Biology subjects

Kharche, S.

Publications and source records attributed to Kharche, S..

3 recordsLinked to original sources

bioRxiv2026

Blood Pressure Regulates Functional Coupling of L-Type Ca2+ Channels: Reimaging the Foundation of Cerebral Blood Flow Control.

The myogenic response is the key autoregulatory mechanism setting cerebral blood flow and its mechanistic foundation is intimately tied to depolarization and the voltage gating of L-type Ca2+ channels (CaV1.2). While critical, this study argues for an additional mechanism, that of pressure itself enhancing CaV1.2 activity via cooperative gating and perimembrane trafficking of channels subunits. These novel insights were pursued at the cell level using patch-clamp electrophysiology and advanced microscopy, and then functionally in pressurized arteries through measures of tone and intracellular [Ca2+]i. Key findings were confirmed in mutant mice with disrupted functional coupling and translated into arteries procured from human brain tissue. From cerebral blood flow simulations of semi-realistic microvascular networks, we predict that loss of this alternative mechanism leads to maldistribution of brain blood flow and potentially a diminishment of cognitive function. This study reveals previously unrecognized pressure-sensitive CaV1.2 regulatory mechanism that advances understanding of cerebral blood flow. SignificanceBlood pressure sets base arterial constriction - a response critical for blood flow control in brain. This response is tied to CaV1.2 channels and their presumptive and exclusive activation by voltage, reasoning now under great scrutiny. We establish herein with advance methods, a second mode of CaV1.2 pressure regulation, that of enhanced functional cooperativity among neighboring channels. This novel mechano-response is tied to PKC and its ability to set channel phosphorylation and CaV1.2 trafficking. CaV1.2 pressure regulation was observed in human tissues and its disruption (mutant mice) impaired myogenic tone in the presence of preserved voltage control. Cerebral microvascular modeling highlights that losing this mechanism destabilizes blood flow distribution in brain, the knock-on effect being comprised cognitive function.

physiology↗

bioRxiv2024

Functional Bias of Contractile Control in Mouse Resistance Arteries

BackgroundConstrictor agonists set vascular tone through two coupling processes, one tied to (electromechanical), the other independent (pharmacomechanical) of membrane potential (VM). This arrangement raises an intriguing query: are they variably recruited such that each agonist elicits a range of vasomotor signatures, functionally biased towards one mechanism or the other? This query underlies this study and our examination of agonist-induced arterial constriction. MethodsMouse mesenteric arteries were exposed to a classic Gq/11 (phenylephrine) or Gq/11/G12/13 (U46619) coupled receptor agonist, and responses monitored in the absence and presence of L-type Ca2+ channel/protein kinase inhibitors. Contractile work was supplemented with measures of protein phosphorylation, VM, and cytosolic Ca2+; conceptual insights were enhanced with computational modeling. ResultsEach constrictor elicited a response curve that was attenuated and rightward shifted by nifedipine, findings aligned with functional bias; electromechanical coupling preceded pharmacomechanical, the latters importance rising with agonist concentration. Ensuing contractile and phosphorylation (CPI-17 & MYPT1 (T-855 & T-697)) measures revealed phenylephrine-induced pharmacomechanical coupling was tied to protein kinase C (PKC), while U46619 was tied to both PKC and Rho-kinase. A switch to pharmacomechanical coupling dominance occurred when agonist superfusion was replaced with discrete application to a small portion of artery. This switch was predicted by electromechanical modeling and supported by direct measures of VM and cytosolic Ca2+. ConclusionsOur work illustrates that constrictor agonists elicit functionally biased responses and that arteries toggle among contractile mechanisms, dependent on receptor signal bias, structural/electrical properties, and how agents are applied. We discuss how hemodynamic control is intimately tied to functional bias in both health and disease states, including but not limited to arterial vasospasm. O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/588016v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@11632b9org.highwire.dtl.DTLVardef@5c1144org.highwire.dtl.DTLVardef@719b08org.highwire.dtl.DTLVardef@795ec3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAgonist-induced constrictor responses exhibit a "functional bias" toward electromechanical or pharmacomechanical coupling dependent on agent concentration and mode of application. C_LIO_LIElectromechanical coupling typically but not exclusively precedes pharmacomechanical, the latter rising to prominence with agent concentration. Pharmacomechanical coupling is mediated through receptor pathways linked to PKC and Rho-kinase, regulatory proteins that target the catalytic and targeting subunit of MLCP. C_LIO_LIFunctional bias is malleable and thus each agonist elicits a range of vasomotor signatures, each presumptively important in controlling blood flow delivery in space and time. C_LI

physiology↗

bioRxiv2020

Conformational plasticity and dynamic interactions of the N-terminal domain of the chemokine receptor CXCR1

Dynamic interactions between G protein-coupled receptors (GPCRs) and their cognate protein partners at the membrane interface control several cellular signaling pathways. An important example is the association of CXC chemokine receptor 1 (CXCR1) with its cognate chemokine, interleukin-8 (IL8 or CXCL8) that regulates neutrophil-mediated immune responses. Although the N-terminal domain of the receptor is known to confer ligand selectivity, the conformational dynamics of this intrinsically disordered region of CXCR1 in particular, and chemokine receptors in general, remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations that were validated by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo-receptor N-terminal region is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we validated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies. Our results indicate that caution should be exercised when chemical shift perturbation is used as a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces. Author summaryHow cells communicate with the outside environment is intricately controlled and regulated by a large family of receptors on the cell membrane (G protein-coupled receptors or GPCRs) that respond to external signals (termed ligands). Chemokine receptors belong to this GPCR family and regulate immune responses. We analyze here the first step of binding of a representative chemokine receptor (CXCR1) with its natural ligand, interleukin 8 (IL8) by an extensive set of molecular dynamics simulations. Our work complements previous mutational and NMR experiments which lack molecular-level resolution. We show that in the inactive state, one of the extracellular domains of the CXCR1 receptor, namely the N-terminal domain, is highly flexible and like a "shape-shifter" can exist in multiple conformational states. However, when IL8 binds, the N-terminal domain undergoes a conformational freezing, and acquires a C-shaped "claw-like" structure. The complex between the receptor and IL8 is still quite dynamic as this C-shaped N-terminal domain forms an extensive but slippery interface with the ligand. We further validated these results by quantitative comparison with NMR and mutagenesis studies. Our work helps clarify the inherent disorder in N-terminal domains of chemokine receptors and demonstrates how this domain can acquire functionally important conformational states in dynamic protein-protein interfaces.

biophysics↗