCN4 and a unique orientation of the HCN4 cAMP transduction center, which is comprised of the N-terminal HCN domain, the C-linker, and the S4-S5 linker. However, it remains unknown if the homologous IRAG requires similar structural features to mimic cAMP-dependent potentiation, or if the site and mechanism of action are different between the two regulators. Using patch clamp electrophysiology, we determined that the initial 43 amino acids of IRAG are necessary and sufficient to confer regulation of HCN4. Similar to LRMP, IRAG also requires a portion of the N-terminus of HCN4 to confer its regulatory effects. Also similar to LRMP, two point mutations in the C-linker region, which are the only sequence differences in that region between HCN4 and the other HCN isoforms, were able to eliminate the effect of IRAG suggesting the unique orientation of the cAMP transduction center in HCN4 is likely important for IRAG function. Taken together, these findings suggest a model whereby IRAG and LRMP interact with the channel in similar regions, although potentially in unique ways, and act on the cAMP transduction center with LRMP inhibiting the coupling of this region to gating and IRAG strengthening it. SUMMARYThe ER transmembrane protein IRAG binds to and potentiates HCN4 channels. This study demonstrates that IRAG regulation of HCN4 requires only the first 43 amino acids of IRAG and involves contributions from the N-terminus and cAMP transduction center of HCN4.

biophysics↗

bioRxiv2023

LRMP inhibits the cAMP-dependence of HCN4 channels by disrupting intramolecular signal transduction

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4 but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here we identify the domains of LRMP essential for regulation. We show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating. And we demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Forster resonance energy transfer (FRET), we showed that the initial 227 residues of LRMP and the N-terminus of HCN4 are necessary for LRMP to interact with HCN4. We found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. And we demonstrate that LRMP-regulation can be conferred to HCN2 by addition of the HCN4 N-terminus along with mutation of 5 residues in the S5 region and C-linker to the cognate HCN4 residues. Taken together, these results suggest that LRMP inhibits HCN4 through an isoform-specific interaction involving the N-terminals of both proteins that prevents the transduction of cAMP binding into a change in channel gating via an HCN4-specific orientation of the N-terminus, C-linker, and S4-S5 linker.

biophysics↗

bioRxiv2022

The funny current If is essential for the fight-or-flight response in cardiac pacemaker cells

The sympathetic nervous system fight-or-flight response is characterized by a rapid increase in heart rate, which is mediated by an increase in the spontaneous action potential (AP) firing rate of pacemaker cells in the sinoatrial node. Sympathetic neurons stimulate sinoatrial myocytes (SAMs) by activating {beta} adrenergic receptors ({beta}ARs) and increasing cAMP. The funny current (If) is among the cAMP-sensitive currents in SAMs. If is critical for pacemaker activity, however, its role in the fight-or-flight response remains controversial. In this study, we used AP waveform analysis, machine learning, and dynamic clamp experiments in acutely-isolated SAMs from mice to quantitatively define the AP waveform changes and role of If in the fight-or-flight response. We found that while {beta}AR stimulation significantly altered nearly all AP waveform parameters, the increase in AP firing rate was only correlated with changes in a subset of parameters (diastolic duration, late AP duration, and diastolic depolarization rate). Dynamic clamp injection of the {beta}AR-sensitive component of If showed that it accounts for approximately 41% of the fight-or-flight increase in AP firing rate and 60% of the decrease in the interval between APs. Thus, If is an essential contributor to the fight-or-flight increase in heart rate.

physiology↗

bioRxiv2021

Bi-directional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current, If, is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. We used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2-5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that {beta}-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously-expressed HCN4 channels and by mathematical models of If. Modelling further suggested that the slow activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions. Significance StatementCardiac pacemaker cells trigger each heartbeat by virtue of spontaneous oscillations in their membrane voltage. Although the funny current (If) is critical for these oscillations and for setting heart rate, its precise role remains an enigma because it activates mostly outside of the physiological voltage range and quite slowly relative to the pacemaker cycle. Here we show that If is persistently active in pacemaker cells; once opened, the small fraction of ion channels that conduct If do not re-close. Consequently, If flows both inward and outward to help propel the voltage oscillations and it paradoxically conducts a large fraction of the net charge movement. These results establish a new conceptual framework for the role of If in driving cardiac pacemaking.

physiology↗

bioRxiv2020

Isoform-specific regulation of HCN4 channels by a family of endoplasmic reticulum proteins

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum transmembrane proteins that interact with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage-dependence of HCN4 activation in response to binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage-dependence of activation in the absence of cAMP. The mechanisms of action of LRMP and IRAG are novel; they are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 availability. Our results suggest important roles for LRMP and IRAG in regulation of cellular excitability and as tools for advancing mechanistic understanding of HCN4 channel function. Significance statementThe pore-forming subunits of ion channels are regulated by auxiliary interacting proteins. Hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channels are critical determinants of electrical excitability in many types of cells including neurons and cardiac pacemaker cells. Here we report the discovery of two novel HCN4 regulatory proteins. Despite their homology, the two proteins -- lymphoid-restricted membrane protein (LRMP) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG) -- have opposing effects on HCN4, causing loss- and gain-of-function, respectively. LRMP and IRAG are expected to play critical roles in regulation of physiological processes ranging from wakefulness to heart rate through their modulation of HCN4 channel function.

physiology↗

bioRxiv2017

A Mixed Periodic Paralysis & Myotonia Mutant, P1158S, Imparts pH Sensitivity in Skeletal Muscle Voltage-gated Sodium Channels

Skeletal muscle channelopathies, many of which are inherited as autosomal dominant mutations, include both myotonia and periodic paralysis. Myotonia is defined by a delayed relaxation after muscular contraction, whereas periodic paralysis is defined by episodic attacks of weakness. One sub-type of periodic paralysis, known as hypokalemic periodic paralysis (hypoPP), is associated with low potassium levels. Interestingly, the P1158S missense mutant, located in the third domain S4-S5 linker of the skeletal muscle voltage-gated sodium channel, Nav1.4, has been implicated in causing both myotonia and hypoPP. A common trigger for these conditions is physical activity. We previously reported that Nav1.4 is relatively insensitive to changes in extracellular pH compared to Nav1.2 and Nav1.5. Given that intense exercise is often accompanied by blood acidosis, we decided to test whether changes in pH would push gating in P1158S towards either phenotype. Our results indicate that, unlike in WT Nav1.4, low pH depolarizes the voltage-dependence of activation and steady-state fast inactivation, decreases current density, and increases late currents in P1185S. Thus, P1185S turns the normally pH-insensitive Nav1.4 into a proton-sensitive channel. Using action potential modeling we also predict a pH-to-phenotype correlation in patients with P1158S. We conclude that activities which alter blood pH may trigger myotonia or periodic paralysis in P1158S patients.\n\nSIGNIFICANCE STATEMENTVoltage-gated sodium channels (Nav) contribute to the physiology and pathophysiology of electrical signaling in excitable cells. Nav subtypes are expressed in a tissue-specific manner, thus they respond differently to physiological modulators. For instance, the cardiac subtype, Nav1.5, can be modified by changes in blood pH; however, the skeletal muscle subtype, Nav1.4, is mostly pH-insensitive. Nav1.4 mutants can mostly cause either hyper-or hypo-excitability in skeletal muscles, leading to conditions such as myotonia or periodic paralysis. P1158S uniquely causes both phenotypes. This study investigates pH-sensitivity in P1158S, and describes how physiological pH changes can push P1158S to cause myotonia and periodic paralysis.

biophysics↗