Lecture Notes

Lecture 1BIPN 100

Tu 4/1/14

Central idea: How does an organism function?

teleological vs. mechanistic:teleological: explain via purposemechanistic: explain via physical propertiesie, neuron fires “to communicate with other cells”, vs “because voltage gated Na/K channels…”

Structure and function are relatedorgans to cells to moleculesstructure/function also compartmentalized

Information flow coordinates functionvia endrocrine and nervous systems

Homeostasis maintains internal stabilityorganism attempts to compensatecompensation succeeds, homeostasis; fails, illnesshomeostasis: regulation of variables so that internal conditions remain stable and relatively constantallostasis: process of achieving homeostasis through physiological or behavioral change

Every organism strives to maintain internal stabilityClaude Bernard (1813-1878): biological systems emerge out of the properties of chemistry and physicsphysiology should be studied experimentallystability of internal environment is essential to health of organism

Internal environment: intracellular fluid, extracellular fluid

3 fluid compartments:1) Intracellular fluid (ICF)2) Interstitial fluid (ECF)3) Plasma (ECF)Ions can diffuse through compartments; proteins and salts cannot

Stability does not mean equilibriumK+, proteins (-) very abundant in cell; lower levels outside cellNa+, Cl-, HCO3- also present in lower levels; very abundant in interstitial fluidwater distributed 2/3 in ICF, 1/3 in ECFcompartments are in disequilibrium

Variables of homeostatic controlWalter B Cannon (1871-1945)Environmental factors:Material needs:Internal secretions: hormones

Law of mass balance: mass balance = existing body load + intake, metabolic production – excretion, metabolic removal

Law of mass action (lechatelier’s principle)r1 = r2: rate of rxn in forward direction = rate of rxn in reverse direction[PL]/[P][L] = Keq

Cannon’s postulates:1. The nervous system preserves the fitness of the internal environment.2. Some systems are under tonic control.3. Some systems are under antagonistic control.4. Signals have different effects on different tissue.

Tonic control: signals that maintain normal level of activitysignals always present but gradedchanges in signal rate

Antagonistic control: signals that have opposing effects on activityeg neurons controlling heart rate

Negative feedback loop: responses counteracting stimulus reduce fluctuations and produce stabilityeg. Initial stimulus + sensor + response - stimulusodd number of negative signs

Reflex steps: stimulus, sensor, input, center, output, target, responsestimulus: regulated variablesensor: cell or membrane receptor that monitors the variableinput signal: signal generated by sensorintegrating center: compares signal with set pointoutput signal: generated by integrating center when variable is out of rangetarget: receives output signal and generates the response

Membranesmade of phospholipid bilayer; hydrophilic outside, hydrophobic inside semipermeable; water and lipophilic molecules can diffuse, larger or lipophobic molecules cannot

Lecture 2BIPN 100

4/3/14

KNOW THE NUMBERS.Math problems about equal level difficulty to the test will be on the problem sets.

Diffusion is faster…at higher temperaturesfor smaller moleculesover shorter distancesand along higher concentration gradients.

Resting cell membranes are most susceptible to K+ ions.

Membranes and resting potential.Membranes give the cell morphology and surface area.

Mechanisms for crossing membranes:diffusion (simply crossing membrane)

what is needed for diffusion?facilitated diffusionactive transportendo/exo/phagocytosis

Diffusionpassivemolecules move down gradient (high [ ] to low [ ])net movement of molecules until equilibriumD a distance^2 (very efficient at short distances)

hemoglobin allows O2 to travel long distances; after breaking apart, O2 then diffuses into cellD a temp D a 1/size (smaller size, more efficient diffusion)

Fick’s Laws of Diffusion:Rate of diffusion ~ surface area ~ concentration gradient ~ membrane permeability1. Diffusion goes down the concentration gradient2. Diffusion changes concentration over time

Which molecules will diffuse across the membrane?lipophilic moleculessmall non-polar molecules (O2, CO2, N)small polar molecules (H2O, urea)Large, polar molecules and charged molecules require mechanisms for transport

Facilitated DiffusionChannels (open, or gated)water-filled poreselectivity filter; only allows specific molecules throughgating mechanism allows for opening and closing of channel

Channel can flux 10^7 molecules per second

Roderick MacKinnon won Nobel Prize for studies on K+ channelfour subunits. One example in detail:protein loops in and out of extracellular face, membrane, and intracellular faceions flow through aqueous center of channel single fileselectivity arises from a-helix “arms” which pull ions into aqueous cavity

How are K+ channels gated?Three physical mechanisms for gating: Ball and chain (physical occlusion at opening) S6 gate (sixth helix protein participates in aiding selectivity) selectivity filter gate (sequence in P loop allows for de-solvation of K+)

Facilitated diffusion: Carrier Proteins(how to transport larger molecules?)water filled pocketbinding changes conformationflux 10^3-10^10^6 molecules per second; slower than channels

Structure/function: sugar transporterssugar molecule coordinated by aromatic and polar AAsvery specific set of AAs creates binding pocket for moleculeslight change in molecule requires change in AA pocket for binding/transport

Lecture 3BIPN 100

4/8/14

Clicker: Steroid hormones act on receptors located in the cytoplasm.Lipophilic hormones diffuse through the cell membrane and act on receptors in the cytoplasm.

Local communication: gap junctionshomo/heteromeric channels connect adjacent cellsvaried selectivity (determined by aperture of the pore)water-filled

Local communication: direct contact

Long distance communicationendocrine: hormones circulate throughout body, act only where receptors arenervous: neurotransmitters, neuromodulators, neurohormones

Signaling molecules bind intracellular receptorsgaseous signaling cascade via No, Co, H2S activating cGMPlipophilic ligands: signaling cascade or directly bind to DNA

Nuclear receptor:A/B: N-terminal domainC: DNA binding domainD: hinge regionE: Ligand binding domainF: C-terminal domain

Signaling molecules bind membrane receptorsReceptor-channelsReceptor-enzymesG Protein-coupled receptorsIntegrin receptors

Common 2nd messengers:cAMP, cGMP, IP3, DAG, Ca2+

Diversity of signaling through GPCRs:G-proteins triheteromeric (three subunits), off statesignal causes conformational change, GDP GTPGai: lower cAMPGas: raise cAMP

Gaq: raise IP# (Ca), DAG productionbeta-gamma: (lipid moiety keeps on membrane) couples to ion channels

Opportunistic organisms disrupt GPCR signaling for own survivalCholera:constitutively active Gas by inactivation of GTPasehyperactivation of AC, elevated cAMP, excess Cl- and H2O into the gutleads to severe diarrhea

Pertussis:inactivates Gai by trapping it in GDP-bound statefailure to inhibit AC, elevated cAMPsuppresses immune response, secretion; whooping cough

Calcium does everything:blood clotting, vesicle fusion, organelle trafficking, co-factor for enzymes, regulates cytoskeleton, muscle contraction, transcription, translation, etcCa and inorganic phosphate form structural compoundvery high [Ca] at channels; microdomains on the order of nanometers

How is the Ca microdomain achieved?Ca immediately catalyzed or diffused

Endocrine systemhormones secreted by cell or group of cellssecreted into blood and circulated (exception: pheromones secreted out of skin)transported to distant targets (though can be used as neuropeptides in other regions)exert effects at low concentration

Peptide hormones:prepackaged: generated from mRNA into proteinTranslated into ER, prohormone packaged into transport vesicle into golgi complexpackaged into secretory vesicles, released into ECF dissolved in plasma (peptides water soluble)

Steroid hormones:cholesterol derivatives (lipophilic) require carrier proteins in plasma

Amine hormones:Thyroid hormones behave like steroid hormones

Lecture 4BIPN 100

Tu 4/10/14

Interactions b/w Thalamus and Pituitary

Posterior Pituitaryhypothalamus projects axons into PPconnected to hypothalamus by infundibulum**unprotected by BBBsecretes vasopressin and oxytocin

Oxytocin:link to behavioral difficulties in autism children

ADH/Vasopressin feedback looposmolarity (high Na) detected by neurons in hypothalamussignals sent to neurons in PPvasopressin released from PP to cells in kidneyactivation of aquaporins facilitates reabsorption to conserve water

Vasopressin in Prairie vs Meadow volesprairie voles mate for life; meadow voles indiscriminant about matesprairie voles secrete vasopressin in reward centers of brain(+) feedback loop for monogamy

Anterior Pituitaryhypothalamic neurons synapse onto portal vessels to endocrine cellscapillary bed has much less blood vol; requires very few hormone molecules portal system maximizes the efficiency of the “tropin” system

Tropic Hormones secreted from hypothalamus into AP via portal system:TRHCRH…

Different hormones expressed by distinct cell populationsie, variable vesicle size and density

AP hormones:1) either activate downstream hormones at target site (TSH, ACTH, LH, FSH)2) or cause effects at site without more hormones (Prolactin, GH)

Hormones act on each other in long feedback loopsHypothalamic neurons secrete GRH into AP, which stimulates release of ACTH onto adrenal cortex, which sends cortisol throughout bodyPresence of ACTH and cortisol inhibit hypothalamic neurons (and GRH production)

Systemic hormone interactions can be superlinear.Epinephrine 5mg/100mL glucoseGlucagon 10mg/100mL glucoseE+G 22mg/100mL glucoseglucose clearance decreases with G+Esuperlinear increase in production

Hormones can have permissive effects.ie, hormones A and B both necessary for relevant effect

Hormones can have antagonistic effects.eg, insulin and glucagon oppose each other’s actions

Loss of homeostasis leads to disease.over/under-production of hormone 1, 2, or 3 can be pathologicaleg, tumor in AP can lead to overproduction of any of 6 hormonesprimary pathology involves hormone affecting tissuesecondary pathology involves hormone secreting hormonedysregulation of receptor signalingdown-regulation of the receptor

Hormone dysregulation leads to pathological symptomslow CRH, low ACTH, high cortisol Cushing’s Syndromelow CRH, low ACTH, low cortisol Addison’s Diseasehigh GH Acromegaly

Lecture 54/15/14

BIPN 100

Clicker: Higher K+ in blood neuron membrane potential more depolarized than typical.Higher reversal potential

Clicker: Action potentials…are all or none electrical signalspropagate more slowly down small diameter axonsmove more quickly via saltatory conductioncan move backwards from dendrites to somado NOT change concentration gradient in/out of cell

Anatomy of a neuronSignal detected in dendrites, processed in soma, propagated through axon/sheaths and to synapseIntegration center (cell body): many potential changes necessary for summationAPMany different types of neurons; anatomy affects function

Hodgkin and Huxley: “Membrane current in Nerve”, 19522 bilateral neurons in giant squid, hundreds of times larger than other axonsH/H hooked up electrodes to axon in order to measure ion flux

1. Ionic current carried through membrane by charging capacitance or moving through resistances2. Current divided into components (Na+, K, leak)3. Current determined by electric potential difference and conductance4. Conductance changes with time; reversal potential does not5. Depolarization influences conductance

a. Potassium conductance is delayed and prolongedb. Sodium conductance is transient

6. Changes in conductance are graded and reversed by repolarization

Ion flux does not change resting potential; maintained by Na/K ATPase

Increasing depolarization, increasing potassium conductanceFourth order equation describes rise of K conductance “potassium ions can only cross membrane when four similar particles occupy certain region of membrane”Sodium conductance also increases with depolarization—but also inactivatesinactivation particle inside p loop of sodium ion channel (1 molecule, 4 subunits)activation gate rapid; inactivation gate slowerdepolarization activates channel, Na ion moves through, depolarization; activates inactivation gate, Na flux blocked, repolarization/refractory period

H/H model equation very closely matches databut scale of time axis is 25% off; why?data recorded for experiment at 6C; different kinetics for each biological process

V-gated sodium channel inactivation gatecloses slowly relative to activation gatedoes not depend on membrane potential; depends on movement of loopdoes not depend on ion permeability; causes ion permeability

Process of propagation requires timethought expt: recording from different sites along axon simultaneouslyeach section of axon experiencing different phase of AP

Biological properties determine biophysical propertieslarge diameter axonlow resistancerapid changes in membrane potential (faster propagation)more advanced mechanism involves myelin sheathessheathe: oligo/schwann wraps membrane over and over around axonvery protective (~10x lipid layers); insulation allows for saltatory conductionWord doc analogy: simple propagation like pressing space bar; myelin is like pressing tab

Demyelination results in diminished AP conductiondegenerated sheathecurrent leak, slowed conduction

How could you get heterogeneity in AP waveform?

Midterm Review4/20/14

BIPN 100

Voltage gated K+ channelsarrangement of specific amino acids selects for K+ due to distribution of chargesa) ball and chain: physical obstruction in poreb) s6 gate: sixth helix swivels based on membrane potential; obstruction physically and electrostaticallyc) selectivity filter gate: aperture of gate prevents large molecules from entering (larger hydration shell)

Na/K ATPaseantiporter; uses ATP for energyknow entire slide (lect 2, slide 21)

SGLT transportersymporterno ATP directly; uses ATP through Na gradient

Cell Membraneinterior: (-) charges

high K+, proteinlow Na+, Cl-

exterior: (+) chargeslow K+, proteinhigh Na+, Cl-

chemical potential via kinetic interactionselectrical potential via changing charges

E Na+ = +60mVE K+ = -90mVcalculate via Nernst equation: E ion = 61/z log ([ion]out / [ion]in)

Membrane voltage (Vm) via GHK equation

G as: Ligand binds to receptor on membrane; G as breaks of from beta and gammaGDP phosphorylated by G as, forms GTPG as bound to GTP activates Adenylyl Cyclase (AC) to form cAMP (use ATP)higher [cAMP] activates PKA

G ai: Ligand binds to receptor on membrane; G ai breaks off from beta and gammaGDP binds to G aiG ai deactivates AC; lower cAMP

G aq:Ligand binds to receptor on membrane; G aq breaks off from beta and gammaG aq bound to GTP activates PLCPIP2 splits into IP3 and DAGDAG activates PKC; PKC phosphorylatescellular responsesIP3 binds to gated Ca2+ channel; influx of Ca2+

Anterior Pituitary:flat pig (F L A T P G)FSH: follicle stimulating hormone LH: leutenizing hormoneACTH: adrenocorticotropic hormoneTSH: thyrotropin PRL: prolactinGH: growth hormone

Post Pit: Vasopressin loopmagnocellular neurons synthesize vasopressin in response to high osmolarity (~280)AP: parvocellular neurons of hypothalamus release trophic hormones into portal system

MEMORIZE Hypothalamus/AP HORMONES!TRH stim TSH stim T3, T4 (thyroid gland)CRH stim ACTH stim cortisol (adrenal cortex)GHRH stim GH (musculoskeletal system)GHIH sup GH (musculoskeletal system)oxytocin stim PRL (mammary glands)dopamine sup PRL (mammary glands)GnRH stim FSH, LH (testes, ovaries)

Different hormones are made by different cell types

Squid giant axonused in Hodgkin Huxley experimentbecause large diameter = low axial resistance = fast AP propagation (and easier to study)

1. Ionic current moves through channels2. Channels pass different ions3. Current determined by electric potential differences and conductance

a. Ohms law (gives a vector!): current = voltage / resistance

b. Or current = (Membrane potential – equilibrium potential) * conductance4. Conductance changes with time, but equilibrium potential does not

a. Concentration gradient not changed by opening of ion channelsb. Neuron would have to fire 1000s of times to change concentration gradient

5. Depolarization influences conductancea. What causes movement of inactivation particle?b. During absolute refractory period, Na+ channels closed; will not activate no matter

how strong the stimulus

Excitatory synapse has asymmetric distribution of vesiclesInhibitory synapse don’t have dense section; same on either sidepresynapse typically has mitochondria, vesicles; glial cells confine signals to synapsepostsynapse has dense scaffolding proteins (receptors)

calcium enters cell, activate synaptotagmin; helices form complex, pull vesicle into cell membrane

toxins cleave SNAREs:botulinum prevents transmission on neuromuscular junction; relaxed musclestetanus shuts down inhibitory neurons; overexcited muscles

neurotransmitter clearance

1. NT returned to axon terminals for reuse, or transported into glial cells2. Enzymes can inactivate NT3. NT can leave synapse by diffusion

NMDAR requires AMPAR for removal of Mg block via depolarizationNMDAR then activates CAMKII to raise Ca

GABA A ionotropic; allows Cl- ions to flowGABA B metabotropic; coupled to GPCRs

Discussion Review4/21/14

BIPN 100

TOPICS:

Neurotransmitters: Glutamate, GABA, Acetylcholine

How is a NT broken down? (ie Ach) (lecture 6 s19)

When would Vm = E? (when cell is completely permeable)

Equilibrium potentials:E K: -90 mVE Na: +60 mVE Cl: -70 mVCa++ not included bc concentration is so low

Heterogeneity in AP waveformrising phase: number of Na+ channels falling phase: number of K+ channelsAP height: Na+ channel speed/activation (how much can get into cell before channel closes)

Vasopressin loopOsmolarity receptors in hypothalamus detect high osmolarityinhibit inhibitory neurons on magnocellular cellsactivated magnocellulars produce ADHADH released into bloodstream, activate aquaporins in collecting ductIncreased water reabsorption, water conserved, osmolarity decreased

Synaptic Vesicles1) ATPase proton pump pumps H+ into cell via primary active transport (using ATP for energy)2) antiporter performs secondary active transport using created proton gradient (using energy released by allowing H+ to travel with its concentration gradient)

Driving force = difference in membrane/equilibrium potentials (Vm – E ion)small driving force, low currentCURRENT = DRIVING FORCE x CONDUCTANCECurrent: I ion = (V m – E ion) x conductancecurrent is how quickly ions are moving through the channelsconductance is how many channels are allowing ion flow (or leading into current)

SGLT transporterlow Na+ inside cell, high glucose

concentration gradient moves Na+ onto transporter; creates glucose binding siteglucose binds; changes protein conformationNa+ and glu released into cytoplasm