Class Note for BME 510 at UA
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Date Created: 02/06/15
BME 510 Lynch Lecture 2 October 24 2008 Calcium Homeostasis and its Measurement Key Concepts 0 Intracellular Ca2 generally refers to cytosolic Ca2 levels which are typically in the 10100 nM range TOTAL cellular Ca2 bound free is ca 35 mM 0 Cytosolic Ca2 levels are maintained through a collaboration between Ca2 channels for Ca entry into the cytosol and CaZATPases that pump Ca2 out of the cell or into intracellular compartments Channels include LOCs and VOCs Ca ATPases have pm and ER isoforms 0 The Nai Ca2 exchanger is near equilibrium in most cells It acts to grossly tune Ca2 levels INTRODUCTION Most living cells maintain membrane potentials on the order of 4080 mV negative inside According to the Nernst equation a cell with a membrane potential of 58 mV at equilibrium will have a concentration of intracellular monovalent cations that is 10X higher than the extracellular concentration For divalent cations the concentration difference would be 100fold Since extracellular Ca is about 1 mM intracellular Ca2 levels should be 100 mM at equilibrium For H the intracellular pH at equilibrium would be 10 unit lower than the extracellular pH which is about 70 However measured Ca2 and H in all cells is invariably lower than that predicted from Nernstian equilibrium indicating that either a Ca and protons are not membrane permeable or b systems exist to pump calcium and protons out of cells and maintain intracellular pH and Ca2 levels out of equilibrium Although Ca2 is relatively imperrneant its inwarddirected ux is increased by channels and hence Ca2 pumps must exist A Regulation of Intracellular Ca2 Berridge M et a1 2003 Calcium Signaling Dynamics Homeostasis and Remodeling Nature Reviews Molecular Cell Biology 4517529 Cell membrane Endoplasmic 7 reticulum ERQA pump BME 510 Lynch Lecture 2 October 24 2008 l Ca2 Channels The electrochemical gradient for Ca2 is large This is because both the electrical gradient and the chemical gradient favor the spontaneous movement of Ca2 from outside of the cell into the cytoplasm of the cell Therefore if they are open Ca2 channels will allow a large in ux of Ca2 into the cytosol Channels fall into two families those which are regulated by Voltage VOCs and those which are regulated by ligand binding to receptors LOCs The different types of Ca2 channels can be distinguished pharmacologically VOCs are activated by depolarization and are found in all excitable and some nonexcitable cells excitable cells can propagate an action potential nerve muscle L Ntype VOCs are tetrameric and are defined by having highaf nity binding sites for dihydropyridine DHP agonists They also bind phenylalkylamines and benzothiazepines and are inhibited by DHP antagonists nifedipine and nitredipine They are also activated by cAMPdependent phosphorylation Ltype VOCs in non muscle cells are structurally distinct from those in excitable cells N Ntype VOCs are probably neuronspecific are insensitive to DHP and are completely inactive at holding potentials more positive than 20 mV Ntype channels are blocked by occonotoxin and are probably responsible for high voltage activated Ca2 currents T type VOCs are found in a variety of excitable and non excitable cells and are also known as lowthreshold channels since they are activated at voltages 3040 mV more negative than are L channels LOCs fall into three categories a those in which the channel is an integral part of a receptor called ROCs b those which are activated by soluble second messengers or SMOCs and c those that are activated by G proteins GOCs ROCs are the bestknown and include the nicotinic acid receptor channel the N methylDaspartate glutamate channel and the extracellular ATP channel Although evidence for GOCs is scanty it is likely that insulinlike growth factor 2 IGF2 induced Ca2 increases occur via Gprotein action SMOCs include the 1P3 sensitive channel Inositol 145 trisphosphate 1P3 is a member of a family of phosphorylated inositols produced via phospholipase C PLC induced hydrolysis of phosphatidylinositol a phospholipid 1P3 is produced from the action of PLC on phosphatidylinositol 45 bisphosphate PLC cleaves between the bridging phosphate and the glycerol moiety thus producing 1P3 and diacylglycerol diAG activates protein kinase C The released 1P3 opens IP3gated SMOCs which are present on the endoplasmic reticulum causing release of Ca2 into the cytosol Cytoplasmic 1P3 is rapidly degraded via phosphatases to inositol and inositol phosphate which are substrates for resynthesis into phosphatidylinositol 2 Ca2 ATPases are found primarily in two cellular compartments the plasma membrane and the sarcoplasmicendoplasmic reticula Both PMATPase and SERATPase SERCA are ElE2 pumps eg they have two states and a phosphorylated intermediate They are thus in the same family as NaK ATPase and Ptype HATPase Hence they are inhibited by vanadate There are multiple isoforms for the PMATPase at least two are expressed in brain and one in erythrocytes Since both N and C termini are intracellular there are an even number of transmembrane domains 8 or 10 There are two ATP binding sites one which is catalytic high affinity and the other which is regulatory low affinity The PMATPase is regulated by calmodulin In reconstituted systems this enzyme is nonelectrogenic with the antiport of 2 H per Ca2 BME 510 Lynch Lecture 2 October 24 2008 There are several isoforms SERCA IIV all having similarity to the PMCa2 ATPase SERCA s are found in muscle and nonmuscle cells In muscle cells SERCA represent 90 of the protein in the SR In nonmuscle cells SERCA levels are lOOfold less and are found mainly in specialized subtypes of ER called calciosomes which contain Ca2 binding proteins calsequestrin or calreticulin In SR the SERCA is regulated by phospholamban PLB a 52 aa amphipathic protein which inhibits Ca2 mobilization in its dephosphorylated form Native PLB has a positive charge It crosslinks and inactivates 2 SERCAs which have numerous acidic negatively charged residues in the juxtamembrane region Upon phosphorylation PLB no longer interacts and inhibition is relaxed 3 Na Ca2 exchange This exchanger is primarily found in muscle and neural cells transporting Na and Ca2 with a stoichiometry of about 31 It is therefore electrogenic and voltage sensitive This exchanger will cause Ca2 in ux upon depolarization and ef ux upon hyperpolarization It therefore works in concert with Voltage gated Ca2 channels during action potentials and in concert with ATPases during repolarization The Km of the exchanger for Ca2 is relatively high 05 2 NM therefore it works to grossly tune Ca2 levels In most cells this exchanger is near equilibrium 4 Ca2 stores and Ca2 induced Ca2 release Calcium storage sites within cells are generically referred to as 39sarcoplasmicendoplasmic reticulum SER These stores are characterized by the presence of several specific proteins a specific Ca2ATPase SERCA is responsible for maintaining the chemical gradient between cell and store space Thapsigargin or cyclopiazonic acid are drugs which inhibit SERCA somewhat specifically Several Ca2 binding proteins calreticulin calbindin calsequestrin buffer Ca2 within the store making the total amount of Ca2 available in the compartment much greater than that which is free in solution Finally there must be a mechanism to release Ca2 from these stores This is accomplished either through activation of ryanodine receptors muscle or 1P3 receptors non muscle 1P3 is generated by activation of Gprotein coupled receptors which activate phospholipase C to produce 1P3 from membrane phospholipids containing inositol Ryanodine receptors are activated primarily by elevation in cytosolic Ca2 but also show voltage sensitivity in striated muscles In addition there is an asyet unknown mechanism that senses the Ca2 levels within storage organelles and if it is too low induces the transplasma membrane uptake of Ca2 through Ca2 channels This phenomenon is known as 39capacitative39 Ca2 entry and is an active area of current research 5 Capacitative Ca2 In ux and the Ca2 release Activated Channel Many vasoactive agonists bind to membranelocalized Gprotein coupled receptors that subsequently activate phospholipase C forming 1P3 and diacylglycerol 1P3 binds to receptors on intracellular Ca2 stores modulating Ca2 release Unloading of Ca2 stores elicits an increase in plasma membrane Ca2 in ux via a Ca2 current referred to as ICRAC The CRAC channel has not been identified although studies are determining the electrical characteristics of the current and its homology to other known channels Members of the transient receptor TRP family of channels are likely to be involved in this store operated in ux Equally elusive has been identification of the mechanism that signals plasma membrane channels to open when stores are depleted Data support the existence of a diffusible messenger but other data suggest a more direct proteinprotein interaction between Ca2 stores and the channels themselves Little is known regarding the physical distribution of Ca2 stores and their possible interaction with specific membrane domains although proposals purporting microdomains organized near or in caveolae where Ca2 regulatory factors coalesce have been presented BME 510 Lynch Lecture 2 October 24 2008 Differential Activation of Signaling Pathways by Phasic vs Tonic Changes in Cell Ca2 Signals generated by changes in cell Ca2 are encoded by both the amplitude of the response and its phase for oscillating changes For pathways that are directly activated such as muscle contraction and secretion immediate changes in Ca2 lead to immediate responses However when Ca2 is activating a pathway cascade that contains multiple components the elevation in Ca2 must be sustained to allow for full pathway activation This explain how low level sustained elevations in Ca2 can lead to changes in growth controltranscription in cells where high amplitude but cyclical changes in Ca2 occur continuously cardiac muscle Caveolae and The Subcellular Organization of Ca2 Signaling The structure of Ca2 stores in striated muscle is clear and evidence from imaging experiments indicates that functional domains within these stores exists with respect to the localization of speci c transport and signaling molecules In smooth muscle and other cell types the localization and organization of these molecules and Ca2 stores themselves is less clear However distinct structures in smooth muscle which form near membrane invaginations referred to as caveolae may represent an organized compartment for Ca2 and in general for second messenger signaling A structural protein similar in function to clathrin has been isolated from these structures and is referred to as caveolin Caveolin appears to assist in the formation of the membrane invagination In the membrane of the caveolae there is an enrichment of cholesterol glycosphingolipids and lipidanchored proteins myristillated In smooth muscle these regions also exhibit high expression levels of PMCa ATPase 1P3 receptors and calmodulin Other proteins such as the CCI channel TRP SOC and the NaCa exchanger also may be enriched in these regions The organization of these structures and their role in Ca2 homeostatis and in smooth muscle excitation contraction coupling is an area of current interest Ac etylcholine Caveolae BME 510 Lynch Lecture 2 October 24 2008 SMOOTH MUSCLE Smooth muscle lines the walls of hollow organs blood vessels and ducts of secretory glands Its role is to regulate luminal diameter Given the diversity of functions these various tissues ful ll it is not surprising that there are many physiological differences between smooth muscle cells of different origins The similarities and differences between smooth muscle types and skeletal muscle are discussed below Structure Smooth muscle cells SMCs are typically 2 10 pm in diameter Vascular SMC s are N4 pm in the nuclear region and of variable length ranging from 20 to 600 um VSMC 20 60 um Due to their small diameter diffusion is not limiting consequently SMCs don t need a t tubule system to conduct the action potential into the interior of the muscle cell Nevertheless SMCs have numerous caveola small invagination of the cell surface that serve to organize the excitation contraction coupling machinery Ca channels and NaCa exchangers are concentrated therein The SR is located adjacent to the plasma membrane especially near caveolae Cells in all types of smooth muscle are well connected by mechanical junctions desmosomes and adherens but gap junction content varies considerably In most smooth muscles e g uterus gut blood vessels gap junctions are numerous and support synchronous contractions of multiple cells Excitation Contraction Coupling Calcium couples activation to contraction in smooth muscle The calcium enters the cytoplasm from the calcium stores SR or from the extracellular space Release of calcium from the SR is induced by one of two mechanisms calciuminduced calcium release CICR or 1P3 induced Ca release CICR involves a Carelease channel similar to that observed in skeletal muscle but it opens in response to elevation of cytosolic calcium rather than a voltageinduced mechanical event mediated by a DHP receptor The Carelease channel activated by 1P3 is a ligandgated channel where the ligand is formed subsequent to activation of the phosphatidyl inositolsignaling cascade Fig l Figure Summary ofmechariismsfor the regulation ofiritracellular calcium in smooth muscle cells Slow Stretch Ion wave Receptor gt C channels a gt and pumps gtDepoarzationgt Channel Ca Na K Ion Fluxes Second messengers Sarcoplasmic Ca2 IP3 cAMP gt reticulum gt IntracellularCa2 Ca2 Contractile regulatory proteins Contraction BME 510 Lynch Lecture 2 October 24 2008 Contraction Unlike skeletal muscle where Ca2 binds to troponin inducing a conformational change in the troponintropomyosin complex that unveils the myosin binding site on actin in smooth muscle Ca2 leads to regulation of myosin activity Smooth muscle and nonmuscle myosin activity is regulated through phosphorylation of the regulatory light chains on myosin In the absence of phosphorylation myosin ATPase is inactive Phosphorylation is permissive to ATPase activity Thus as illustrated in Figure 4 Ca2 mediates myosinactin interaction in an indirect fashion Ca2 binds to calmodulin CAM to form Ca CAM which subsequently binds to and activates myosin light chain kinase MLCK This kinase phosphorylates the light chain presumably causing a conformational change making possible activation of the myosin ATPase by actin Phosphorylation of the light chain is both necessary and sufficient for activation of contraction MLCK activity and Ca2 levels decrease after peak tension is attained yet tension can be sustained This maintenance phase of smooth muscle contraction is less well understood but is an essential feature of smooth muscle physiology The mechanistic basis for tension maintenance is an area of active research The activity of MLCK can be modulated by a MLCKkinase which is a substrate for cAMP dependent protein kinase PKA In the phosphorylated state the affinity of MLCK for Ca CAM is diminished decreasing myoson phosph 0rylati0n and thereby myosinactin interaction and force cAMP also stimulates CaA T Pase activity and calcium sequestration while cGMPdependent kinase activates Myosinphosphatase both of which diminish contractile force Thus multiple mechanisms activated through second messenger pathways are available for the modulation of MLCK activity and light chain phsophorylation state M LCK Pinact Phosphatase PMLCK PKA lt gt kinaseact MLCK39k39naseanact MLCKact 3 SR CM lt MLCKCM lea2 i cak CaCM gt CaCMMLCK MLCK Myosin MyosinP Actin Phosphatase l I CGMPM AMP M AM latch Figure Regulation of contraction by calcium in smooth muscle Note the involvement of calmodulin CM in activating myosin light chain kinase MLCK which subsequently phosphorylates myosin light chain See Hartshome DJ and T Kawamura Regulation of Contractionrelaxation in Smooth Muscle News in Physiol Sci NTPS 7 5964 1992
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