Sodium-Calcium Exchanger (NCX) in Cardiac Muscle Transports PDF

Transzportok a szívizmon Myocardial transports Repetition types v vs. cc. Divisions: according to: 1. energy demand 2. charge movement 3. number of transported materials Transport in detail...

Transzportok a szívizmon Myocardial transports Repetition types v vs. cc. Divisions: according to: 1. energy demand 2. charge movement 3. number of transported materials Transport in details Evolution Transport on cardiac muscle Na / K pump  subunit: 7 TM, 100-110 kDa (catalitic) : 1 TM, 40 kDa glycoprot. () ? only in renal cells Binding sites In HUMAN heart: 1, 2, 3 1, (2) Atrium Ventricle 3 < 1 < Stochiometry: 3 Na+ : 2 K+ Electrogen: 3+:2+  Na / K pump operation Albert-Post model Conformations: E1: high energy, high affinity for ATP, low aff. for K+ E2: low energy, low affinity for ATP, high aff. for K+ Modes of operation  Measuring pump activity 1. Direct flux measurements with radioactive isotopes Adv.: direct, good in membranes Disadv.: Na movement through other ways, complicated geometry ic. organelles ec. glycocalix bad in cardiac muscle. 2. Ionselective electrodes K+ selective ec. electrode disadv.: limited ec. K+ diffusion, passive K+ conductances  ic. Na+ selective microelectrode adv.: simultaneous MP measurement disadv.: Na activity, not cc. Na redistribution might disturb 3. Pump current measurements in VC adv.: constant MP, disadv.: Rb+ is not K+  Regulation of pump activity 1. Ic. Na+ ↑  pump activity ↑ linear or sigmoid relationship steep curve 2. Ec. K+ EC50= 1-2 mM  fully active, BUT in hypokalemia digitalis intoxication 3. ATP, ADP, Pi ratio of ATP/ADP+Pi dual role of ATP: 1 M phosphorilation, catalysis, high affinity binding site 200 M regulatory, increase of activity ATP hydrolysis: 61 kJ/mol energy release in normal case; in hipoxia 49 kJ/mol, BUT only 47 kJ/mol is needed for the transport 4. MP, electrogen transport In model: Upon depolarization: Na+ efflux ↑, BUT K+ entry ↓ opposite roles of the two cations 5. Number of pumps Turnover rate: 5 hours 6. catecholamines, T3, aldosterone, corticosteron increases pump activity (cAMP and PKA phosphorilation) 7. ANP inhibits  8. Effect of cardiac glycoside Strophantus: Ouabain = Strophantin, cymarin Digitalis: Digoxin, digitoxin Scilla glykoside: Proscillaridin (from plants) Peruvoside EC50=10-9 – 10-7 M on pump current activate in small dose potential explanations: catechoamine release from nerve endings competitive inhibition of an endogenous blocker (EDLF from the adrenal gland) Endogeneous cardiotonic steroids marinobufagenin  Role of Na/K pump in cardiac muscle 1. Creation and maintenance of: Na and K gradients membrane potential excitability 2. Providing driving force for secondary active transports (NCX) Ouabain induced DAD Fülöp et al. Gen. Physiol. Biophys. 2003, 22(3), 341-353.  Localization of Na/K pump in cardiac myocytes Less regulated by PLM  Regulation of Na/K pump by phospholemman  subunit  subunit Regulation of Na/K pump by phospholemman  Na/K pump in cardiac diseases Reduced NKA expression and/or function in hypertrophy / heart failure → → [Na+]i ↑ → activation of mitochondrial (inner membrane) NCX → → [Ca2+]i ↑ and [Ca2+]m ↓ → ATP production ↓ → metabolic insufficiency Phospholemman hypo-phosphorylation also occurs  Experimental evidence suggesting NCX Force and resting tension are linearly related with [Ca2+]e / ([Na+]e)2  Structure of NCX -1 and -2 involved in ion transport NCX1.1 in the cardiac form CBD1 and 2 Ca2+ binding domains 10 TM -1 -2 Found as dimers Stochiometry: Na+ : Ca2+ 2 : 1 3 : 1 4 : 1 Electrogen transport Munekazu Shigekawa et al., Circulation Research 2001 Modes of operation …………………... during AP Donald M. Bers, Nature 2002  Regulation of NCX 1. Ions, Na, Ca in ec., ic. space competitive binding 2. MP ENCX=3ENa – 2ECa NOT constant if MPENCX  reverse mode, hyperpolarize 3. pH acidosis inhibits both modes alkalosis stimulates 4. Blockers: ions: La3+, Cd2+, Ni2+ (5-10 mM) non specific amiloride, 2-4 dichlorobenzamil (10-100M), KB-R 7943, SEA 0400 ORM-10103, ORM 10962 5. High dose of ATP (0,1-1 mM) stimulates as a lubricant  Role of NCX in calcium extrusion  85 %  15 % 0 100 200 300 400 500 Time (ms) Donald M. Bers, Nature 2002  Role of NCX in calcium extrusion 0 100 200 300 400 500 Time (ms) Donald M. Bers, Nature 2002 Role of NCX in pacemaking Role of NCX in pacemaking Role of NCX in force-frequency relationship  Role of NCX in arrhythmogen events Ischemia-reperfusion, Ca paradox Hypocalcemia  Na permeability of Ca channel ↑  [Na+]i ↑ + reperfusion ([Ca2+]e ↑) Ca overload Reverse mode NCX ↑ Spontaneous Ca2+ release from the SR NCX replaces Ca2+ to Na+ DAD EAD SAP Depolarization (EAD, DAD) upon exceeding the threshold of INa SAP Zsolt A. Nagy et al., British Journal of Pharmacology 2004 Zsolt A. Nagy et al., British Journal of Pharmacology 2004  PMCA Structure 10 TM, 140 kDa Binding sites Stochiometry: 1 ATP : 1 Ca2+ Splice variants by alternative splicing Nowadays more than 30 isoforms  Regulation of PMCA 1. C terminal inhibition (analogy with Plb) 2. PKC, but not PKA stimulates with phosphorilation of CaM binding site Ca-CaM complex activates (Ca sensitivity and Vmax ↑) 3. Blockers: La3+, Vanadate ions negative feed-back  Role of PMCA Fine tuning of Ca extrusion during diastole 80% NCX, high capacity, low affinity 20% PMCA low capacity but high affinity Donald M. Bers, Nature 2002 SERCA Monomer of 10 TM, 100-110 kDa Binding sites Stochiometry: 1 ATP : 2 Ca2+ 3 genes with alternative splicing gives 5 isoforms Function Conformations: E = E1: high energy, ADP sensitive, high affinity for Ca2+ *E = E2: low energy, ADP insensitive, low affinity for Ca2+ Rate limiting step in systole: E2 → E1 in diastole: Ca binding Dual role of ATP: low dose (1 M), phosphorilation high dose (100 M), regulatory, lubricant  Regulation 1. Mg2+ 2. Phospholamban (pentamer) 1975 Slows turnover, decrease Ca sensitivity Greater inhibition of SERCA in heart failure due to Reduced phosphorylation and increased PLB/SERCA2 ratio  M2, M6, M9 31 aa 52 aa SLN Uncouples Ca2+ transport from ATP degradation ↓ Heat production ↑