Cell signalling

 Cell Signaling: 

Cell Signaling Mechanisms

The sympathetic and parasympathetic branches of the autonomic nervous system have distinct effects on the heart

• Sympathetic postganglionic neurons increasing heart rate and force of contraction primarily via beta-1 adrenergic receptors. 

• parasympathetic system: 

  •  especially through the vagus nerve,  innervates various heart structures

  • heart activity (slow heart rate ) by affecting various heart regions, including the sinoatrial and atrioventricular nodes, and his bundle .

The autonomic nervous system's sympathetic and parasympathetic branches mediate distinct effects on the heart.

Autonomic Nervous System Overviews.The heart rate is primarily modulated through the autonomic nervous system, particularly via the activation of muscarinic M2 receptors

When there is Increased tissue demand for blood flow:  activates the sympathetic arm is going to be activate, leading to elevated cardiac output, while the parasympathetic branch is suppressed. 

This interplay highlights how different organs, particularly the heart and skeletal muscle, are influenced by the nervous system to maintain homeostasis.

Sympathetic vs Parasympathetic Effects

The heart's function is innervated  by parasympathetic nerves primarily from the vagus nerve (cranial nerve 10), which innervates ganglia surrounding the heart. 

• These nerves originate from ganglia situated in the fat pads surrounding the heart,

• These nerves target specific regions such as the SA node, AV node, atria, and the His bundle, 

  • not affecting ventricular muscles.  Explains lack of effect of parasympathetic nervous system on ventricular but not aortic  contractility 

  • This localized innervation allows for distinct regulatory effects on cardiac function.

Innervation of the Heart

The parasympathetic nervous system primarily affects the atrial region and nodal areas of the heart, as it does not innervate ventricular muscle, leading to a lack of impact on ventricular contractility.

 In contrast, sympathetic innervation is widespread throughout the heart, including the ventricular regions, resulting in different effects on heart function. Consequently, the autonomic nervous system plays a crucial role in regulating cardiac activity, with distinct pathways for parasympathetic and sympathetic signals.

Vagal Innervation and Cardiac Function

The balance between sympathetic and parasympathetic innervations at the SA and AV nodes is crucial for regulating heart rate and conduction rate. 

While sympathetic activation enhances atrial muscle contractility, parasympathetic effects reduce it, showcasing the importance of their interplay

. In response to stress or exercise, the heart can rapidly adjust its ionotropic, chronotropic, and lusitropic properties to meet physiological demands.

Sympathetic Regulation of Heart

When it has to increase force development/increase of of sacromere length or get into the ideal physiological range: 

• Increasing preload in the cardiac system utilizes the cardiac reserve significantly affected by enhanced beta adrenergic signaling through cyclic AMP and protein kinase A, responding to sympathetic stimulation. 

• The study of beta adrenergic signaling, particularly using agonists like isoproterenol, illustrates the relationship between contractile force and calcium transient  

  • rising intracellular calcium precedes the increase in contractile force. This dynamic is crucial for understanding excitation-contraction coupling and the heart's adaptive response to stress.

Beta Adrenergic Signaling

As isoproterenol dose increases, both the amplitude of calcium transients and force production during muscle contraction rise progressively, demonstrating a positive ionotropic effect. 

Additionally, a leftward shift in the curve suggests an increased rate of response, indicative of positive chronotropy, which signifies that heart rate accelerates with higher isoproterenol levels.

Positive Inotropic and Chronotropic Effects

• Increased intracellular calcium levels during isoproterenol (iso) interaction enhance both the rate of muscle contraction and relaxation, demonstrating a positive lusitropic effect. 

• This results in a steeper decline in force production, highlighting increased contractile dynamics. 

The discussion will further explore how beta adrenergic receptor activation from sympathetic nerve activity leads to these inotropic, lusitropic, and chronotropic effects.

Intracellular Calcium Dynamics

The activation of the beta adrenergic receptor signaling pathway leads to an increase in cyclic AMP (cAMP) and protein kinase A (PKA) activity. 

• Results: phosphorylation of crucial components involved in excitation-contraction coupling, such as L-type calcium channels and ryanodine receptors, which enhances the physiological response associated with the fight-or-flight mechanism. 

• Previous knowledge of target proteins and their modulation through phosphorylation is reinforced as these interactions are further explored.

fo

13:45

Beta Adrenergic Receptor Activation

• Adrenal receptors, including alpha and beta types, play a significant role in calcium release and reuptake within smooth muscle cells, influenced by the autonomic nervous system. 

• These receptors are classified as G protein-coupled receptors and particularly focus on beta adrenergic receptor subtypes (beta 1, beta 2, and beta 3), each linked to distinct G proteins that generate unique cellular effects. 

G Protein Coupled Receptors

B-ADERNERGIC RECEPTORS ARE also g coupled receptors 

.G protein-coupled receptors (GPCRs), including beta adrenergic receptors, consist of heterotrimeric protein complexes with G alpha, beta, and gamma subunits, where G alpha subunits are

 further classified into four main types of interest:

•  G alpha s (heart) 

•  G alpha i/o (heart) 

• , G alpha q/11,  (heart) 

•  G alpha 12/13. ( found in blood vessel) 

G Protein Signaling Pathways

• G protein-coupled receptors (GPCRs) play a significant role in cell signaling, particularly in the heart and blood vessels

•  various G alpha subunits activate different signaling pathways. 

  • The mechanism involves the exchange of GDP for GTP, facilitating conformational and functional changes in the receptors. 

  • Understanding the steps of this signaling process, such as the interaction between ligands and receptors like beta adrenergic receptors, is crucial for comprehending how cellular communication occurs.

17:50

G Protein Signaling Mechanism

6 steps: 

1. Isoproterenol binding to beta adrenergic receptors (ligand binding) 

2. Induces a conformational change in G-protein coupled receptors (GPCRs)

3. leading to the exchange of GDP for GTP. 

• Bound in relaxed state 

4. Dissociation of the GTP-bound alpha subunit (g beta and g gamma) 

• , which activates downstream signalling cascades 

5.  Hydrolysis of GTP restores the inactive state by allowing reassociation with beta and gamma subunits, 

• LIGAND COMES OFF once hydrolysis occurs 

7. enabling the process to repeat, with g beta 1 and beta 2 adrenergic 

8, reassosciate g alpha and g beta 

6. receptors being the predominant subtypes in the human heart.

Beta Receptor Subtypes

• Beta 1 and beta 2 adrenergic receptors have greater sensitivity to catecholamines

 like isoproterenol compared to norepinephrine, highlighting their role in mediating the autonomic nervous system's effects on the heart. 

• The unique structure of catecholamines, characterized by a benzene ring with 2 hydroxyl groups + intermediate ethyl chain and amine group, i

• Influences their activity and the necessity for beta blockers in managing excessive sympathetic activation in cardiovascular disorders. (BLOCK ACCESS TO RECEPTORS) 

• Each beta receptor subtype exhibits different affinities for agonists and antagonists, emphasizing the complexity of their signaling mechanisms.

B1/B2 IS PRESENT IN ABOUT 60=-705 in the atria and 705-805 in ventricles

B3 is not that much

Catecholamine Effects on Heart

Different adrenergic receptor subtypes exhibit variable affinities for agonists like isoproterenol, with beta 3 displaying the highest affinity compared to other beta pathways. This variability influences the activation of distinct signaling pathways when catecholamines are increased. The canonical beta 1 adrenergic signaling pathway, prominently involving adenylate cyclase and cyclic AMP, is central to understanding these downstream effects.

Downstream Signaling Pathways

The beta-2 adrenergic receptors exist in several conformational states:  active (drug bound to receptor) , inactive, and inactivated states, influencing their activity. 

Understanding these conformations is crucial as they relate to the receptor's structure and function, particularly in the context of drug binding.

28:44

Beta 2 Receptor Conformational States

The conformational changes in the transmembrane regions, particularly TM5 and TM6, are crucial for receptor activation, especially when ligands bind to the beta-2 adrenergic receptor.

 This binding induces a significant change in the G alpha s subunit, activating signaling cascades by facilitating GDP to GTP exchange. The structural dynamics of the G alpha s subunit, including its ras-like and alpha helical domains, play a vital role in this process, allowing the nucleotide binding pocket to open and enable GTP binding.

G Protein Activation

ONCE BETA ADNERGIC ACTIVATED CUSE CONF CHANGE IN G ALPHA SUBUNITS  

The G alpha s subunit is composed of two key domains:

1.  g ALPHa ras-like domain interacting with the beta 2 adrenergic receptor and a G beta gamma subunit, 

2. alpha helical domain crucial for activating the signaling cascade. The G alpha s ras unit engages directly with the receptor and interacts with the G beta gamma subunit, forming a complex that drives cell signaling.

Structural changes in GAS following B2AR acTIVATION 

• The activation of the G alpha s subunit leads to a rotational movement of its alpha helical domain, which occurs upon GDP binding or when GTP replaces GDP. 

• This rotation opens the nucleotide binding pocket, allowing GTP to bind and induce further movement of the alpha helical domain (gasah) . Consequently, the transition from GDP to GTP is essential for the functional dynamics of the G alpha s subunit.

Nucleotide Binding Pocket Dynamics

The intrinsic rate of GTP hydrolysis by the G alpha subunit is influenced by the duration the GTP is bound to it.

 Once GTP replaces GDP, the G alpha subunit is released from G beta gamma subunits and acts as an effective vector to trigger downstream signaling cascades. This dynamic emphasizes the importance of the GTP-bound state in regulating cellular signaling processes.

34:25

GTP Hydrolysis Rate

Experimental measurements of GTP hydrolysis indicate that its rate is too slow to account for the rapid deactivation kinetics of G-protein signaling observed in vivo, which is 10 to 100 times faster than expected. 

While GTP hydrolysis is thought to be crucial in turning off G-protein pathways by reverting GTP to GDP, the actual deactivation dynamics suggest other factors are contributing to this faster process. Specifically, the involvement of guanine nucleotide exchange factors plays a role in explaining these observations.

35:54

Regualtors of G-protein Signalling

1. GEF 

2. GDI

Guanine nucleotide exchange factors (GEFs): play a crucial role in cell signaling by facilitating the replacement of GDP with GTP, thus activating the G alpha subunit. 

guanine dissociation inhibitors:  prevent GDP from dissociating and regulators of G protein signaling (RGS) help in deactivating the signaling pathway by promoting the reverse process.

 Together, GTPase-activating proteins (GAPs) and regulators of G protein signaling (RGS) help in deactivating G proteins, highlighting the dynamic regulation of cell signaling pathways.

RGS Proteins Function

RGS proteins play a crucial role in regulating G protein-coupled receptor signaling by facilitating two main mechanisms: they accelerate the hydrolysis of GTP on the G alpha subunit, thus turning off the signaling, and they compete with effector proteins to drive the inactive state of the G alpha subunit by binding to the GTP-bound form. This regulation ensures that downstream signaling is effectively controlled, highlighting the importance of RGS proteins in cellular signaling pathways.

Role of Regulatory Proteins

Regulatory proteins, particularly RGS (Regulator of G Protein Signaling), play a crucial role in the activation and inactivation of G protein coupled receptors by influencing the hydrolysis of GTP on the G alpha subunit. T

1. They accelerate this hydrolysis, leading to the transition of G alpha from its active GTP-bound form back to its inactive GDP-bound state, thus preventing downstream signaling.

2.  RGS proteins can compete with effector proteins, further driving the system towards inactivation.

#2 is affecting other ones which can affect the isgnalling cascade 

01:23

G Protein Activation

- Adenylyl cyclase (AC):  catalyzes the conversion of ATP to cyclic AMP (cAMP), 

- two transmembrane regions and two cytoplasmic domains (C1a and C2a) that form the catalytic domain essential for this conversion.

- CA1 AND Ca2 is WHERE  where atp is cyclized to cAMP   

- nine isoforms of AC, with AC5 and AC6 being predominantly expressed in cardiomyocytes. The structural configuration of this enzyme is critical for its catalytic activity.

Coupled in the g complex

2 6 trasnmembrane regions, 2 cytoplasmic domains, c1a and c2A 

Adenylyl Cyclase Structure

Cyclic AMP production begins with the breakdown of ATP, losing two inorganic phosphates to form a cyclic monophosphate, which occurs in roughly two seconds, slower than the initial activation of the receptor and G protein that happens within 50 milliseconds.

 In the heart, the rate-limiting step for cyclic AMP production by the beta adrenergic receptor pathway is the expression level of adenylyl cyclase, which is crucial for driving the rate of this reaction.

Cyclic AMP Production

The rate-limiting step in the G protein-coupled receptor signaling pathway determines the speed of the overall reaction, which primarily depends on the expression levels of adenylyl cyclase. The process involves GDP activation leading to the conversion of ATP into cyclic AMP, facilitated by GTP-bound alpha subunits of the G protein. This increase in intracellular cyclic AMP is crucial for cell signaling processes.

05:54

Rate Limiting Step

INCREASE IN camp LEADS TO  Activation of protein kinase A (PKA),: a cyclic AMP-dependent protein kinase, involves the conversion of its inactive form (apoenzyme) to its active form (haloenzyme). 

This process requires cyclic AMP to bind to the regulatory subunits, leading to the release of the catalytic subunits and activating the enzyme. 

WE NEED A INCREASE IN CAMP TO CONVERT TO IRTS ACTIVE FORM BC PKA IS IN AN INACTIVE FORM 

Protein Kinase A Activation

• Phosphorylation of target proteins plays a crucial role in modulating their activity, enhancing the function of signaling cascades. 

• In beta-2 adrenergic receptor signaling, it activates activation or inhibition of adenylyl cyclase, depending on the associated G protein subunit, illustrating the complexity of pathway outcomes based on receptor interactions

• Classical pathway: gs, ac, camp, pka 

• Activate another g coupled: gi, pde4, camp, pka (inhibition) 

Beta-2 Adrenergic Signaling

Different mechanisms exist to modulate signaling cascades, particularly through the inhibition of adenylyl cyclase via the g alpha i subunit, 

which activates specific phosphodiesterases (PDEs) that degrade cyclic AMP. 

• This degradation process inactivates the signaling pathways involving cyclic AMP and cyclic GMP. 

Way to be modulated for signalling cascade 

Phosphodiesterases Role

Phosphodiesterases (PDEs), of which there are over 50 types, play a crucial role in modulating cyclic AMP (cAMP) and protein kinase A (PKA) signaling by breaking phosphodiester bonds, effectively turning off the signaling cascade

. Additionally, phosphatases (PPs) contribute to this process by catalyzing the dephosphorylation of proteins, thus further regulating the cyclic AMP/PKA signaling pathway.

Phosphatases Function

Phosphatases play a crucial role in cell signaling by dephosphorylating amino acids like tyrosine, serine, and threonine, as well as lipids, and are primarily associated with G protein coupled receptors through phosphatase 1, 2A, and P2B (calcineurin). These signaling molecules are organized into macromolecular complexes near their target proteins rather than being randomly dispersed within the myocyte, ensuring that phosphorylation and dephosphorylation processes are efficiently modulated. The interplay between kinases and phosphatases is essential for regulating cellular activities.

13:27

Macromolecular Complexes

Signalling moelecules form macromolecualr complexes 

Alpha kinase anchoring proteins (A-KAPs) : assembling signaling complexes that enhance the regulation of various cellular activities, including those of cyclic AMP and PKA in cardiomyocytes.

 By localizing regulatory molecules close to their targets,A-KAPs help achieve distinct physiological responses even when cyclic AMP levels are similar. The presence of these complexes is essential for mediating functional changes within cells and influencing signaling cascades.

A Kinase Anchoring Proteins (AKAP)

Kinase anchoring proteins (AKAPs) play a crucial role in organizing signaling complexes that regulate activities such as excitation-contraction coupling in cardiomyocytes. 

They ensure that protein kinases (like PKA and PKC) and phosphatases are localized near the L-type calcium channel, facilitating modulation of calcium influx by either enhancing or downregulating signaling.

 This structural organization enables efficient regulation of cellular functions.

Excitation-Contraction Coupling

Rianidine receptors at the sarcoplasmic reticulum (SR) interact with a macromolecular signaling complex that responds to when cytosolic calcium levelsi increase  

This complex, comprising protein kinase A (PKA), phosphodiesterase, and phosphatase 2A, regulates the phosphorylation of rianidine and influences calcium release. 

Additionally, proteins such as phospholamban and SIRCA2 facilitate the active transport of calcium back into the SR, optimizing cell signaling through localized interactions.

In orderto change the reuptake of calcium into the SR 

17:40

Compartmentalization of Signaling

G-couple protein receptors and AKAPS play a vital role in the targeting and regulation of calcium signaling, providing tight spatial and temporal control. 

Different phosphodiesterases and phosphatases are localized in specific cell regions, contributing to efficient signaling through compartmentalization. 

compartmentalization affects beta adrenergic signaling, particularly noting the predominance of beta one adrenergic receptors in the human heart.

Beta Adrenergic Receptor Distribution

Hwo do we study compartmentalization ??? 

B1 and b2 IS WORTH A LOT COMPARED TO B3 

In wild-type mice, which possess both beta 1 and beta 2 adrenergic receptors, administration of isoproterenol leads to an expected increase in the rate of contraction due to ionotropic and chronotropic effects. 

This raises questions about the individual contributions of each receptor, particularly in the context of signaling pathways. 

• When examining mouse hearts expressing only beta 1 adrenergic receptors, researchers aim to understand the compartmentalization and regulation of these distinct signaling pathways.

• If it only expresses b1: b2 will be knocked out 

20:23

Receptor Knockout Studies

The impact of beta-adrenergic receptor knockout on contraction rates reveals distinct responses to isoproterenol stimulation. 

• In beta 2 knockout mice, a sustained increase in contraction rate occurs, but is less pronounced than in wild-type mice, 

• beta 1 knockout mice show only a small, transient response. This highlights the different roles of beta 1 and beta 2 receptors in mediating cardiac contraction dynamics.

•  loss of both beta 1 and beta 2 adrenergic receptors results in no change in contractile rate when isoproterenol is administered, illustrating the distinct regulatory roles of these receptors in heart signaling. 

Differential Responses

 Compartmentalization of different beta adrenergic subtypes allows for unique targets and pathways to be activated by the same signaling molecule, promoting varied activation profiles.

 The specific localization of beta receptors around t-tubules and the plasma membrane enhances their accessibility to circulating catecholamines and underscores the complexity of beta adrenergic signaling in cardiomyocytes.

24:29

Beta Adrenergic Signaling Complexity

WHERE In the cardiomyocytes where they are located? 

Immunostaining of beta adrenergic receptors in cardiomyocytes reveals distinct distribution patterns: 

beta-1 receptors show increased density at the cell periphery and within t-tubules, beta-2 receptors are primarily located inside the cell along the t-tubules

. This highlights the compartmentalization and preferential localization of different beta adrenergic receptor isoforms, showcasing their functional significance within cardiac cells.

25:54

Immuno Staining of Receptors

Subcompartmentalization: Preferential localization and interaction w basic adrenergic receptors 

Adenylyl cyclase isoform 6 (AC6) interacts with beta 1 adrenergic receptors on the plasma membrane and in T-tubules, while 

• isoform 5 (AC5) is located in T-tubules, specifically coupled with beta 2 adrenergic receptors.

  •  This localization exemplifies subcompartmentalization, highlighting the distinct roles of different adenylyl cyclase isoforms in heart signaling. 

  • The anchoring of AC5 to the T-tubular membrane further illustrates the specificity of these interactions.

CAV3= present in many cell types and signalling ,molecules 

27:14

Adenylyl Cyclase Interaction

Cavia linen 3: compartments in the cell membrane that are rich in signaling molecules and facilitate transmembrane transport, playing a crucial role in regulating signaling dynamics, particularly with beta-adrenergic receptors. 

Analysis of beta-adrenergic signaling illustrates that beta-1 receptors lead to a quicker and more sustained contraction response compared to the slower, transient responses associated with beta-2 receptors. This understanding enhances insights into the role of specific receptor isoforms in cardiovascular signaling.

Caveolae and Signaling Molecules

In the context of compartmentalization, t

he response seen in hearts with only beta2 adrenergic receptors and lacking beta1 receptors indicates distinct signaling mechanisms. 

The transient enhancement in contraction upon isoproterenol stimulation is attributed to the involvement of phosphodiesterases (PDEs) with beta2 receptors, which are absent in the beta1 receptor complex. This difference highlights how the presence or absence of PDEs impacts cyclic AMP degradation, thus influencing cardiac contractility.

No phosphodiesterase cannot break camp but its bad bc its necessary or else it cannot subcaprtmentalize 

29:53

Differential Responses of Receptors

Differential modulation of beta adrenergic receptors affects cyclic AMP signaling by compartmentalizing and regulating pathways uniquely. 

Specifically, beta 2 phosphodiesterases reduce the duration of PKA signaling by quickly inactivating cyclic AMP. The study of compartmentalization, exemplified by adenyl cyclase five anchored to the T tubular membrane linked to beta adrenergic receptor two, illustrates how these interactions mediate signaling outcomes.

31:22

HWO TO STUDY COMPARTMENTALIZATION

Modulation of Signaling Duration

The response to cyclic AMP activation is concentration-dependent, particularly with isoproterenol stimulation. Low doses lead to a transient response, while higher doses result in prolonged activation of cyclic AMP and PKA activity, highlighting differences in stimulation of beta adrenergic receptors. The concentrations examined range from sub-nanomolar to micromolar, demonstrating varying degrees of response.

32:47

Concentration dependant 

Log from a low concentration of isoprotenol: go from a low transcient repsonse to a higher pak activity 

Stimulation of adeenrgic stiumualtion (green) those elicit a rapid trasicent increase up initally then rapid decline (graph a ) 

gRAPH C

If we odnt get activation of cyclic am

Cyclic AMP Activation

Molar concentrations of isoproterenol lead to a rapid but transient increase in cyclic AMP levels, followed by a decline. At sub micromolar to micromolar concentrations, both cyclic AMP and PKA activity increase more significantly and sustain longer. This differential response is attributed to the dual regulation of cyclic AMP and PKA by adenylate cyclase and phosphodiesterases, influenced by isoproterenol concentration.

Differntial concentration response 

Dual mechanistic reguaotion of camp and pka by adenyl cyclase and phosphodiesterases

1NM isoprotoernoL(low levels)

34:12

Concentration-Dependent Responses

Activation of PKA around the receptor leads to two main effects: 

1. phosphorylation and inactivation of adenylyl cyclase, 

2. activation of phosphodiesterases (PDEs). 

• This results in decreased cyclic AMP levels due to increased breakdown of cyclic AMP, effectively acting as a negative feedback mechanism that reduces signaling magnitude and duration, especially noticeable at low isoproterenol levels. At higher concentrations (10 micromolar), a dose-dependent dissociation of PDE from the beta-adrenergic receptor occurs.

Isoprotenol is also activating pka im pretty sure? 

35:42

Negative Feedback Mechanism

HIGH LEVELS OF ISOPRTENOL 

• The dissociation of PDE from the complex at higher levels leads to sustained elevation of cyclic AMP in myocytes, enhancing PKA activation on the sarcoplasmic reticulum. 

• This activation phosphorylates phospholamban and troponin I, contributing to increased cardiac contractility

•  sustained isoproterenol levels might indicate a modulating role for beta 2 adrenergic phosphorylation and internalization.

37:00

Sustained Activation Effects

 receptor desensitization: Sustained isoproterenol exposure leads to beta-2 adrenergic receptor phosphorylation and internalization, 

This mechanism is crucial for reducing signaling persistence, preventing continuous activation. The desensitization occurs in caveolae, involving phosphorylation by kinases such as protein kinase A and GRK2, which provide negative feedback.

38:24

Internalization of Receptors

MECHANISMS TO HELP DECRESASE SIGNALLING SO IT DOESNT GO ON FOREVER 

Receptor Desensitization Mechanism

Receptor desensitization occurs when a receptor, phosphorylated at the C-terminal end, binds to beta-arrestin, leading to its internalization with clathrin and AP2 involvement. This process diminishes the receptor's ability to bind ligands, reducing their effectiveness in triggering an intracellular response over time. Consequently, even with continued agonist stimulation, the response wanes due to these feedback mechanisms.

38:24

Receptor is phosphroylated at c terminal end, target it w beta arresrtin which leasds to receptor internalization 

Receptor Desensitization Mechanisms

Receptor desensitization involves the phosphorylation of receptors at the C-terminal end, promoting the binding of beta-arrestin and other molecules like clathrin and AP2, leading to receptor internalization.

 This process reduces the receptor's ability to bind ligands on the cell membrane, limiting the effectiveness of agonists and causing a diminished response over time. Ultimately, despite ongoing stimulation, the feedback mechanisms ensure that the cellular response to the agonists decreases.

Even if receptors bind it limits affetciveness or abikity of agnes to promote repsonse overtime 

45:24

Clathrin-Mediated Endocytosis

The binding of beta arrestins to G protein-coupled receptors (GPCRs) facilitates receptor sequestration through clathrin-coated pits, resulting in the internalization of these receptors. 

Vesicles interact w plasma membrane to transport molecules into the cell thru endocystosis. Pinch off form membrane and into the cell 

Once internalized, GPCRs exhibit two pathways for beta arrestin interaction, leading to rapid ligand dissociation and receptor dephosphorylation before being recycled back to the plasma membrane. This process serves to temporarily down-regulate signaling, ensuring receptors are available for future ligand binding.

48:03

Receptor Recycling vs Degradation

Receptor management involves two main pathways after internalization: class A receptors can be recycled back to the membrane, while class B receptors primarily face degradation. Additionally, G protein-coupled receptors bound to beta arrestin can initiate alternative signaling pathways, adding complexity to the signaling cascades that regulate vital responses like cardiac function during stress. This intricate regulatory system is crucial for managing the fight-or-flight response and ensuring adequate performance in both cardiac and skeletal muscles.

50:52

Complex Signaling Pathways

The discussion focuses on sympathetic innervation and the modulation of l-type calcium channels, expanding on previous course topics. Additional layers of cell signaling cascades will be explored, enhancing understanding of complex signaling pathways. Participants are encouraged to ask questions for clarification.

B1 snd B2 receptor desensitization 

1. Homologus d sesnsitazation of g coupled receptors resutls in binding of beta arrestins to agonsist occupied receptors , following phosphrylations of refctor by GRK. phosphorylation at the sites of c terminal end are targets for beta arrestin binding

Binding of beta arrstin is occldue coupling between receptor and hetertrimeric protein. LEd to termination of signaling thru g protein defectors? 

It induces a confirmational change and prevents coupling btw g prteoin and g receptor:

2.  Receptor bound beta arestins play as n adaptor protein and target other ones 

AP2? Apart of the clatherin machine which bring vesicles intp and out of cell 

Targeting receptor in order to initiate pits for membrane to seal off (INTERNALIXATION)

Clatherin pits (check 45min) brings clatherin in the cell

3. Once internalized the g coupled receptors experience 2 pathways 

1.. Class a genes: rapid dissociation of beta arrestin, trafficked to an acidfied endysmmal compartment where lignadd is dissociated 

Recycle through plasma ,membrane and recycle all back to the membrane 

1:26

Beta Adrenergic Signaling

Sympathetic innervation of the heart primarily:  postganglionic axons connecting to the cardiac nerve, which coordinates with regions such as the atria and ventricles. 

02:46

Sympathetic Innervation of Heart

Changes in heart rate are influenced by sympathetic and parasympathetic stimulation, affecting the firing rate of sinoatrial nodal cells.

 Increased parasympathetic activity lowers the firing rate, 

increased sympathetic activity raises it

factors:HCN (alter slope of diastolic potential), L type calcium, 

04:07

Action Potential Firing

Sympathetic activation leads to a steeper slope in diastolic potential, resulting in more rapid depolarization and quicker action potential firing in pacemaker cells. Additionally, reduced repolarization means the membrane potential remains closer to the threshold, making it easier to reach it during increased sympathetic stimulation.

07:04

Sympathetic Activation Effects

A steeper slope in depolarization leads to a quicker threshold attainment, increasing firing rates when sympathetic activation predominates. Conversely, during parasympathetic activation, with inhibited sympathetic activity, the rate of firing decreases despite the threshold remaining unchanged. The contrasts between these states highlight the differing impacts of sympathetic and parasympathetic nerve activities on neuronal firing rates.

08:18

Parasympathetic Activation Effects

Parasympathetic activation results in a less steep slope in depolarization, leading to a prolonged period before reaching threshold. This mechanism is influenced by the binding of neurotransmitters like acetylcholine to muscarinic receptors, which alters sodium influx and impacts cyclic AMP levels. Additionally, parasympathetic stimulation causes hyper-repolarization, enabling more potassium channels to activate, thereby enhancing the degree of repolarization beyond the typical -60 millivolts.

09:35

Ion Channel Modulation

Hyperpolarizing: greatier degree of potassium channel activation 

Repolarization and a greater difference in membrane potential slow heart rate by affecting pacemaker cells' firing rates. Hyperpolarizing funny currents (HCN) are influenced by both sympathetic and parasympathetic nervous systems, notably via cyclic AMP modulation. Increased HCN currents speed up depolarization, increasing sodium influx and thus elevating the pacemaker cells' firing rate and heart rate.

12:15

Excitation-Contraction Coupling

Excitation-contraction coupling involves several key components regulated by beta-adrenergic stimulation, including L-type calcium channels (Cav 1.2), ryanodine receptors, and various regulatory proteins like phospholamban. The activation of beta-adrenergic receptors leads to a cascade that ultimately increases protein kinase A (PKA) activity, which phosphorylates the L-type calcium channels, enhancing their activity and resulting in a greater calcium influx. This process is crucial for the regulation of cardiac contractility.

Regualtion of CAV1.2

• Regulated through pka dependatn phosphorylation 

• Norepinephrine see gdp and gtp bound, cyclic amp will increase 

15:00

GTP Binding and PKA Activation

Phosphorylation significantly enhances inward calcium current through l-type calcium channels when norepinephrine, or isoproterenol, is present, resulting in increased calcium influx into the cell. This process involves activation of beta adrenergic receptors, which boosts calcium movement. Additionally, a protein phosphatase plays a crucial role in deactivating this signal by removing the phosphate group, allowing the system to return to its baseline function.

16:24

Calcium Current Increase

Increased stimulation of beta adrenergic receptors does not directly correlate with an increase in individual ion channel current, yet it leads to a higher overall whole cell current, particularly under isoproterenol conditions. This effect is primarily due to enhanced open probability of l-type calcium channels, contributing to an elevated inotropic state of the heart by raising intracellular calcium levels and facilitating calcium-induced calcium release.

17:45

Inotropic State of the Heart

Increasing availability of calcium enhances myocardial contractility, leading to an elevated inotropic state. The alterations in beta adrenergic pathway signaling play a crucial role in the regulation of l-type calcium channels, particularly via isoproterenol, which activates the signaling cascade and increases cyclic AMP, resulting in enhanced l-type calcium current.

19:11

Beta Adrenergic Pathway Signaling

Increasing isoproterenol leads to a rise in l-type calcium current due to heightened intracellular cAMP levels, which activate the PKA catalytic subunit to phosphorylate target proteins. This process is primarily mediated by the beta-adrenergic signaling pathway, but inhibition of this pathway can reverse the effects. Employing a PKA inhibitor effectively blocks PKA activity, demonstrating the pathway's regulatory mechanisms.

20:25

L-Type Calcium Current Amplitude

Activation of signaling pathways, such as those initiated by isoproterenol, can be reversed by phosphatases which remove phosphate groups. The functionality of L-type calcium channels is enhanced through the formation of a macromolecular complex that necessitates the close proximity of specific components, including beta-adrenergic receptors and the Cav 1.2 channels. This structured assembly is critical for efficient signaling and calcium channel activity.

21:48

Phosphatase Role in Signaling

The macromolecular complex involves the g alpha s subunit as the main effector, activating adenylate cyclase to increase cyclic AMP and subsequently activate PKA. The importance of this complex, particularly the role of aCAP in anchoring components, is highlighted by the consequences of inhibiting these interactions, which disrupts normal signaling responses such as those induced by isoproterenol in beta-adrenergic signaling.

Compartmentalization with akap is important? 

• Wehn e prevent akap with other interactions 

• No augmentation 

23:07

Macromolecular Complex Formation

Phosphorylation in the signaling pathway enhances the open probability of L-type calcium channels, resulting in increased whole cell current. However, inhibiting acap disrupts this process, preventing isoproterenol from augmenting calcium currents through these channels, as acap's absence means beta adrenergic signaling cannot effectively activate proximity to calcium channels. This illustrates the importance of macromolecular complexes in facilitating the signaling cascade required for channel amplitude augmentation.

Compartmentalization with akap is important? 

• Wehn e prevent akap with other interactions 

• No augmentation!!

• Akap is not there so the cascade fro cascade signalling so its not in proximity of calcium channel thus no augmentation 

24:32

Inhibition of Calcium Channel Activity

• Beta adrenergic receptors regulate the ryanodine receptor 2 (RyR2) in the heart by activating a signaling pathway that increases protein kinase A (PKA) activity. 

• This activation leads to the phosphorylation of RyR2,

  •  promoting calcium release from the sarcoplasmic reticulum. The process involves complexes of RyR2 with PKA and phosphatases that either phosphorylate or dephosphorylate the receptor to manage its activity.

25:53

Ryanodine Receptor Phosphorylation

Beta adrenergic receptor stimulation enhances the calcium sensitivity of ryanodine receptors by increasing their open probability through reduced interaction with calmodulin and associated proteins. This effect leads to a left shift in calcium concentration requirements for receptor activation, making them more responsive to lower levels of intracellular calcium, particularly during stress or exercise.

27:23

Excitation-Contraction Coupling Regulation

During systole, phosphorylation influences the opening of ryanodine receptors, enhancing calcium release from the sarcoplasmic reticulum (SR), while sympathetic activation increases calcium entry and release, impacting systolic function. Additionally, beta-adrenergic signaling modifies excitation-contraction coupling by regulating SR calcium ATPase (SERCA2a), leading to improved calcium reuptake into the SR, which ultimately supports diastolic function. The activity of phospholamban and sarcolipin plays a crucial role in regulating SERCA2a, with phosphorylation modifications enhancing calcium re-uptake.

During systole

27:23

Calcium Sensitivity

During systole, phosphorylation destabilizes the channel, enhancing the opening probability of ryanodine receptor 2 and increasing calcium release from the sarcoplasmic reticulum, particularly in response to sympathetic activation. Beta adrenergic signaling also affects excitation-contraction coupling, impacting calcium entry and reuptake into the sarcoplasmic reticulum, thus influencing both systolic and diastolic function.

27:23

Calcium Sensitivity Increase

During systole, phosphorylation destabilizes the ryanodine receptor, leading to increased channel opening and enhanced calcium release from the sarcoplasmic reticulum (SR). Additionally, beta-adrenergic signaling impacts excitation-contraction coupling, affecting both calcium entry and SR reuptake, which ultimately influences systolic and diastolic functions. These mechanisms highlight the role of sympathetic activation in modulating cardiac calcium dynamics.

30:16

Calcium Reuptake Mechanisms

To enhance calcium dynamics, there is a need to increase sarcoplasmic reticulum (SR) calcium load, achieved through increased calcium release and reuptake. This leads to a larger calcium transient and stronger muscle contraction, influenced by factors such as the sensitivity of ryanodine receptors to luminal calcium and the phosphorylation of sarcolipin which reduces inhibition on calcium reuptake. Furthermore, activation of beta adrenergic pathways results in the phosphorylation of cardiac troponin I, decreasing calcium sensitivity in contractile proteins, which modifies the force generation for a given level of calcium.

33:17

Contractile Protein Regulation

Contractile protein regulation is influenced by phosphorylation, specifically of cardiac troponin I, which decreases calcium sensitivity, allowing for quicker calcium dissociation from troponin C. This mechanism enhances relaxation (lusitropy) and simultaneously increases force generation through calcium transients. Additionally, phosphorylation of myosin binding protein C improves the proximity of the myosin head to actin, increasing the likelihood of cross-bridge formation and contributing to muscle contractility.

40:52

Dromotropic Effects

Conduction velocities of electrical signals in the myocardium vary, influencing the timing and synchronization of heart contractions. The fastest conduction occurs in the Purkinje system, ensuring that the atria have time to contract before the ventricles. Additionally, beta adrenergic receptor signaling can regulate this conduction velocity, impacting the overall timing of cardiac activity.

42:16

Conduction Velocity Factors

Sympathetic stimulation via beta adrenergic signaling enhances conduction velocity in the heart by increasing the upstroke velocity (phase zero) of action potentials, primarily through the activation of voltage-gated sodium channels. This relationship indicates that as sodium current amplitude increases due to elevated cyclic AMP levels, both the upstroke velocity and conduction velocity rise, reflecting improved dromotropic effects. Additionally, conduction velocity is also influenced by gap junctions, which facilitate electrical signal transmission between cardiac cells.

48:31

Gap Junction Function

Beta adrenergic receptor stimulation enhances the opening of connexin 40 while decreasing the opening of connexin 45, impacting conduction velocity by promoting faster electrical signaling in the heart's conduction system. This selective alteration allows for increased rates of pacemaker firing and calcium influx, leading to positive chronotropic and inotropic effects. The result is a more efficient movement of electrical signals through the heart, effectively directing the signal along faster pathways and optimizing cardiac function.

01:26

Beta Adrenergic Receptors

Healthy hearts exhibit a positive inotropic response, which is significantly reduced in diseased hearts, as discussed in earlier lectures. The use of cardiac glycosides enhances this intrinsic contractility by inhibiting sodium calcium ATPase. In contrast, stimulation of beta 3 adrenergic receptors with a specific agonist shows a favorable impact, further illustrating the differences in cardiac response between healthy and diseased states.

02:46

Inotropic Response in Healthy Hearts

An increase in brl37344 concentration leads to a negative ionotropic effect observed as reduced contraction amplitude, particularly in healthy non-failing hearts. This effect raises questions about the role of beta adrenergic receptors, as stimulation of beta 3 receptors triggers this unexpected decline in contractility. Understanding this involves examining the beta 3 adrenergic signaling pathway, which activates endothelial nitric oxide synthase via a specific g alpha subunit.

04:12

Negative Ionotropic Effect of Beta 3

Activation of beta-3 adrenergic receptors leads to the production of nitric oxide via endothelial nitric oxide synthase, which subsequently stimulates guanylate cyclase to produce cyclic GMP. Cyclic GMP then binds to the regulatory subunits of protein kinase G, releasing its catalytic subunits, which phosphorylate L-type calcium channels, reducing their sensitivity. This process results in a differential response compared to the effects observed with cyclic AMP and protein kinase A in beta-2 adrenergic signaling.

05:46

Beta 3 Signaling Pathway

The activation of the beta-3 adrenergic receptors results in decreased calcium current and sensitivity of l-type calcium channels in myocardial contractility, although their role is minor in healthy hearts due to the predominance of beta-1 and beta-2 receptors. However, in heart disease, beta-3 adrenergic receptor expression increases significantly, as evidenced by higher levels observed in heart failure compared to normal hearts.

07:15

Role of Beta 3 in Heart Disease

In heart failure, there is a notable decrease of approximately 50% in beta-1 adrenergic receptor expression, alongside the desensitization of beta-2 receptors, while beta-3 adrenergic receptors show increased expression. This shift emphasizes the growing importance of beta-3 adrenergic receptors in heart disease, as they lead to reduced contractility through the activation of the eNOS pathway and decreased L-type calcium channel activity. Overall, the altered dynamics of these receptors illustrate their critical roles in the pathophysiology of heart failure.

08:42

Increased Beta 3 Expression in Heart Failure

In heart failure, the effects of beta 1 and beta 2 signaling are antagonized by increased cGMP and PKG-dependent reductions in contractility, leading to an overall inhibition of heart function. Notably, the expression of beta 3 signaling increases, which was demonstrated in experiments where beta-3 adrenergic stimulation yielded less reduction in contractility in heart disease patients compared to healthy individuals. This counterintuitive outcome can be explained by examining how heart failure impacts the molecules that are part of the beta 3 adrenergic signaling pathway.

10:08

Beta 3 Antagonism of Beta 1 and 2

In non-failing hearts, eNOS activity facilitates beta-3 adrenergic signaling which influences contractility; however, in heart failure, this activity decreases, leading to reduced signaling and less inhibition of contractility. The lecture also explores skeletal muscle types, highlighting that fast-twitch and slow-twitch fibers utilize different energy sources, with beta-2 adrenergic receptors predominating across muscle types. Notably, these receptors vary in density across muscle fibers, particularly in the vastus lateralis.

13:09

Skeletal Muscle Fiber Types

The adrenergic signaling pathway increases protein kinase A activity, enhancing contractility in cardiac muscle, but not in skeletal muscle, raising the question of how force of contraction is achieved in the latter. Instead of increasing contractility directly, beta-2 adrenergic signaling in skeletal muscle regulates metabolism by activating enzymes involved in glycogen breakdown through a cascade initiated by agonist binding to beta adrenergic receptors. This mechanism highlights the distinct roles of adrenergic pathways in muscle function.

14:46

Beta 2 Signaling in Skeletal Muscle

Beta adrenergic receptors play a crucial role in modulating energy pathways, particularly in the Krebs cycle, by facilitating the breakdown of sugar molecules into usable forms such as pyruvate. When a ligand binds to these receptors, it triggers a cascade that activates the G protein alpha subunit, leading to the activation of adenylate cyclase. This activation results in increased cyclic AMP levels, which subsequently activate protein kinase A, impacting various signaling pathways.

16:11

Skeletal Muscle Metabolism Regulation

In skeletal muscle, protein kinase A phosphorylates and activates phosphorylase kinase, which then activates glycogen phosphorylase, converting glycogen to glucose 1-phosphate. This precursor undergoes further transformation by phosphoglucomutase to produce glucose 6-phosphate, enabling energy production through glycolysis and the Krebs cycle. The breakdown of glycogen serves as a rapid energy source during anaerobic conditions, significantly enhancing ATP generation in muscle, while beta adrenergic stimulation plays a crucial role in increasing cardiac contractile activity during exercise.

19:12

ATP Generation in Skeletal Muscle

During a cardiac cycle, approximately two percent of ATP in cardiac myocytes is consumed, leading to rapid turnover of ATP in less than a minute. The majority of ATP production (about 90%) occurs through oxidative phosphorylation in the mitochondria, which comprise about one third of the myocyte volume, while the remaining ten percent derives from creatine phosphate and adenylate kinase reactions. As the heart's metabolic demands increase with higher inotropy and chronotropy, efficient ATP production and mitochondrial adaptation become crucial.

22:13

Mitochondrial Role in Cardiac Energy Production

Mitochondria, situated close to the sarcoplasmic reticulum, are able to detect fluctuations in calcium levels, which allows them to modulate energy production in response to the heart's metabolic demands. ADP and calcium act as crucial regulators of the tricarboxylic acid cycle, linking sarcoplasmic reticulum calcium levels to ATP production. This relationship is particularly evident during increased intracellular calcium levels that occur during heightened physical activity.

23:46

Calcium Sensing by Mitochondria

Calcium dynamics in the cardiac myocyte are crucial as changes in cytosolic calcium levels directly affect mitochondrial signaling and energy production. When l-type calcium channels are blocked by varapamil, there is a noticeable reduction in both cytosolic and mitochondrial calcium levels, resulting in decreased contractility and energy generation. Conversely, when intracellular calcium is increased through isoproterenol, it enhances mitochondrial calcium uptake, demonstrating the importance of the sarcoplasmic reticulum's physical coupling to mitochondria in sensing metabolic demands.

29:43

Mitochondrial Calcium Microdomain

The mitochondrial calcium uniporter (MCU) on the inner mitochondrial membrane facilitates the entry of calcium ions into the mitochondrial matrix, driven by a strong electrochemical gradient. Elevated levels of calcium stimulate key enzymes in the tricarboxylic acid cycle, ultimately enhancing ATP production. This discussion also touches on parasympathetic signaling through muscarinic receptors, highlighting acetylcholine's role in mediating responses at target organs.

32:54

Calcium's Role in TCA Cycle Activation

Muscarinic receptors, particularly M2 and M4, are G protein-coupled receptors that can be activated by nicotine, influencing parasympathetic signaling pathways. These receptors are coupled with G alpha subunits that, depending on their type, can initiate different signaling cascades, specifically inhibiting adenylate cyclase through G alpha i o. The interplay of these receptors is crucial for understanding their various roles in cellular signaling and heart function.

32:54

Parasympathetic Nervous System Overview

Muscarinic receptors, crucial for parasympathetic signaling, include five types (M1 to M5) that couple with diverse G-proteins, leading to distinct signaling pathways. Notably, M2 and M4 receptors are linked to the G-alpha i/o subunit, which inhibits adenylate cyclase, impacting various physiological responses. The interaction of nicotine with muscarinic receptors further elucidates the complexity of these signaling mechanisms.

34:27

Muscarinic Receptor Function

Muscarinic receptors M1, M3, and M5 are coupled with G alpha q and activate phospholipase C, while M2 and M4, coupled with G alpha i/o, can regulate potassium channels. The antagonistic nature of the sympathetic and parasympathetic nervous systems means that acetylcholine primarily slows the heart rate, with excessive doses potentially leading to complete cardiac arrest by preventing action potentials. This overview sets the stage for further exploration of the signaling pathways involved.

35:50

M2 Receptor Effects on Heart Rate

M2 receptors play a crucial role in regulating heart rate by mediating the effects of acetylcholine on pacemaker cells in the sinoatrial node. Activation of M2 receptors leads to the inhibition of adenylyl cyclase, decreasing cyclic AMP levels and attenuating the HCN current, which ultimately slows heart rate. Additionally, higher concentrations of acetylcholine activate potassium channels that shorten the action potential, particularly in atrial cells due to their higher expression, leading to enhanced repolarizing currents.

40:13

Action Potential Shortening

The mechanism involving repolarizing currents makes the membrane potential more negative and is influenced by phosphatidylinositol-4,5-bisphosphate (PIP2), which is synthesized from PIP. PIP2 plays a crucial role in the activation of acetylcholine-sensitive potassium channels, but it is eventually hydrolyzed, affecting its regulatory function. The interplay between PIP2 processing and muscarinic receptors is essential for understanding its impact on cellular metabolism.

41:45

Role of PIP2

Phospholipase C (PLC) is an enzyme that cleaves phospholipids, specifically hydrolyzing PIP2 into inositol trisphosphate (IP3) and diacylglycerol (DAG) with calcium as a co-factor. IP3 binds to its receptor while DAG activates protein kinase C, initiating a signaling cascade that enhances contractility via beta-1 signaling. This process highlights the critical role of PIP2 in cellular signaling pathways.

43:16

Phospholipase C Activation

The study of the regulatory mechanisms of acetylcholine-sensitive potassium channels is explored through patch clamping experiments. Activation of g beta gamma subunits increases the open probability of these channels, highlighting their role in the signaling pathway. Additionally, the activation of phospholipase C leads to the hydrolysis of PIP2, further contributing to the modulation of potassium channel activity.

44:42

Patch Clamping Experiment

The interaction of g beta gamma with potassium channels is significantly influenced by the presence of pip2, as its hydrolysis by (PLC) reduces the activation effectiveness of g beta gamma. Specifically, pre-exposure to PLC diminishes the channels' open probability, indicating the critical role of pip2 in regulating these channels. This highlights the necessity of pip2 for the g beta gamma mediated activation of potassium channels.

46:03

G Beta Gamma Interaction

The studies highlight that the activation of acetylcholine-sensitive potassium channels by G beta gamma is dependent on the presence of PIP2; without PIP2, G beta gamma cannot activate these channels. Providing PIP2 can restore channel activity, illustrating its crucial role in this interaction. Furthermore, recent crystallization of the Kr 3.2 subunit reveals a direct binding site for PIP2 on the channel, emphasizing the structural basis for this modulation.

47:33

PIP2 Requirement

The channel's functionality is governed by a selectivity filter and two gating mechanisms: the inner helix gate and the cytoplasmic gate. When G beta-gamma is expressed with the acetylcholine-sensitive potassium channel, it activates the opening of the cytoplasmic domain gate, while PIP2 alone facilitates the opening of the inner helix gate, indicating distinct regulatory roles for these elements in channel gating.

49:01

Channel Gating Mechanisms

The binding of G-gate beta gamma and PIP2 to our potassium channel is essential for fully opening the channel, as each binding event alone only partially activates the gates. Both molecules must act together to open the cytoplasmic domain gate and the inner helix gate, allowing potassium to flow through. Additionally, mechanisms such as channel desensitization are necessary to regulate signaling and prevent indefinite activation of this pathway.

50:26

Complete Channel Opening

Desensitization in cells involves the initial spike followed by a decrease in current when exposed to choline or vagal stimuli due to decreased receptor binding. This process is linked to the hydrolysis of pip2, which mediates desensitization of the GTP-binding protein-stimulated potassium current. Crosstalk between different muscarinic signaling pathways is emphasized, highlighting the role of G protein beta-gamma subunits in stabilizing acetylcholine-sensitive potassium channels and the phospholipase C pathway's effect on limiting channel activity.

52:06

Receptor Desensitization Effects

Desensitization of the channel is primarily due to the downregulation and hydrolysis of PIP2, as seen when a set of choline leads to reduced responsiveness upon reapplication. The inhibition of PLC prevents this desensitization, suggesting that PIP2 breakdown is key to the channel's reset mechanism, especially noted in cells expressing both M1 and M2 muscarinic receptors. Specifically, while M2 receptors show little desensitization, M1 and M3 receptors contribute significantly to the desensitization process, highlighting distinct signaling pathways.

52:06

Desensitization of Potassium Channels

The desensitization of potassium channels is attributed to the downregulation and hydrolysis of PIP2. Under control conditions, activation by sedicoline initially increases channel response, but this diminishes over time as the system reaches a quasi-steady state. A subsequent application of sedicoline after recovery reveals that channel responsiveness is reduced, as indicated by a lower second peak compared to the first, demonstrating receptor desensitization.

52:06

Desensitization Mechanism

Desensitization of the channel occurs due to the down regulation and hydrolysis of PIP2, leading to a decrease in channel responsiveness over time. Following the initial activation by acetylcholine, a subsequent washout and reapplication results in a reduced response, indicating receptor desensitization. Consequently, the second peak of response to acetylcholine is less than the first, showing that the same dosage does not elicit the same effect after desensitization.