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CVS Physiology lecture 3.5

Kathryn Wardle English 140 cards

Cards in this deck (140)

  1. 1Blocking Na+/K+-ATPase =loss of electrochemical gradient
  2. 2Ion channel functionallow flow of ions through the pore; about 10^7/sec/channel
  3. 3Gating of channels =pore alternates between being open and closed; only passes current when pore is open
  4. 4Channel gating controlled by:membrane potential (voltage-gated); binding of a chemical (ligand gated); membrane deformation (mechanical gating)
  5. 5Can classify ion channels by:selectivity, type of gating, site of occurrence, using novel structural information
  6. 6Reversal potential of ion channels depends ontheir relative permeability
  7. 7A channel completely selective for 1 ion has a reversal potential ofthe equilibrium potential for that ion
  8. 8Reversal potential =potential at which, if the channels are open, there will be no net flow of charge across the membrane
  9. 9Equal permeability of 1 ions through a channel, reversal potential =0 (halfway between Ena and Ek)
  10. 10No net flow of charge =ions flowing but they charge is balanced so there is no net flow of charge
  11. 11Flows flow down theirelectrochemical gradient
  12. 12An open ion channel can be compare toa resistor; will obey Ohm's law
  13. 13A single channel I-V curve showswhat the current flowing through the channel would look like if the channel was open
  14. 14Whole cell I-V curve would showcurrents produced by the whole cell; these would depend on the amount of channels open
  15. 15Can measure reversal potential on an I-V curve bywhere the curve crosses the x axis
  16. 16BKca channels =large conductance calcium-activated K+ channels
  17. 17Patch clamp techniqueelectrodes have small holes on the tip so when they touch the cell, the circumference of the electrode will become attached to the membrane and can be removed
  18. 18Conductance of channel equation =i/(V-Erev); i = single channel current, V-Erev = driving force; units in siemens
  19. 19NCX functionregulates smooth and cardiac muscle; uses sodium gradient to move calcium from low -> high concentration; less able to work closer to Ena as there is less Na+ gradient so less energy
  20. 20NCX reversal potential depends onconcentration of Na+ and Ca2+; this dictates which way the exchanger works
  21. 21NCX forward modeCALCIUM EFFLUX (normal mode); low [Ca2+]o and high [Ca2+]i, i.e. during calcium-induced calcium release and [Ca2+]i is high
  22. 22NCX forward mechanismcalcium binds to the exchanger and induces a conformational change so it is released to the outside of the cell; 3 Na+ bind and another conformational change releases them into the cell
  23. 23Why does NCX enter reverse mode?driving force for Na+ decreases as you approach Ena
  24. 24NCX reverse mode generatesan outward current
  25. 25NCX forward mode generatesan inward current
  26. 26NCX reverse mechanismcalcium is brought in from the higher [Ca2+] outside into low [Ca2+] inside; 3 Na+ delivered to the outside
  27. 27When does NCX enter reverse mode?peak of the cardiac action potential to increase calcium influx into the heart cell
  28. 28Encx calculated using3Ena - 2Eca
  29. 29Effect of increasing [Na+]i on Enabecomes less positive
  30. 30Ena under normal conditions =+66mV
  31. 31Encx at typical [Na+] =-50mV
  32. 32NCX at -80mV =pump will want to move membrane potential up towards Encx at -50mV; must work in forward mode to produce a depolarisation and achieve this
  33. 33NCX during peak of cardiac APthere is a positive difference between the AP and Encx, so the NCX works in reverse mode to bring calcium into the heart in return for Na+; after repolarisation it becomes more negative again so the NCX will work in the forward mode again to remove calcium from the cytoplasm
  34. 34Whole-cell current =N x i x Popen
  35. 35N =total number of channels
  36. 36i =single channel current
  37. 37Popen =open probability
  38. 38Voltage clamp is used becauseneed to stop the membrane potential from changing as this will affect the activity of the ion channels
  39. 39Voltage clamp enables you toclamp the membrane potential at a chosen voltage so you can measure the currents flowing through the membrane at that potential
  40. 40Function of voltage-clamp amplifiercompares the voltage of the cell with the voltage you want to clamp the cell at and minimises this, whilst measuring the current flowing across the cell membrane
  41. 41To eliminate the Na+ current from recordings, canchange [Na+]o so that Ena = 0mV or with a channel blocker, then step Vm to 0mV so there will be no net flow and you will only see the K+ current
  42. 42Ek =-90mV
  43. 43Opening K+ channels results inK+ flowing out of the cell = outward current
  44. 44Opening Na+ channels results incurrent entering the cell = large inward current
  45. 45Current from voltage-gated channels gets smaller after a while due togetting closer to the reversal potential decreases the driving force, so the current will change direction and flow outward at potentials higher than the reversal potential
  46. 46States of channels =closed, open and inactivated
  47. 47Activation =channels move from being closed to being open following depolarisation
  48. 48Deactivation =channels move from open to closed following hyperpolarisation
  49. 49Inactivation =channels enter a non-conducting state that is different from the closed state, recovery from which requires hyperpolarisation
  50. 50At negative potentials, voltage-gated ion channels will beclosed
  51. 51When voltage is stepped to a positive potential, channelsopen; more open as potential increases
  52. 52Availability curve showswhat % of channels are closed or inactivated (so cannot be opened readily)
  53. 53How to measure an availability curvestart at -100mV so all channels are shut, then step to 0mV; repeat but start at increasingly more positive potentials. At negative potentials, most channels are closed so can open, but by -50mV there is hardly any current produced as all the channels are inactivatedcgvbvb
  54. 54Normalisation =divide each of the currents by the maximum current
  55. 55To measure recovery of Na+ channels from inactivation:will see a current at an initial voltage pulse to 0mV which will wane as the depolarisation is maintained and the channels enter the inactive state; after a second pulse a smaller current will be produced; increasing time between pulses = increased size of second current as more channels can recover
  56. 56To calculate the number of channels recovered from inactivation:divide amplitude of the second current produced by the amplitude of the initial current
  57. 57Recovery from inactivation is dependent onmembrane potential; faster recovery at -100mV compared to -70mV, and the length of the refractory period which allows them to recover
  58. 58Measure activation =seeing how many channels are open at a given voltage
  59. 59Relative activation will increase withincreased depolarisation of the voltage
  60. 60Relative activation is the same asrelative Popen
  61. 61Relative Popen =Popen/maximum Popen
  62. 62Tail currentscurrents seen when you activate a channel and then step down to a more hyperpolarised current; a little current is seen at the end of the first pulse
  63. 63Tail currents used formeasuring how active channels are at a given voltage
  64. 64Nature of single channel currentscan see the channel open and close randomly; will look different if repeated
  65. 65Effect of stepping down from V1 to V2 on the single channel currentwill decrease as there is less driving force as the electrochemical gradient is smaller
  66. 66If a channel is open at the start of V2, it willalso be open at the beginning of V2
  67. 67The amount of channels open at the end of the first pulse isequal to the amount of channels open at the start of the second pulse
  68. 68A whole cell current is made fromsumming up all of the individual channel openings
  69. 69Vm being further from Ena =larger currents generated due to a larger driving force
  70. 70Effect of larger voltage step on Popen =increases faster and reaches a higher level of as the voltage is more depolarised
  71. 71Effect of maximal activation on tail currentsPopen increases faster but no extra channels are opening so the size of the tail current is the same as at lower potentials which maximally activate the channels
  72. 72Stepping down at V2 effects recordings bushowing that even though the same number of channels are open, there is a reduction in the single channel current
  73. 73Na+ channels single channel recordingbulk of openings are at the beginning of the pulse as they inactive as time passes
  74. 74K+ single channel recordingsdon't inactivate so when summed into an ensemble they show a large outward current
  75. 75Selectivity of Na+ channelsnot hugely selective, lets mostly Na+ through
  76. 76Selectivity of K+ channelsmore selective than Na+ channels
  77. 77Selectivity of calcium channelshighly selective; they can distinguish between Na+ and Ca2+ with a high calcium selectivity and flux; some other divalent ions can permeate/block; in absence of calcium, channel is permeable to monovalent cations
  78. 78Assumptions of the GHK current equations:ions do not interact with each other or the channel pore; voltage field within membrane is constant; number of open channels per area of membrane is constant
  79. 79An equal concentration of each ion on either side of the membrane =straight line according to GHK which passes through 0
  80. 80GHK equation predictsthe single channel current as you change the voltage with varying ionic concentrations on either side of the membrane
  81. 81Theory of how ions move through channelsions must enter the channel before they bind, so there is an interaction between the pore and the ions; there is an energy barrier for the ion to get into the channel as the ion needs to lose its hydration shell
  82. 82Hydration shell of ionsions in solution are surrounded by water molecules so these need to be lost before they can bind to the channel
  83. 83Energy profiles of different ionsdifferent ions have different energy profiles; some have higher energy barriers which prevent the ion entering certain channels as they are rejected
  84. 84Effect of a deeper well on energy profile on single channel currentdeeper well = energetically favourable for ions to stay in the channel so less current will flow = smaller single channel current produced
  85. 85Rules for a 2 ion channel:ion cannot enter a site already occupied by another ion; ion cannot pass over a site without binding to it
  86. 86Another ion Ca2+ channels are permeable to isBa2+
  87. 87Current if Ba2+ goes through calcium channelsis larger than the calcium current; used to investigate these channels as they create larger currents
  88. 88KD of calcium's single channel current =13.9mM
  89. 89KD of calcium's single channel current = with barium =27.9mM; goes through channel quicker so doesn't build as well to the channel
  90. 90Blockers of the calcium channel =many divalent cations: cadmium, cobalt etc.; they enter the channel and block it before leaving again
  91. 91Activator of calcium channels =Bay K 8644
  92. 92Calcium chelator =EDTA
  93. 93How to remove calcium from a solution =use EDTA (calcium chelator) as a buffer to remove contamination
  94. 94When no calcium present, the channels willpass monovalent cations; this creates a larger current which a very small amount of calcium will block
  95. 95Membrane topology of calcium channelsimilar structure to voltage-gated Na+ channels; 4 glutamate residues in each of the pore regions which face into the pore
  96. 96Mutations in calcium channels' glutamate residues =how well calcium can block the flow of monovalent current through the channel is impaired; these residues are key for allowing a small amount of calcium to block monovalent cations flowing (especially when all 4 are changed to alanine)
  97. 97How many affinities for calcium do calcium channels have?2: calcium flux saturates above 10mM; the 4 glutamate residues in the pore form a high affinity binding site within the channel too
  98. 98How does calcium block monovalent cations flowing through calcium channels?1-10um of calcium is enough for calcium to enter the pore and coordinate with the 4 glutamate resides to bind strongly to the pore and prevent Na+ entering; 2 calcium molecules can enter but these will repel each other so don't bind as strongly as one molecule would
  99. 99Kv3.1 channelhigher single channel current
  100. 100Kv2.1 channellower single channel current
  101. 101Difference in structure between Ca2+ and K+ channelsCa2+ = 4 glutamate residues facing pore; K+ channels have a backbone of carbonyl oxygen atoms which points into the channel and is crucial for enabling K+ flux
  102. 102Does Na+ go through K+ channels?K+ is larger than Na+, but Na+ doesn't pass because, when K+ loses its hydration shell, the distance between the oxygen atoms in the pore region exactly matches the shape of K+ whereas Na+ is too small to be coordinated perfectly
  103. 103Calcium interacts with glutamate becauseglutamate is negative and calcium is positive
  104. 104Manner of K+ flux through K+ channel =they go through in single file, but it is likely there are still some water molecules between these, as according to the knock-on model
  105. 105Knock-on modelthere is simultaneous occupancy of the selectivity filter with 2 K+ ions, with water in between them; they can fit through the pore with a water molecule between each ion
  106. 106Detection of voltage field occurs incharges within the S4 membrane spanning domain of voltage-gated ion channels
  107. 107Kv channel subunit structure =6 TM domains and a P region, one of which is the S4 region; 4 subunits form 1 functional channel
  108. 108Structure of the S4 regionevery 3rd amino acid has a positive charge on it which detects a change in the voltage field, causing a conformational change which opens the channel
  109. 109Why is it hard to get structural information about conformational change of channels?because they are constantly flipping between states
  110. 110How can we get information about K+ channels' structures easier?using bacterial channels which also have a selectivity filter and a gate
  111. 111What have we found from bacterial structural information?within the alpha helix there is a glycine molecule which enables helices to bend quite easily (glycine helix) and opens the gate; in the closed state K+ cannot enter the channel due to the presence of its hydration shell which won't be lost until it enters the pore, in open configuration the channel is much wider so K+ ions can enter
  112. 112Kv1.2 structure =4 alpha subunits; S1-S4 are voltage-sensing, S5-S6 are pore-forming; T1 domain is what K+ goes through to get into the channel; Kv(beta) can associated with the channel and alter its gating/inactivation
  113. 113Kv(beta) subunit potentiatesinactivation of K+ channels
  114. 114Does Kv1.1 inactivate?no
  115. 115Does Kv1.4 inactivate?yes - this is an A-type K+ channel which is important in the heart for the transient outward current just after the AP peak
  116. 116Why do Kv1.1 and 1.4 behave differently ?in the alpha subunit of Kv1.4, the N terminal has a particular sequence of amino acids which can get into the pore of the channel once the channel is open; if this is removed the inactivation is also removed
  117. 117Effect of a beta subunit on inactivationincreases rate of inactivation
  118. 118N-type inactivationtype of ion channel block; when the N-terminal of the alpha subunit in Kv1.4 enters the pore and blocks it, thus inactivating it
  119. 119N-terminal of the beta subunitsimilar to that of the Kv1.4 subunit; can also block the pore; will therefore impart an inactivation on the Kv1.1 which doesn't usually inactivate
  120. 120Methods of how blockers block ion channels:slow block, intermediate block, fast block
  121. 121Slow block =remains on the channel, inhibiting for a long time e.g. toxins, only need a small amount as they bind so tightly
  122. 122Intermediate block =the mean open time of the channel has been reduced
  123. 123Fast block =channels open and block very quickly, often so fast that there is a reduction in single channel current rather than discrete blocking events
  124. 124Channels are often blocked in which state?open
  125. 125Channel recordings when blockers are presentchannels flick between being open and blocked, eventually enter the closed state
  126. 126KATP channels =voltage-independent K+ channels which open over a range of voltages
  127. 127Blocking of KATPin control, mean open time was 12ms; blocker causes much briefer opening times of 0.7ms
  128. 128Blocking rate constant is dependent onmembrane potential; suggests the blocker's binding site is deeper within the pore so to get there the blocker has to cross part of the membrane voltage field
  129. 129Channels can also be blocked naturally byintracellular ions, such as Mg2+ and polyamide ions
  130. 130Inward rectifier currentscurrent is not necessarily inward; they will pass current in the inward direction much easier than will pass current in the outward direction
  131. 131Inward current in the inward rectifier K+ channelswould have to be more negative than Ek, which doesn't occur very often under physiological conditions
  132. 132Kir2.1 =strong inward rectifier
  133. 133Inward rectifier gating process linked tothe permeation of the channel; altering Ek causes a shift in inward rectifier current
  134. 134Inward rectifier structure =much smaller than voltage-gated K+ channels; only 2 TM domains and 1 charged amino acid within the cell membrane region
  135. 135Charged amino acid within the inward rectifier channel =aspartate D172; removal reduces the amount of rectification the channel performs
  136. 136What is crucial in producing the strong inward rectification?polyamines; most potent is spermidine
  137. 137How did researchers discover that polyamines are important for inward rectification?expressed inward rectifier in oocytes and found that when they removed patches, rectification was gradually lost so they thought the oocyte was producing something necessary for inward rectification; washing inside of patches also removed this; when spermidine applied the rectification gradually returned
  138. 138Inward rectifiers are blocked strongly bypolyamines
  139. 139Polyamide block of inward rectifierscan enter the channel but not pass through; has to move 4 positively charged ions out of the channel so that 2 molecules can block the pore, thus making block voltage-dependent
  140. 140Inward rectifier currents predominant duringcardiac AP; as heart cell is repolarising, the inward rectifiers currents come into play during the late stage

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Flashcards on CVS Physiology lecture 3.5, created by Kathryn Wardle on 01/05/2019.
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Kathryn Wardle · May 2019
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May 1, 2019
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