Basics of muscle contraction

Control of intracellular Ca²⁺ - principal mechanism that initiates contraction and relaxation in smooth and striated muscle

Regulatory pathways:

striated muscle-Ca²⁺ activates contraction by binding to thin filament associated protein, troponin

smooth muscle-Ca²⁺ binds to calmodulin, which then associates with the catalytic subunit of myosin light chain kinase-phosphorylates serine 19 on the regulatory light chain of myosin (rMLC). Phosphorylation of Ser19 allows the myosin ATPase to be activated by actin and the muscle to contract.

Basics of muscle contraction

Calcium regulation is vital

In smooth muscle, the cytosolic free Ca²⁺ concentration is ~ 0.1 mM in basal state; ~ 10,000 times lower than that present in the extracellular space (mM)

Activation of cells induces an increase in cytosolic concentration up to ~1-10 mM.

Ca²⁺ diffuses in cell much more slowly than predicted from its small volume; Ca²⁺ atom migrate 0.1-0.5 mm, lasting only ~ 50 ms before being bound.

Ca²⁺ used by different vasoactive agents comes from extracellular and/or intracellular space.

Intracellular Ca²⁺ is localized in the mitochondria and SR

Location is most important

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"The degree of interaction is..."

The degree of interaction is determined by the net level of phosphorylation of the 20 kDa regulatory light chains of myosin II (rMLC).

MLC is regulated by MLC kinase (MLCK) and MLC phosphatase (MLCP or PP1M).

The extent of the rMLC phosphorylation and the amplitude of force production depends on the balance of the activities of MLCK and MLCP.

Under certain conditions, force is also regulated independent of the changes in rMLC phosphorylation levels perhaps by thin filament associated proteins (caldesmon and calponin), which can be phosphorylated by MAP kinase and/or other kinases.

Thin filament associated proteins might modulate the effect of rMLC phosphorylation, which is alone sufficient to initiate and maintain contraction.

MLCP is a trimer comprising a 130 kD regulatory myosin binding subunit (MBS), a 37 kD catalytic subunit (PP1c), and a 20 kD protein of uncertain function (M20).

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"Well-established that cAMP and cGMP..."

Well-established that cAMP and cGMP decreases Ca²⁺ sensitivity of contraction in both intact and permeabilized smooth muscle.

In vitro, PKA phosphorylates MLCK at two sites; site A decreases affinity of MLCK for Ca²⁺/calmodulin complex.

However, agents that elevate PKA have negligible effects on phosphorylation of site A and Ca²⁺ activation of MLCK; suggests that cAMP/PKA desensitizes smooth muscle by an alternate mechanism.

Phosphorylation of MLCK by PKG has no effect on activity.

Endogenous nitric oxide and related nitrovasodilators regulate blood pressure by activation of soluble guanylate cyclase, elevation of cGMP, activation of cGMP dependent kinase (cGKIa or PKG). cGMP-mediated vascular smooth muscle cell relaxation is characterized by a reduction in intracellular calcium concentration and activation of PP1M, which reduces the sensitivity of the contractile apparatus to intracellular calcium.

The mechanism by which cGMP increases PP1M activity and myosin light chain dephosphorylation was elucidated in a series of experiments published by Surks et al.

"Y2H used to identify potential..."

Y2H used to identify potential cGKIa binding proteins.

2.5 x 10⁶ clones from human activated T cell library

Clone AL9 encoded the COOH terminal 181 amino acids of myosin binding subunit of myosin phosphatase. MBS is a 130 kD regulatory subunit of PP1M that confers the specificity of PP1 for MLC and is the site on PP1M that is regulated by rho kinase.

The COOH terminal 181 amino acids of MBS includes a leucine zipper domain.

"MBS targets cGKIa to the..."

MBS targets cGKIa to the SMC contractile apparatus and activation of cGKIa increases PP1M activity, the cGKIa increases PP1M activity.

Thromboxane analog U46619 caused an increase in myosin light chain phosphorylation from 10 to 68% in both vector and cGK_1-59 transfected vascular smooth muscle cells.

In vector alone transfected SMC, 8 Br-cGMP inhibited U46619 mediated myosin light chain phosphorylation.

Expression of cGK1-59 diminished the ability of 8 Br-cGMP to inhibit myosin light chain phosphorylation following U46619 stimulation.

MBS assembles a multienzyme complex tethering a phosphatase and at least two kinases (Rho, cGK) with counter-regulatory effects.

"PKG phosphorylates RhoA"

PKG phosphorylates RhoA

Phosphorylation may inhibit RhoA by (1) increasing association with guanine nucleotide dissociation inhibitor leading to termination of RhoA activation. (2) ? reduced interaction with Rho kinase

Decreased RhoA/ROK activity would favor MLCP activity, leading to relaxation.

Telokin-identical to the C-terminus MLCK is PKA/PKG phosphorylated. Phosphorylated telokin may increase MLCP activity, thereby mediating PKG mediated relaxation.

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"CamKII has been reported to..."

CamKII has been reported to phosphorylate site A of MLCK; Note: although PKA phosphorylates same site in vitro, no evidence that it phosphorylates in vivo.

Phosphorylation was associated with a decrease in Ca²⁺ sensitivity of rMLC phosphorylation.

Suggested that this represents a negative feedback to inhibit high levels of rMLC phosphorylation.

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Ion channels in smooth muscle

"Excitation-contraction coupling in smooth muscle..."

Excitation-contraction coupling in smooth muscle is believed to occur by two mechanisms-electromechanical and pharmacomechanical coupling.

Electromechanical coupling operates through changes in surface membrane potential; typically resting membrane potential= -40 to -70 mV.

Primary drive for the rise in intracellular calcium is membrane depolarization, with the consequential opening of voltage operated calcium channels; neurotransmitters or hormones acting to depolarize the membrane will cause contraction while those producing membrane hyperpolarization will cause relaxation.

Like cardiac muscle, the influx of Ca²⁺ likely causes release of Ca²⁺ from sarcoplasmic reticulum.

"Drugs that block calcium entry..."

Drugs that block calcium entry through VOCC will inhibit electromechanical coupling-thus the use of calcium channel blocking agents to relax vascular smooth muscle, thus producing vasodilatation and a decrease in blood pressure.

Cell-type dependent; for instance, in asthma, Ca²⁺ blocking drugs are not effective in promoting relaxation of muscle.

Electromechanical coupling appears to play a predominant role in phasic smooth muscle in which the membrane potential often displays marked oscillations upon which are superimposed calcium spikes

The plasma membranes contain numerous ion channels and the distribution and properties vary among different tissues, contributing to the diversity of smooth muscle.

"Pharmacomechanical coupling- does not depend..."

Pharmacomechanical coupling- does not depend upon changes in membrane potential or calcium entry via the VOCC.

The rise of intracellular Ca²⁺ is brought about by a combination of Ca²⁺ release from intracellular stores and Ca²⁺ entry through non-voltage gated channels, primarily receptor operated calcium channels or store operated Ca²⁺ channels

Ca²⁺ signal often similar to that seen in many non-excitable cells, consisting of an initial rise in [Ca ²⁺]_i followed by a smaller, but sustained increase dependent upon Ca²⁺ entry from the extracellular space.

This secondary influx of Ca²⁺, in association with the process of Ca²⁺ sensitization whereby the contractile apparatus may be activated by near-resting levels of [Ca²⁺]_i, allows muscles to maintain tone over prolonged periods in the presence of an agonist; occurs in tonic smooth muscle.

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"The relative importance of electromechanical..."

The relative importance of electromechanical or pharmacomechanical coupling for any given smooth muscle preparation can be estimated by determining the effects of inhibitors of VOCC’s on the contraction to agonists.

For example, in guinea pig ileum, dihydropyridines such as nifedipine will virtually abolish all contractions, suggesting that electromechanical coupling predominates

However, both mechanisms probably occur to some extent in all smooth muscle. In addition, the opening of ROCC and SOCC also produce membrane depolarization, thus activating electromechanical coupling.

"Approximately 20 years ago,"

Approximately 20 years ago, it was hypothesized that receptor activation could lead to Ca²⁺ entry by a mechanism independent of membrane depolarization in smooth muscle

Receptor operated currents have been described as non-selective cation currents rather than Ca²⁺ channel

In the rabbit ear artery, externally applied ATP produced a rapid, transient depolarization of muscle, shown to result from activation of a non-selective cation conductance with significant Ca²⁺ permeability. Similar responses were reported to ATP in rat vas deferens, rabbit portal vein, and human saphenous veins.

In addition to ATP, Noradrenaline, Acetylcholine, Histamine, Endothelin-1, Neurokinin A, Substance P, and Vasopressin have been shown to activate a receptor-operated cation current.

Store-operated calcium channels/currents

In the late 1980’s, Putney proposed the model for “capacitative calcium entry” in which intracellular Ca²⁺ store depletion stimulated Ca²⁺ influx across the plasma membrane to maintain a raised [Ca²⁺]_i in the face of prolonged agonist application and to aid in refilling of the stores on agonist withdrawal.

It is not the Ca²⁺ released from the stores that activates SOCC. Thus, if the rise in [Ca²⁺]_i is prevented by inclusion of a Ca²⁺ buffer, then the store operated current would still be present. It is the fact that the stores are empty of Ca²⁺ that drives the response by an as yet unknown mechanism.

Many of the neurotransmitters which activate ROCC simultaneously activate phospholipase C, liberating IP3. Therefore, SOCC is activated due to IP3 mediated depletion of the sarcoplasmic reticulum.

"Molecular evidence suggests that store-operated..."

Molecular evidence suggests that store-operated and receptor-operated channels may be formed from proteins belonging to the same family, being the mammalian homologues of the transient receptor potential (TRP) channels.

Less clear whether they form the channels in native smooth muscle

One putative model is that TRPC proteins may fall into two classes; one responsive to receptor activation but not store depletion and the other responsive to store depletion.

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Sarcoplasmic reticulum in smooth muscle

The SR is the physiological intracellular source and sink of Ca²⁺ in smooth muscle, as in striated muscle.

The Ca²⁺ pump of the SR is a SR/ER Ca²⁺-ATPase of 100 kDa with isoforms 2a and 2b.

The SR also contains phospholamban, which regulates Ca²⁺ uptake by the SR.

Central SR appears to form a continuous system connected with the peripheral SR.

The peripheral SR can form surface coupling with the plasma membrane; regions where the SR and plasma membranes come to within 8-10 nm of each other and are connected by elctron-dense bridging structures.

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Jaggar et al, Am J Physiol Cell Physiol (2000) 278:C235

Ryanodine receptors recorded in planar lipid bilayer;

Note Ca²⁺ dependence.

Jaggar et al, Am J Physiol Cell Physiol (2000) 278:C235

Hypothetical modulation of Ca²⁺ spark frequency.

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"Elevation of [Ca²⁺]..."

Elevation of [Ca²⁺]_SR can cause increased spark and transient K_Cafrequency that should lead to membrane hyperpolarization, decrease in voltage-dependent Ca²⁺ channel activity, reduction in global [Ca²⁺]_i and dilation.

May also increase the driving force for sarcolemma extrusion mechanisms that are located in the vicinity of the release site, such as Na⁺-Ca²⁺ exchanger and Ca²⁺-ATPase. May also inactivate sarcolemmal voltage dependent Ca²⁺ channels.

“Superficial buffer barrier hypothesis- Ca²⁺ entering SMC is buffered by the SR and is discharged vectorially towards the sarcolemma, without any effect on global [Ca²⁺]_i.

Jaggar et al, Am J Physiol Cell Physiol (2000) 278:C235

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"Myogenic tone refers to the..."

Myogenic tone refers to the ability of vascular smooth muscle to alter its state of contractility in response to changes in intraluminal pressure

The vessel constricts in opposition to an increase in intravascular pressure and dilates when the pressure decreases

Behavior observed in a variety of vascular tissues, including veins and conduit arteries, but especially prevalent in resistance vasculature.

Classically described as being a Ca²⁺ dependent process where pressure evoked depolarization and Ca²⁺ entry through voltage gated Ca²⁺ channels play obligatory roles

Consistent with a role for pressure-induced depolarization, blockers of voltage gated Ca²⁺ channels have been shown to reduce myogenic responses.

"Arteriolar SMC possess ion channels..."

Arteriolar SMC possess ion channels sensitive to cell membrane stretch that may be activated by vessel distension arising from an increase in intraluminal pressure.

Have relative permeability: K⁺>Na⁺>Ca²⁺

Ca²⁺ influx would be relatively small- generally believed that stretch activation of these channels mainly contributes to membrane depolarization with subsequent opening of voltage gated calcium channels.

K_Ca currents have been shown to attenuate the stretch-induced changes in membrane potential and myogenic constriction.

Mechanical perturbation of cell membranes may release factors that modulate the activity of such channels.


	Control of intracellular Ca²⁺ - principal mechanism that initiates contraction and relaxation in smooth and striated muscle
	Regulatory pathways:
	striated muscle-Ca²⁺ activates contraction by binding to thin filament associated protein, troponin
	smooth muscle-Ca²⁺ binds to calmodulin, which then associates with the catalytic subunit of myosin light chain kinase-phosphorylates serine 19 on the regulatory light chain of myosin (rMLC). Phosphorylation of Ser19 allows the myosin ATPase to be activated by actin and the muscle to contract.


	Calcium regulation is vital
	In smooth muscle, the cytosolic free Ca²⁺ concentration is ~ 0.1 mM in basal state; ~ 10,000 times lower than that present in the extracellular space (mM)
	Activation of cells induces an increase in cytosolic concentration up to ~1-10 mM.
	Ca²⁺ diffuses in cell much more slowly than predicted from its small volume; Ca²⁺ atom migrate 0.1-0.5 mm, lasting only ~ 50 ms before being bound.
	Ca²⁺ used by different vasoactive agents comes from extracellular and/or intracellular space.
	Intracellular Ca²⁺ is localized in the mitochondria and SR
	Location is most important


	The degree of interaction is determined by the net level of phosphorylation of the 20 kDa regulatory light chains of myosin II (rMLC).
	MLC is regulated by MLC kinase (MLCK) and MLC phosphatase (MLCP or PP1M).
	The extent of the rMLC phosphorylation and the amplitude of force production depends on the balance of the activities of MLCK and MLCP.
	Under certain conditions, force is also regulated independent of the changes in rMLC phosphorylation levels perhaps by thin filament associated proteins (caldesmon and calponin), which can be phosphorylated by MAP kinase and/or other kinases.
	Thin filament associated proteins might modulate the effect of rMLC phosphorylation, which is alone sufficient to initiate and maintain contraction.
	MLCP is a trimer comprising a 130 kD regulatory myosin binding subunit (MBS), a 37 kD catalytic subunit (PP1c), and a 20 kD protein of uncertain function (M20).


	Well-established that cAMP and cGMP decreases Ca²⁺ sensitivity of contraction in both intact and permeabilized smooth muscle.
	In vitro, PKA phosphorylates MLCK at two sites; site A decreases affinity of MLCK for Ca²⁺/calmodulin complex.
	However, agents that elevate PKA have negligible effects on phosphorylation of site A and Ca²⁺ activation of MLCK; suggests that cAMP/PKA desensitizes smooth muscle by an alternate mechanism.
	Phosphorylation of MLCK by PKG has no effect on activity.
	Endogenous nitric oxide and related nitrovasodilators regulate blood pressure by activation of soluble guanylate cyclase, elevation of cGMP, activation of cGMP dependent kinase (cGKIa or PKG). cGMP-mediated vascular smooth muscle cell relaxation is characterized by a reduction in intracellular calcium concentration and activation of PP1M, which reduces the sensitivity of the contractile apparatus to intracellular calcium.
	The mechanism by which cGMP increases PP1M activity and myosin light chain dephosphorylation was elucidated in a series of experiments published by Surks et al.


	Y2H used to identify potential cGKIa binding proteins.
		2.5 x 10⁶ clones from human activated T cell library
		Clone AL9 encoded the COOH terminal 181 amino acids of myosin binding subunit of myosin phosphatase. MBS is a 130 kD regulatory subunit of PP1M that confers the specificity of PP1 for MLC and is the site on PP1M that is regulated by rho kinase.
		The COOH terminal 181 amino acids of MBS includes a leucine zipper domain.


	MBS targets cGKIa to the SMC contractile apparatus and activation of cGKIa increases PP1M activity, the cGKIa increases PP1M activity.
	Thromboxane analog U46619 caused an increase in myosin light chain phosphorylation from 10 to 68% in both vector and cGK_1-59 transfected vascular smooth muscle cells.
	In vector alone transfected SMC, 8 Br-cGMP inhibited U46619 mediated myosin light chain phosphorylation.
	Expression of cGK1-59 diminished the ability of 8 Br-cGMP to inhibit myosin light chain phosphorylation following U46619 stimulation.
	MBS assembles a multienzyme complex tethering a phosphatase and at least two kinases (Rho, cGK) with counter-regulatory effects.


	PKG phosphorylates RhoA
	Phosphorylation may inhibit RhoA by (1) increasing association with guanine nucleotide dissociation inhibitor leading to termination of RhoA activation. (2) ? reduced interaction with Rho kinase
	Decreased RhoA/ROK activity would favor MLCP activity, leading to relaxation.
	Telokin-identical to the C-terminus MLCK is PKA/PKG phosphorylated. Phosphorylated telokin may increase MLCP activity, thereby mediating PKG mediated relaxation.


	CamKII has been reported to phosphorylate site A of MLCK; Note: although PKA phosphorylates same site in vitro, no evidence that it phosphorylates in vivo.
	Phosphorylation was associated with a decrease in Ca²⁺ sensitivity of rMLC phosphorylation.
	Suggested that this represents a negative feedback to inhibit high levels of rMLC phosphorylation.


	Excitation-contraction coupling in smooth muscle is believed to occur by two mechanisms-electromechanical and pharmacomechanical coupling.
	Electromechanical coupling operates through changes in surface membrane potential; typically resting membrane potential= -40 to -70 mV.
	Primary drive for the rise in intracellular calcium is membrane depolarization, with the consequential opening of voltage operated calcium channels; neurotransmitters or hormones acting to depolarize the membrane will cause contraction while those producing membrane hyperpolarization will cause relaxation.
	Like cardiac muscle, the influx of Ca²⁺ likely causes release of Ca²⁺ from sarcoplasmic reticulum.


	Drugs that block calcium entry through VOCC will inhibit electromechanical coupling-thus the use of calcium channel blocking agents to relax vascular smooth muscle, thus producing vasodilatation and a decrease in blood pressure.
	Cell-type dependent; for instance, in asthma, Ca²⁺ blocking drugs are not effective in promoting relaxation of muscle.
	Electromechanical coupling appears to play a predominant role in phasic smooth muscle in which the membrane potential often displays marked oscillations upon which are superimposed calcium spikes
	The plasma membranes contain numerous ion channels and the distribution and properties vary among different tissues, contributing to the diversity of smooth muscle.


	Pharmacomechanical coupling- does not depend upon changes in membrane potential or calcium entry via the VOCC.
	The rise of intracellular Ca²⁺ is brought about by a combination of Ca²⁺ release from intracellular stores and Ca²⁺ entry through non-voltage gated channels, primarily receptor operated calcium channels or store operated Ca²⁺ channels
	Ca²⁺ signal often similar to that seen in many non-excitable cells, consisting of an initial rise in [Ca ²⁺]_i followed by a smaller, but sustained increase dependent upon Ca²⁺ entry from the extracellular space.
	This secondary influx of Ca²⁺, in association with the process of Ca²⁺ sensitization whereby the contractile apparatus may be activated by near-resting levels of [Ca²⁺]_i, allows muscles to maintain tone over prolonged periods in the presence of an agonist; occurs in tonic smooth muscle.


	The relative importance of electromechanical or pharmacomechanical coupling for any given smooth muscle preparation can be estimated by determining the effects of inhibitors of VOCC’s on the contraction to agonists.
	For example, in guinea pig ileum, dihydropyridines such as nifedipine will virtually abolish all contractions, suggesting that electromechanical coupling predominates
	However, both mechanisms probably occur to some extent in all smooth muscle. In addition, the opening of ROCC and SOCC also produce membrane depolarization, thus activating electromechanical coupling.


	Approximately 20 years ago, it was hypothesized that receptor activation could lead to Ca²⁺ entry by a mechanism independent of membrane depolarization in smooth muscle
	Receptor operated currents have been described as non-selective cation currents rather than Ca²⁺ channel
	In the rabbit ear artery, externally applied ATP produced a rapid, transient depolarization of muscle, shown to result from activation of a non-selective cation conductance with significant Ca²⁺ permeability. Similar responses were reported to ATP in rat vas deferens, rabbit portal vein, and human saphenous veins.
	In addition to ATP, Noradrenaline, Acetylcholine, Histamine, Endothelin-1, Neurokinin A, Substance P, and Vasopressin have been shown to activate a receptor-operated cation current.


	In the late 1980’s, Putney proposed the model for “capacitative calcium entry” in which intracellular Ca²⁺ store depletion stimulated Ca²⁺ influx across the plasma membrane to maintain a raised [Ca²⁺]_i in the face of prolonged agonist application and to aid in refilling of the stores on agonist withdrawal.
	It is not the Ca²⁺ released from the stores that activates SOCC. Thus, if the rise in [Ca²⁺]_i is prevented by inclusion of a Ca²⁺ buffer, then the store operated current would still be present. It is the fact that the stores are empty of Ca²⁺ that drives the response by an as yet unknown mechanism.
	Many of the neurotransmitters which activate ROCC simultaneously activate phospholipase C, liberating IP3. Therefore, SOCC is activated due to IP3 mediated depletion of the sarcoplasmic reticulum.


	Molecular evidence suggests that store-operated and receptor-operated channels may be formed from proteins belonging to the same family, being the mammalian homologues of the transient receptor potential (TRP) channels.
	Less clear whether they form the channels in native smooth muscle
	One putative model is that TRPC proteins may fall into two classes; one responsive to receptor activation but not store depletion and the other responsive to store depletion.


	The SR is the physiological intracellular source and sink of Ca²⁺ in smooth muscle, as in striated muscle.
	The Ca²⁺ pump of the SR is a SR/ER Ca²⁺-ATPase of 100 kDa with isoforms 2a and 2b.
	The SR also contains phospholamban, which regulates Ca²⁺ uptake by the SR.
	Central SR appears to form a continuous system connected with the peripheral SR.
	The peripheral SR can form surface coupling with the plasma membrane; regions where the SR and plasma membranes come to within 8-10 nm of each other and are connected by elctron-dense bridging structures.


	Ryanodine receptors recorded in planar lipid bilayer;
	Note Ca²⁺ dependence.


	Elevation of [Ca²⁺]_SR can cause increased spark and transient K_Cafrequency that should lead to membrane hyperpolarization, decrease in voltage-dependent Ca²⁺ channel activity, reduction in global [Ca²⁺]_i and dilation.
	May also increase the driving force for sarcolemma extrusion mechanisms that are located in the vicinity of the release site, such as Na⁺-Ca²⁺ exchanger and Ca²⁺-ATPase. May also inactivate sarcolemmal voltage dependent Ca²⁺ channels.
	“Superficial buffer barrier hypothesis- Ca²⁺ entering SMC is buffered by the SR and is discharged vectorially towards the sarcolemma, without any effect on global [Ca²⁺]_i.


	Myogenic tone refers to the ability of vascular smooth muscle to alter its state of contractility in response to changes in intraluminal pressure
	The vessel constricts in opposition to an increase in intravascular pressure and dilates when the pressure decreases
	Behavior observed in a variety of vascular tissues, including veins and conduit arteries, but especially prevalent in resistance vasculature.
	Classically described as being a Ca²⁺ dependent process where pressure evoked depolarization and Ca²⁺ entry through voltage gated Ca²⁺ channels play obligatory roles
	Consistent with a role for pressure-induced depolarization, blockers of voltage gated Ca²⁺ channels have been shown to reduce myogenic responses.


	Arteriolar SMC possess ion channels sensitive to cell membrane stretch that may be activated by vessel distension arising from an increase in intraluminal pressure.
	Have relative permeability: K⁺>Na⁺>Ca²⁺
	Ca²⁺ influx would be relatively small- generally believed that stretch activation of these channels mainly contributes to membrane depolarization with subsequent opening of voltage gated calcium channels.
	K_Ca currents have been shown to attenuate the stretch-induced changes in membrane potential and myogenic constriction.
	Mechanical perturbation of cell membranes may release factors that modulate the activity of such channels.