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ACh Receptors

There are two broad classes of cholinergic receptors: nicotinic and muscarinic. This classification is based on two chemical agents that mimic the effects of ACh at the receptor site nicotine and muscarine.

Table I summarizes some of the properties of nicotinic and muscarinic receptors.

Table I Nicotinic and Muscarinic Receptors and their Actions
Nicotinic	Muscarinic
Bind nicotine	Bind muscarine
Blocked by curare (tubocurarine)	Blocked by atropine
Linked to ionic channels	Linked to 2nd messenger systems through G proteins (see below)
Response is brief and fast	Response is slow and prolonged
Located at neuromuscular junctions, autonomic ganglia, and to a small extent in the CNS	Found on myocardial muscle, certain smooth muscle, and in discrete CNS regions
Mediate excitation in target cells	Mediate inhibition and excitation in target cells
Postsynaptic	Both pre- and postsynaptic

The Nicotinic Receptor is an Ion Channel

Figure 11.8 Schematic of the five subunit nicotinic ACh receptor in the postsynaptic membrane at the NMJ. ACh binds to the two a subunits. The bottom half shows the molecular structure of each a subunit of the nicotinic receptor based on cDNA derived amino acid sequence. The β, γ and δ subunits have an analogous structure to the α subunit.

As indicated in Table I, nicotinic receptors are located at the NMJ, autonomic ganglia and sparsely in the CNS.

The NMJ nicotinic ACh receptor consists of five polypeptide subunits: two α subunits and one each of β, δ, and γ (see Figure 11.8). A funnel-shaped internal ion channel is surrounded by the five subunits. The binding surface of the receptor appears to be primarily on the α subunits, near the outer surface of the molecule. The subunits contain recognition sites for agonists, reversible antagonists, and α-toxins (cobra α-toxin and α-bungarotoxin).

Whereas the NMJ nicotinic receptor is composed of four different species of subunit (2 α, β, γ, δ), the neuronal nicotinic receptor also is composed of only two subunit types (2 α and 3 β).

 

 

 

 

 

The Muscarinic Receptor is Coupled to G-Proteins

Figure 11.9 ACh released into the extracellular space interacts with muscarinic receptors on both the innervated cell and the ACh nerve ending.

Muscarinic receptors, classified as G protein coupled receptors (GPCR), are located at parasympathetic autonomically innervated visceral organs, on the sweat glands and piloerector muscles and both post-synaptically and pre-synaptically in the CNS (see Table I). The muscarinic receptor is composed of a single polypeptide. Seven regions of the polypeptide are made up of 20-25 amino acids arranged in an α helix. Because each of these regions of the protein is markedly hydrophobic, they span the cell membrane seven times as depicted in Figure 11.9. The fifth internal loop and the carboxyl-terminal tail of the polypeptide receptor are believed to be the site of the interaction of the muscarinic receptor with G proteins (see right). The site of agonist binding is a circular pocket formed by the upper portions of the seven membrane-spanning regions.

ACh has excitatory actions at the neuromuscular junction, at autonomic ganglion, at certain glandular tissues and in the CNS. It has inhibitory actions at certain smooth muscles and at cardiac muscle.

Figure 11.10 Muscarinic receptors are seven transmembrane proteins that mediate their signals through G proteins.

The biochemical responses to stimulation of muscarinic receptor involve the receptor occupancy causing an altered conformation of an associated GTP-binding protein (G protein). G protein is made up of the three subunits α, β and γ. In response to the altered conformation of the muscarinic receptor, the a subunit of the G protein releases bound guanosine diphosphate (GDP) and simultaneously binds guanosine triphosphate (GTP). The binding of GTP "activates" the G protein, allowing dissociation of the α subunit from the trimeric complex and for it to interact with effector systems to mediate specific responses. An inherent GTPase catalytic activity of the G protein hydrolyzes the GTP back to GDP. This hydrolysis terminates the action of the G protein. The rate of hydrolysis of the GTP thus dictates the length of time the G protein remains activated.

The responses mediated by muscarinic receptors through G proteins include:

Figure 11.11 Activated G protein interacts with adenylyl cyclase to either activate or inhibit its activity.

Inhibition of Adenylate Cyclase: The muscarinic receptor, through interaction with an inhibitory GTP-binding protein, acts to inhibit adenylyl cyclase. Reduced cAMP production leads to reduced activation of cAMP-dependent protein kinase, reduced heart rate, and contraction strength.

 

 

 

 

 

 

Figure 11.12a G protein stimulation of PLCβ generates DAG and IP3. The DAG stimulates PKC and IP3 frees Ca2+ from smooth ER.		Figure 11.13b Stimulation of IP3 receptors by four molecules stimulates the release of Ca2+ from the smooth ER.

Stimulation of Phospholipase C: The muscarinic receptor activates phosphoinositide-specific phospholipase C (PLC_β) through interaction with a GTP-binding protein. As shown in Figure 11.12a, the hydrolysis of phosphatidylinositol bis-phosphate yields two second messengers; inositol tris-phosphate (IP₃) and diacylglycerol (DAG). The DAG activates protein kinase C (not shown). Cellular responses are influenced by PKC's phosphorylation of target proteins. As shown in Figure 11.12b, the IP₃ diffuses to the smooth endoplasmic reticulum (ER) where it interacts with IP₃ receptors to increase Ca²⁺ release from the intracellular storage site.

 

 

Figure 11.14 G protein directly increases K+ conductance by interacting with the K+ channels.

Activation of K⁺ Channels: In response to muscarinic cholinergic receptor stimulation, a GTP binding protein also can interact directly with K⁺ channels to increase K⁺ conductance, (Figure 11.13). This conductance increase increases the resting membrane potential in myocardial and other cell membranes leading to inhibition.