INTRACELLULAR FLUID (ICF) – fluid inside the cells

EXTRACELLULAR FLUID (ECF) – occupies extracellular

Plasma and interstitial fluid = same composition
compartment
- Major difference is the absence of plasma
CELL MEMBRANE – barrier that separates these two
proteins from interstitium
THE INTRACELLULAR AND EXTRACELLULAR FLUIDS
- Plasma proteins affect solute distribution
Total Body water is the sum of ICF and ECF volumes
because of their VOLUME and ELECTRICAL
Total Body water (TBW) – ~60% of total body weight
CHARGE
(males);
Volume Occupied by Plasma Proteins
~50% total body weight
 Proteins (and lipids) occupy ~7% of total plasma
(females)
volume
~65-75% in an infant
 Plasma composition of ions is reported in units
Variability in amount of adipose tissue can influence the
of milliequivalents (meq) per liter of plasma
fraction
solution
Acute changes detected simply by monitoring the body
 Milliequivalents per liter of protein-free plasma
weight
solution – more meaningful unit for cells bathed
Assuming 70kg male has ~42L TBW:
in interstitial fluid
- 60% (25L) is intracellular
o Because only protein-free portion of
- 40% (17L) is extracellular
plasma can equilibrate across capillary
Plasma Volume
wall (PLASMA WATER)
 20% of ECF (~3L)
 Plasma water fraction is 93%
 Blood volume – total volume of intravascular
Effect of Protein Charge
component (6L)
 Plasma proteins carry net negative charge
 Plasma volume 3L
 Tend to retain cations in plasma
 Cell elements of blood 3L
 Cation concentration in protein-free solution is
o Hematocrit – Fraction of blood occupied by
lower by ~5%
these cells
 Anion concentration of protein-free solution is
Interstitial fluid
higher by ~5%
 75% of ECF
All body fluids have approximately the same
 Bathes non-blood cells of the body
osmolality, and each fluid has equal number of
 Two smaller compartments communicate only
positive and negative charges
slowly:
Osmolality
o Dense connective tissue
- All cell compartments have approximately the
o Bone matrix
same osmolality
 Capillaries – barrier that separates intravascular
- Describes the total concentration of all particles
and interstitial compartments
that are free in a solution
Transcellular fluids
o Particles bound to macromolecules do
 5% (~1L)
not contribute
 Space surrounded by epithelial cells
- Expressed as the number of osmotically active
 Includes synovial fluid and CSF
particles per kilogram of water
ICF is rich in K+, whereas ECF is rich in Na+ and Cl-
- Plasma proteins contribute: ~14mEq/L
Cell maintains relatively high K+ concentration and low
o Measured in grams per liter is very low
Na+ by using Na-K pump
o Contribute only slightly to total
- Actively extrudes Na
osmolality (~1 mOsm)
- Transports K actively into cell
- Total solute concentration of the intracellular
Transcellular fluids differ greatly in composition from
compartment higher than in interstitium
each other and from the plasma because they are
o Considerable fraction of ions is bound
secreted by different epithelia
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1
 to proteins or complexted to other
small solutes
o Proteins themselves attach to materials
that are out of solution
o Mg and Ca are either complexed or
bound
Electroneutrality
- All solutions must respect principle of BULK
NEUTRALITY
- The excess positive charge is balanced out by
intracellular macromolecules as well as organic
phosphates
- Anion gap – usually 9-14mEq/L
o Difference between ignored anions and
ignored cations
SOLUTE TRANSPORT ACROSS CELL MEMBRANES
In passive, noncoupled transport across a permeable
membrane, a solute moves down its electrochemical
gradient
 A substance can passively move across a
membrane when there is both a favorable
driving force and an open pathway
 Permeable – when a pathway exists
 Driving force determines passive transport
 Electrochemical gradient – energy difference
acting on a solute between two compartments
 Electrochemical potential energy difference
includes concentration of concentration
(chemical), and voltage(electrical)
 Noncoupled transport – movement of X not
directly coupled to the movement of any other
solute or to any chemical reaction
 Concentration gradient will act as a driving
force but voltage difference will also drive net
movement of X
 Electrochemical gradient for X is the only
driving force that contributes to transport of X
 Must always be “downhill”
 Unidirectional flux – movement of X across the
membrane in one direction
 Net flux – algebraic sum of the two
unidirectional flux (or net transport rate)
o Only occurs if unidirectional fluxes are
unequal
 Equilibrium – when no net driving force is acting
on X
o A special case of steady state wgherein
there is no net driving force thus no net
transport
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o Steady state – both rate of transport
and driving force are constant with time
 Net transport only takes place when net driving
force is displaced from equilibrium
At equilibrium, the chemical and electrical potential
energy differences across the membrane are equal but
opposite
[𝑋]𝑖
∆𝜇𝑥 = 𝑅𝑇𝑙𝑛 + 𝑧𝑥 𝐹(𝜑𝑖 − 𝜑𝑜)
[𝑋]𝑜
First term on right hand side – difference in potential
energy
- Energy change as X moves across the
membrane
Second term – difference in electrical potential energy
- Energy change as a mole of charge particle
moves across the membrane
- (𝜑𝑖 − 𝜑𝑜)- Voltage difference across the
membrane OR Vm/membrane potential
- X is at equilibrium if electrochemical potential
energy difference is zero
- ∆𝜇𝑥 - net driving force (unit: joules/mole)
- Two special cases of equilibrium:
o When eiehter chemical or electrical
term is zero the other must also be zero
o When neither chemical nor electrical is
zero, equilibrium can occur only when
the two terms are equal but of opposite
sign
𝑅𝑇 [𝑋]𝑖
𝑉𝑚 = 𝐸𝑥 = − 𝑙𝑛
𝑍𝑥 𝐹 [𝑋]𝑜
- NERNST equation – describes the conditions
when an ion is in equilibrium
- X can be in equilibrium only if voltage difference
across the membrane equals Equilibrium
potential (EX)
- EX – value that membrane voltage would have
to have for X to be in equilibrium
(Vm – EX) is the net electrochemical driving force acting
on an ion
- When net driving force is negative – cations will
enter the cell and anions will exit
o When Vm is more negative than EX (cell
is too negative for X to be in
equilibrium), a cation will tend to enter
the cell
In simple diffusion, the flux of an uncharged substance
through membrane lipid is directly proportional to its
concentration difference
2
 - Only factor that determines the direction of net
transport is the driving force
- Kinetics – the rate of transport
o Requires knowing actual mechanism
that mediates passive X transport
- Flux (JX) – how fast X moves
o Units of moles/(square
centimeterxsecond)
- Partition coefficient – how fast X can dissolve in
membrane lipid
- Diffusion coefficient – how fast X moves once it
is in the membrane
- Membrane thickness – Distance to traverse is
short
- Permeability coefficient of X (PX) – combining
above three factors
- Fick’s Law
𝐽𝑥 = 𝑃𝑥 ([𝑋]𝑜 − [𝑋]𝑖)
- Net flux – difference between unidirectional
fluxes
- Unidirectional influx is proportional to outside
concentration
- Unidirectional efflux is proportional to outside
concentration
- Next flux is proportional to concentration
difference
- Kinetic behavior of transport system cannot
violate laws of energetic or thermodynamics
- When concentration gradient for neutral
substance is zero, the flux is zero
- Law of simple diffusion: when concentration
gradient for neutral substance is zero, flux is
zero
Some substances cross the membrane passively
through intrinsic membrane proteins that can form
pores, channels, or carriers
Pathways are formed from integral proteins; three
types:
1. Pore – always opens
a. Porins in outer membrane of
mitochondria
b. Aquaporin water channels
2. Channel – alternately open and closed; equippd
with movable barrier or gate
a. Virtually all ion channels
b. Gating – process of opening and closing
of the barrier
c. Channel is a gated pore
3. Carrier – never offers continuous
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transmembrane path
a. Two gates never open at the same time
b. If two gates are both closed, transiting
particles is trapped, or OCCLUDED
Water-filled pores can allow molecules, some as large
as 45 kDa, to corss membrane passively
 Pores – provide aqueous transmembrane
conduit
 Porins – large-size pores
 Mitochondrial porin allows solutes as large as 5
kDa to diffuse passively
 One mechanism cytotoxic T lymphocytes kill
target cells  release monomers of pore-
forming protein called PERFORIN
o Perforin assemble like staves of a barrel
to form doughnut-like channels (16nm
diameter)
 Binding of antibodies (“classic” pathway) or
presence of native polysaccharides
(“alternative” pathway) triggers complement
cascade
o Doughnut-like structure with diameter
of 10nm; monomers of C9
 Nuclear pore complex (NPC) – 30 different
proteins
o Molecular mass of 108 Da; Outer
diameter of 100nm
o Involves ATP hydrolysis but also has
passive component
o Contained within NPC is simple aqueous
pore with diameter of ~9nm
 Allows molecules <45 kDa but
completely restricts proteins
>60kDa
 Aquaporin 1 (AQPI) – first water channel to be
studied
o 28 kDa protein; belong to larger family
of aquaporins (AQP)
o Exist as tetramers in lipid bilayer
 Each monomer = six membrane
spanning helices + two shorter
helices that dip into plane of
membrane
Gated channels, which alternately open and close,
allow ions to cross the membrane passively
 Gated ion channels = one or more polypeptide
subunits with a-helical spanning membrane
segments
 Functional components:
3
 o Gate – determines whether channel is Move H+ into the
open or closed; each state reflecting cells (normally);
different conformation Hv1 channel tend
o Sensors – respond to changes in to be closed
membrane voltage, second-messenger normally;
Mediates H+ extrusion
systems, or ligands (neurohomal Proton Activates when:
during states of strong
agonists) channel membrane
membrane
 Regulates transitions b/n open (Hv1 H+ depolarizes, or
depolarization, or severe
and closed states channel) cytoplasm
intracellular acidification
o Selectivity filter – determines the acidifies (when
classes of ions or particular ions that driving force
have access favors
o Open channel pore – provides a OUTWARD
continuous pathway b/n two sides movement)
 During each channel opening, many ions flow = Anion
Modestly Transepithelial
sufficient to be detected as small current channels
negative (move movement of Cl from
(Vm – EX) (Cl- or
out) lumen to blood
Channel Electrochemical Physiologic functions HCO3-)
driving force
(1) Transmission of Some carriers facilitate the passive diffusion of small
information – solutes such as glucose
responsible for action Carrier-mediated transport systems
Strongly negative
Na+ potential - Specific affinity for binding one or a small
(large INWARDLY
channel (2) Renal tubule and number of solutes
directed)
intestine – ENaC Na+; - Simplest – mediates facilitated diffusion
allow Na to enter - Cotransporters – carry two or more solutes in
epithelial cell the same direction
Fairly close to - Exchangers – move solutes in opposite
zero or Generates resting directions
somewhat membrane voltage that Solute Carrier (SLC) Superfamily – all carriers that do not
K+
POSITIVE is inside negative; help hydrolyze ATP or couple to an electron transport chain
channel
(equilibrium or terminate action - 52 SLC families contains up to 53 genes
moves OUT of potentials - Differ in:
cell) o molecular mechanism
Transmembrane o kinetic properties,
signaling for both o regulation,
Strongly negative
Ca2+ excitable and o sites of membrane targeting,
(tends to move
channel nonexcitable cells; o tissues in which they are expressed, or
INTO the cell)
generating action o developmental stage they are
potential in some cells expressed
Carrier-mediated transport behave according to a
general kinetic scheme for facilitated diffusion in a cycle
of SIX steps
- Can only mediate downhill, passive transport of
X
- Mediates only facilitated diffusion
- Fixed number of carriers is available and they
have limited speed
- If extracellular X concentration is increased,

DO NOT DISTRIBUTE © RAT 4

 influx of X will eventually reach a maximal value
once carriers have become loaded
- Simple diffusion – no maximal rate of transport
- Relationship describing carrier-mediated
transport follows MICHAELIS-MENTEN kinetics
as do enzymes
o Velocity of enzymatic reaction depends
on substrate concentration, Michaelis
constant, and maximal velocity
o The lower the Km – the higher the
AFFINITIY of the transporter for the
solute (transporters are easily filled
because they bind easily)
- Ionophores – “ion carriers”; bind to an ion on
one side, diffuse across lipid phase, and release
ion on the opposite side
o Valinomycin – K ionophore
- GLUT glucose transporters – members of SLC2
family
o 12 membrane spanning segments
o Multiple hydrophilic polypeptides loop
facing ECF or the ICF – form a
permeation pathway through lipid
bilayer
 7, 8, 11 spanning segments
 Also acts as gates and solute-
binding sites
- GLUT1 – expressed constitutively on the surface
- GLUT4 – predominantly present in membranes
of intracellular vesicles (storage pool for
transporters)
o Insulin recruits GLUT4 isoform to the
plasma membrane from storage pool
- Urea transporter (UT) – SLC14 family
- Organic cation transporter – SLC22 family
o Electrogenic – moves an electrical
charge (or carries current)
The physical structures of pores, channels, and carriers
are quite similar
 All have segments surrounding a solute
permeation pathway
 Differ kinetically
o Pore – continuously open
o Channel – undergo conformational
transitions between closed and open
states
 When open – open to both ECF
and ICF simultaneously
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o Carrier – complete cycle of
conformational changes
 Transports only small, fixed
number
 Mediates COUPLED modes of
transport
The Na-K pump, the most important primary active
transporter in animal cells, uses the energy of ATP to
extrude Na and take up K
Active transport – can transfer solute uphill
Primary Active transport – driving force comes from
favorable energy change; associated with exergonic
chemical reactions (ATP hydrolysis)
 Pumps – all energized by ATP hydrolysis hence,
are ATPases
Secondary Active transport – driving force provided by
coupling uphill movement to the downhill movement of
one or more solutes
Na-K pump – first enzyme recognized to be an ion pump
 Has α and β subunits
o α – 10 transmembrane segments;
catalytic subunit; mediates active
transport
o β – 1 transmembrane segment;
essential for proper assembly and
membrane targeting of the Na-K pump
 Couples extrusion of 3 Na for uptake of 2 K ions
with hydrolysis of 1 ATP molecule
 Transport steps of Na-K pump are energetically
uphill
o Can be reversed and forced to
synthesized ATP
o ATP releases so much energy – brings
about net active exchange for Na for K
in desired directions
 ONLY PRIMARY ACTIVE TRANSPORT FOR Na
 MOST IMPORTANT PRIMARY ACTIVE
TRANSPORT FOR K
 Restricted to basolateral side of epithelial cells
 Responsible for maintaining a low [Na]I and a
high [K]I relative to ECF
 Exists in two major conformational states:
o E1 – binding sites for the ions face
inside of cells
o E2 – binding sites face the outside
 Member of E1-E2 ATPases OR P-type ATPases
 Ordered cycling that underlies action of the
pump
5
 Pores Channels Carriers
+
Example Water channel (AQP1) Shaker K channel Glucose transporter (GLUT1)

Conduit through membrane Always open Intermittently open Never open

None (continuously Cycle of conformational
Unitary event Opening
open) change
Particles translocated per
---- 6x104 1-5
event
Particles translocated per
Up to 2x109 106-109 when open 200-50,000
second
Eight stages of Na-K pump:
Stage 1: ATP-bound E1 * ATP state – just after the
pump has released its bound K+ to the ICF; Na+
binding sites have high affinities for Na+

Stage 5: Empty E2-P state – 3 bound Na ions dissociate

 protein undergoes MINOR change to empty
E2-P form; HIGH affinity binding for
extracellular K
Stage 2: Na-bound E1 * ATP * 3Na state – Three Na
ions bind

Stage 6: K-bound E2-P * 2K state – two K ions bind

Stage 3: Occluded E1-P * (3NA) state – ATP previously

bound phosphorylates the pump at an
aspartate redisue  triggers MINOR change in
E1 form  now occludes 3 bound Na ions 
INACCESIBLE TO BOTH ICF and ECF; ADP leaves
Stage 7: Occluded E2 * 2K state – hydrolysis of
acylphos-phate bond releases inorganic
phosphate  minor change happens  pump
occludes the two bound K ions

Stage 4: Deoccluded E2-P * 3Na state – MAJOR change

shifts pump from E1 to E2 conformation; has
two effects:
o Pump now communicates to ECF
Stage 8: Deoccluded E1*ATP*2K state – Binding of ATP
o Affinity for Na decreases causes MAJOR change  shifts pump from E2
back to E1. Change has two effects:
o Pump now communicates with ICF
o Affinity for K decreases

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 Coppe
r Mutated in
pump Wilson’s
P1B
(ATP7 disease
B)
Stage 1: ATP-bound E1*ATP state – returns the pump to *Plasma-membrane Ca ATPase
its original state ** SERCAS – Sarcoplasmic and endoplasmic reticulum
calcium ATPases
 Stoichiometry of the pump – 3 Na to 2 K ***Along with Na-K pump, only member of P2C
 Each cycle – next extrusion of one positive subfamily with B subunit
charge from the cell The F-type and V-type ATPases transport H+
o ELECTROGENIC F-type or FoF1 ATPases
 Rate of active transport – saturable function  Of the inner membrane mitochondria
o Number of pumps is finite  Final step in ATP synthesis pathway
o Also saturable function of ATP  Like a lollipop held in your hand
 Depends on metabolic rate of o Hand-like: Fo portion embedded in the
cell membrane
 HALLMARK of Na-K pump – blocked by cardiac  Pathway for H transport
glycosides (Ouabain and digoxin)  Three subunits (a, b, c)
o Compounds have high affinity for E2-P  ab2c10-12
state which also have high affinity for o F1: points into mitochondrial matrix
extracellular K  “Stick” – γ subunit with
 Binding of K competitively attached ε subunit
antagonized  “Candy” – has ATPase activity
o Hypokalemia potentiates digitalis  Three alternating pairs
toxicity in patients of a and Bs ubunits
Besides the Na-K pump, other P-type ATPases include  δ subunit
H-K and Ca pump  α3β3γδε
Family of P-type ATPases share significant similarity o Molecular mass: 500 kDa
with alpha subunit  The ‘hand’ acts as a turbine that rotates in the
Structu plane of the membrane driven by H ions that
Localizati Stoichiom
Pump re/ Regulation flow through the turbine
on etry
Family  Stick is an axle (Y and E subunits of F1) rotates
Parietal with turbine
cells of Phosphoryl  Stationary chemical factory is energized by
Extrusion A and
gastric ation rotating axle
of H, ***B
H-K
gland;
uptake of subunit
through E1 o One ATP synthesized for 120o turn of
Kidney and E2 turbine
K (2:2); s;
and intermediat  The Fo a and b subunit and δ – stator that holds
1 ATP P2C
intestine es candy in place
s Under physiologic conditions – F-type ATPases runs as
Most an ATP synthase
cells;
1 H: 1 Ca:  Citric acid cycle captures energy as electrons
sarcoplas and transfer these to NAD and FAD
Ca 1 ATP P2A
(*PM
mic
**SERCAS subfam ==  NADH and FADH2 transfers electrons to ETC
CA)
reticulu
- 2 H: ily  As electrons are transferred, electrons gradually
m in lose energy
2Ca: 1ATP
muscle o Finally combine with 2H and ½ O2 to
cells form H2O
 Complex I, III, IV pump H across the inner

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 membrane glycoprotein) cationic DE-
o Established large out-to-in H gradient metabolite BINDING
across the membrane s and DOMAIN
 Complex V (FoF1 ATPase) use this large drugs; (NBD)
electrochemical potential energy Pumps binds ATP
o H ions flow backward through FoF1 anticancer
ATPase drugs out
 Generates ATP in matrix space of cell
from ADP and Pi 170 kDa
o Chemisomotic hypothesis
o One ATP for every three H+ 2
 Will function as ATPase in reverse membrane
V-type H pump -spanning
 Membranes surrounding organelles contain domain
vacuolar-type (or V-type) H-ATPase (MSD1 and
o Pumps H from cytoplasm to the interior MSD2)
MRP/CFTR
of the organelle composed
Subfamily
 Important for sorting proteins, dissociating Apical of 6
(cystic Low
ligands, optimizing activity of acid hydrolases, membrane membrane
fibrosis conductanc
accumulating neurotransmitters in vesicles of spanning
transmembra e Cl
 Apical membranes of renal tubule cells also epithelial segments;
ne channel
have V-type H pumps cells 2 NBDs
conductance
o Independent of K
regulator)
o Similar to hand-held lollipop Cytoplasmi
 V-type pump has only six subunits but each c
twice as large as a c subunit in F-type ATPase regulatory
ATP-binding cassette transporters can act as pumps, domain (R)
channels, or regulators separates
Subfamilies named ABCA through ABCG two halves
Some are pumps that hydrolyze ATP to provide for of CFTR
solute transport, but some do not couple energy to
perform active transport CFTR
Location Function Structure - R domain contains multiple PKA and PKC
Mediates phosphorylation sites
efflux of - Neurohumoral agents phosphorylate sites 
ABCA Macrophag
phospholipi -- promotes activation of CFTR
Subfamily es
ds and - Binding of ATP to NBDs also controls opening
cholesterol and closing of the channel
Multidrug- ATPases 6 - Two regulatory mechanisms: Protein
resistance and 1o membrane phosphorylation and interaction with NBDs
Liver,
transporters active -spanning
kidney, GIT
(MDRs) transporter and
MDR1 (p- Extrudes NUCLEOTI
Cotransporters, one class of secondary active - Two major classes:
transporters, are generally driven by energy of the o Cotransporters or symporters – move
inwardly directed Na gradient the driving solute and driven solute in
2o transporters can move a solute uphill the same direction
- Fuel it by coupling to downhill movement o Exchangers or antiporters

Location Family Structure Stoichiometry Driving force Function

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 Inwardly
directed Na
can drive
uphill
accumulation
Useful for
Apical Single subunit 1 Na: 1 of glucose into
DM type 2:
Na- membrane of with 14 Glucose the cell
Reduce
GlucoseCotransporter proximal tubule SLC5 membrane- SGLT1 isoform:
glucose
(SGLT) and small spanning 2 Na: 1 10:1 Na con’cn
reabsorption
intestine segments Glucose gradient = 10-
in kidney
fold glucose
gradient
-60mV=
another 10-
fold
Na+-Driven
Cotransporters for
Organic Solutes
Na-driven amino-acid SLC 6
Proximal tubule --- ---- ---- ----
transporters SLC 38
and small
Na-coupled
intestines
cotransporters for
monocarboxylates, SLC5 ---- ---- ---- ----
dicarboxylates, and
tricarboxylates
Electrogenic
HCO3 efflux Acid-base
NBCS
(into the transporters;
1:3 (Na:HCO3)
Basolateral blood) regulation of
Na/HCO3
membrane of intracellular
Cotransporters SLC4 1:2
certain HCO3 influx pH, for
(NBCs)
epithelial cells epithelial
Electroneutral
HCO3-
NBCs
HCO3 influx secretions
1:1
Na=-Driven Cotransporter of Other Inorganic Anions
Na/I cotransporter SLC5A5
Sulfate cotransporter SLC13
SLC17,
Inorganic phosphate
SLC20,
cotransporter (NaPi)
SLC34

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 NKCC1:
nonepithelial Inhibited by
cells, furosemide
basolateral and
Na/K/CL membrane of Inwardly umetanide
cotransporter (NKCC) some epithelial directed Na to (loop
Or Bumetanide- cells SLC12 drive diuretics) –
sensitive accumulation increase
cotransporter (BSC) NKCC2: apical of Cl and K urine flow by
membranes of inhibiting
cells in thick transport at
ascending loop LoH
of Henle
Na/Cl Cotransporter Apical
Blocked by
(NCC) or Thiazide- membrane of
SLC12A3 K independent thiazide
sensitive dital tubule of
diuretics
cotransporter kidney
H-driven Cotransporters Driven by downhill inward movement of H
Uptake of
Renal proximal
H/Oligopeptide small
tubule and SLC15
cotransporter (PepT1) peptides;
small intestine
Electrogenic
H-driven amino-acid
SLC36
cotransporters (PAT1)
Moves
Either net
lactate out of
inward or net
cells that
Monocarboxylate outward
produce
cotransporters direction
lactate and
(MCT1) depending on
into cells that
lactate and H
consume
gradient
lactate
Couples influx
Kidney and of H to influx
Divalent metal-ion proximal of Ferrous iron
SLC11
cotransporter (DMT1) portions of or other
small intestines divalent
metals
Exchangers, and another class of secondary active transporters, exchange ions for one another
Transporters exchange cations for cations and anions for anions
Location Family Stoichiometry Driving Force Function
Helps maintain
Inwardly directed steep, inwardly
3 Na: 1 Ca
Na-Ca Exchanger Na drives uphill directed
Ubiquitous SLC8
(NCX) extrusion of Ca electrochemical
Electrogenic
from cell potential energy
difference for Ca

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 pH regulation and
Almost every cell
Inwardly directed cell volume
Na-H Exchanger Na drives uphill
NHE3: apical SLC9 1:1
(NHE) extrusion os H from NHE3: acid
membranes of
cell; raises pH secretion and Na
several epithelia
absorption
Multidrug and Renal proximal Uptake of H in
toxin extrusion tubule cells, SLC47 exchange for
transporter (MATE) hepatocytes organic cations
Inwardly directed pH regulation
1 Na: 2 HCO3 (in
Na-driven Cl-HCO3 Na to drive uphill (keeps it alkaline);
SLC4 one direction): 1 Cl
Exchanger (NDCBE) entry of HCO3 into also Na
(opposite direction)
cell reabsorption
AE1: transport
All cells express
HCO3 into RBC in
one of the three
the lung and out of
electroneutral SLC Function
lung in peripheral
4 CL-HCO3 independently of
tissues
exchangers – anion Na
SLC4 or
Cl-HCO3 exchanger exchangers (AE1 to
SLC26 AE2 and AE3
AE3) Inwardly directed
(acidifies cells)
Cl drives HCO3 out
SLC26 family: can of cell
Uptake of Cl =
be electrogenic
regulation of cell
(not 1:1)
volume
Other Anion Exchangers
Cl-formate Secondary active
Renal proximal
exchange and Cl- SLC26A6 uptake of Cl and
tubule
oxalate exchange secretion of oxalate
Transport I (for
Pendrin
thyroid gland)
Organifc anion- Mediate uptake of
transporting bile acids, bilirubin,
SLC21
polypeptides and test substrate
(OATPs) bromosulphthalein
Prostaglandin Mediates uptake of
SLC21
transporter (PGT) prostanoids
Mediate uptake of
endogenous
organic anions,
Organic anion
SLC22 drugs, penicillin,
transporters (OATs)
and p-
aminohippurate
(test substrate)
Renal proximal Mediates urate
URAT1 SLC22
tubule transport

REGULATION OF INTRACELLULAR ION high

CONCENTRATIONS - Sodium – 145 mM
The Na-K pump keeps [Na+] inside the cell low and [K+] o predominant cation in ECF

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 o Maintained by Na-K pump
- Potassium – 4.5 mM
o Predominant cation in ICF
- Oubain – inhibits Na-K pump
- -60 mV – inside-negative membrane voltage
generated by Na-K pump
- Electrogenic pump – causes a net outward
current of a positive charge
o Generates an inside negative Vm
o Small contribution to negative Vm
- Active K accumulation favors the exit of K
through K channels
o Tendency of K to exit (with unmatched
negative charges left behind) is the
MAIN cause of inside-negative
membrane voltage
- Ba2+ - inhibitor of K channels
o Vm becomes less negative (cell
depolarizes)
- Principal pathway for current flow is through K
channels
- Inside-negative Vm + large concentration
gradient for Na = strongly favors passive entry
of Na
- Cell harness the energy of Na entry for three
major purposes:
o Amiloride-sensitive Na channels (ENaCs)
– restricted to apical or luminal surface;
Na-K pump restricted to basolateral
 Transepithelial Na transport
takes place
o Excitable cells – passive Na entry
occurs; role in generation of action
potential
 Na is cycled at high energy cost
for information transfer
o Every cell – to drive the secondary
active transport of nutrient and ions
The Ca pump and the Na-Ca exchanger keep
intracellular [Ca2+] four orders of magnitude lower
than extracellular [Ca2+]
- Calcium - ~1mM in ECF but 100 nM in ICF
- Negative membrane voltage and large chemical
gradient – inwardly directed electrochemical
gradient for Calcium FAR LARGER THAN ANY
OTHER ION
- Calcium channels gated by voltage or by
humoral agents
o Rapid entry occurs only in short bursts
- What transport mechanism keep Calcium low?
Ca Pump (SERCA) in Organelle Membranes
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- Sarcoplasmic reticulum and endoplasmic
reticulum – actively sequester cytosolic Ca in
intracellular stores
- Stores of Ca released in bursts as part of signal-
transduction process
- There is a limit to how Ca a cell can store;
extrusion must balance passive influx of
Calcium
Ca pump (PMCA) on the plasma membrane
- Most cells contain Ca pump – major role in
extruding Calcium
- Rising levels of intracellular Ca stimulate Ca
pump to extrude Ca
- Pump itself is incapable of feedback control –
has a high Km for Clacium (virtually inactive at
physiological Ca)
o High levels of Ca  bind to calmodulin
o Ca-CaM binds to Ca pump  lowers Km
 stimulates Ca extrusion
o Low levels of Ca  Ca-Cam dissociates
from Calcium pump
- At levels of ~100nM, Ca pump is major route of
Ca extrusion
Na-Ca Exchanger (NCX) on the Plasma Membrane
- Extrudes Clacium only when [Ca]I rises
substantially above normal levels
- Restores low Ca when large influxes of Ca occur
- Most notable in excitable cells such as neurons
and cardiac muscle
In most cells, [Cl-] is modestly above equilibrium
because Cl uptake by Cl-HCO3 exchanger and Na/K/Cl
cotransporter balances passive Cl efflux through
channels
- Cl ICF< Cl ECF
- All cells have ANION-SELECTIVE CHANNELS – Cl
can permeate passively
- Cl-HCO3 exchanger – Most common pathway
for Chloride uptake
o Outwardly directed energy for HCO3 –
driving force for entry of Cl
- Na/K/Cl cotransporter – stimulated by low Cl;
uphill
- Passive Cl efflux through anion-selective
channels opposes Cl uptake mechanisms
- K/Cl cotransporter – another factor; driven by
outward K gradient; moves Cl out of cell too
o K gradient promotes Cl EFFLUX by
generating negative Vm and by driving
K/Cl cotransport
The Na-H exchanger and Na-driven HCO3 transporters
keep the intracellular pH and HCO3 above their
12
equilibrium values
Extracellular pH ~7.4
- HCO3 ~24mm
- PCO2 ~40mm Hg
Intracellular pH ~7.2
- pHi – actually much more alkaline if H and
HCO3 were passively distributed across the cell
membrane
- H can enter and HCO3 can exit passively
o Both occur at low rate
o H – cation channels and H selective
channels
o HCO3 – through most Cl channels
- -60mV – 10-fold concentration gradient for H
o 10-fold higher within cell – corresponds
to pH that is 1 pHi more acidic than pHo
- Acid extruders – transport acid out of cell;
functions in acid extrusion
o Secondary active transporters
energized by Na gradient
 Na-H, Na-driven Cl-HCO3 ex-
changers, Na/HCO3
cotransporters – most
important
 Na/HCO3 – 1:1 or 1:2
o Sensitive to changes in pH – stimulated
when cell is acidified and inhibited
when cell is alkalinized
o V-type, H pumps or H-k pumps on
collecting duct and the stomach
- Because of acid extruders pHi is not far more
alkaline than 7.2
o Acid loaders balance acid extrusion
- Passive leakage of H and HCO3 through
channels tend to acidify cells
- Transporters move HCO3 out of cells
o Cl-HCO3 exchanger – most common
o Electrogenic Na/HCO3 cotransporter
(1:3) – moves HCO3 across renal
proximal tubules
WATER TRANSPORT AND THE REGULATION OF CELL
VOLUME
Water transport is driven by osmotic and hydrostatic
pressure differences across membranes
- H2O molecules can dissolve in lipid bilayers at
low but finite rate by simple diffusion
o Ease of diffusion depends on lipid
composition
o Low fluidity membrane (PLUS
cholesterol) = further reduce H2O
permeability
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- AQPs – increase membrane H2O permeability
- Erythrocytes and renal proximal tubule – AQP1
always present
- Collecting duct regulate H2O permeability by
inserting AQP2 channels under control of
arginine vasopressin
- Nonsaturable function
- Two passive driving force:
o Chemical potential energy difference –
difference in water concentration
o Energy difference per mole of H2O –
results from difference in hydrostatic
pressure
- At equilibrium osmotic pressure difference is
equal to hydrostatic pressure difference
- Animal cells cannot tolerate any significant
hydrostatic pressure difference w/o derofrming
o Thus it is always near zero = no
significant driving force
- Movement of H2O out of cells driven by
osmotic gradients only
- Osmosis – movement of H2O driven by osmotic
gradient
- Hydrostatic pressure differences – important for
driving fluid out of across the walls of capillaries
- Presence of greater concentration of proteins
sets up difference in osmotic pressure that pull
fluid back into capillary
o Called COLLOID OSMOTIC PRESSURE or
ONCOTIC PRESSURE
- Hydrostatic pressure = Oncotic pressure 
equilibrium
- Hydrostatic > oncotic = ULTRAFILTRATION
(movement ouf ot capillary)
Because of the present of impermeant charge proteins
within the cell, Donnan forces will lead to cell swelling
- Relative exclusion of NaCl from intracellular
space is vital for maintaining H2O content
- No Na-K pumps = cells swell
- Gibbs-Donnan equilibrium
[𝑁𝑎]𝑜 [𝐶𝑙]𝑖
= =𝑟
[𝑁𝑎]𝑖 [𝐶𝑙]𝑜
- r = donnan ratio
- Total number of positive charges must balance
total number of negative charges (bulk
electroneutrality)
The Na-K pump maintains cell volume by doing
osmotic work that counteracts the passive Donnan
forces
- Distribution of ions toward Donnan equilibrium
 progressive water entry, swelling, then
13
 bursting
o Negative charge on impermeant
intracellular solutes will lead to bursting
- Largely excluding NaCl to counter act – make
NaCl functionally impermeable
o Offsets osmotic effects of intracellular
negative charges
o Steady state maintained by active
transport
- Inside Na, K, Cl are constant because:
o Active extrusion of 3 N a for 2 K
o Net flux of Cl is zero
- If Na-K pump inhibited = depolarization
o Passive entry of Na ions exceed efflux of
K major role in raising intracellular osmolality; Particularly
o Continuous outward K
o Inside-negative Vm driving force for
excluding Cl
 Cl now enters
o Increase in osmotically active particles
o Leads to cell swelling
o Pathways dependent on Na-K pump are
de-energized
Cell volume changes trigger rapid changes in ion
channels or transporters, returning volume toward
normal
Response to Cell Shrinkage
- Addition of mannitol – solution becomes
Hyperosmolal
o Draws H2O out of cells
- Many types of cells respond by activating solute
uptake process known as Regulatory Volume
Increase (RVI)
- Shrinkage activates ubiquitous NHE1 isoform –
Na-H exchanger
o Increased uptake of Na, extrusion of H
 alkalinize cell  activates Cl-HCO3
 net effect is entry of NaCl  Na-K
pump extrude Na  FINAL: KCl net gain
- Resulting increase in intracellular osmoles
draws water into the cell
- RVI response may be mediated by NKCC1
isoform and the Na/K/Cl cotransporter
Response to Swelling
- Hypo-osmolal – extracellular osmolality is
decreased
o H2O moves in cells
- Many cells respond by activating efflux
pathways known as Regulatory Volume
Decrease (RVD)
o Activates Cl or K channels (or both) –
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generally directed outward
o Net efflux of K and Cl – lowers
intracellular solute
o Initiated by activating K/Cl
cotransporter
Cells respond to long-term hyperosmolality by
accumulating new intracellular organic solutes
Acute response – uptake of salts
Long term adaptation – involves accumulation of
organic solutes (Osmolytes) within the cell; include:
1. 2 Impermeant alcohol derivatives of common
sugars (Sorbitol and inositol)
2. 2 amines (betaine and taurine)
Generation of organic solutes (idiogenic osmoles) –
true in brain cells
Sorbitol  Glucose catalyzed by ALDOSE REDUCTASE
Cells can also transport them into cytosol from outside
- Use distinct Na-coupled cotransport systems
The gradient in tonicity-or effective osmolality-
determines the osmotic flow of water across a cell
membrane
TBW – distributed in interstitial, intracellular,
transcellular fluids, and blood plasma
Water Exchange Across Cell Membranes
- Increasing hydrostatic pressure in interstitial
space will cause cell to compress – intracellular
hydrostatic increases similarly
o H2O does not enter
- Increase ECF osmolality – cause H2O to move
out of cell
- If cell does not have RVI, cell volume will be
reduced indefinitely
- Urea penetrates slowly than H2O does
o Initial effect is to shrink cell
o As it gradually equilibrates across cell
membrane cell reswells to its initial
volume
- Effective osmolality – also known as tonicity
o Tonicity – includes both Na and glucose
but not BUN
- Isosmolal – 290 mOsm
- Hyperosmolal - >290 mOsm
- Hypo-somolal - <290 mOsm
- Hypertonic, isotonic, hypotonic – comparing one
solution with another solution across well-
defined membrane
o Isotonic – effective osmolality is same
with reference solution
o Hypertonic – higher effective osmolality
o Hypotonic – solution has lower effective
14
 osmolality
- Shifts of H2O result from alterations in effective
ECF osmolality or tonicity
o Changes in tonicity usually caused by
decreases in Na in ECF and plasma,
increase in Na, or increase in glucose
Water Exchange Across the Capillary Wall
- Freely permeable to solutes smaller than
plasma proteins
- Only net osmotic force that acts by asymmetric
distribution of proteins in plasma VS interstitial
fluid
- Protein osmotic pressure, colloid osmotic
pressure, oncotic pressure
- Oncotic pressure difference – tends to pull H2O
from the interstitium
- Net movements of H2O accompanied by small
solutes
o Do not contribute significantly to
osmotic driving force
Adding isotonic saline, pure water, or pure NaCl to the
ECF will increase ECF volume but will have divergent
effects on ICF volume and ECF osmolality
Adding solute-free water or NaCl will alter volume and
composition of fluid compartments
Infusion of Isotonic Saline
- NO CHANGE OCCURS
- Efficient way to expand ECF
Infusion of “Solute-Free” Water
- Infusing glucose is equivalent to infusing pure
water because it is metabolized to CO2 and H2O
- Infusing pure H2O  cause cells near the point
of infusion to burst (UNWISE)
- Lowers ECF osmolality
- Relatively Ineffective means of expanding ECF
o More of added water ended up
intracellularly
- Dilutes osmolality of body fluids
Ingestion of Pure NaCl Salt
- Adding or removing Na will mainly affect ECF
volume
- Adding or removing solute-free H2O affects
osmolality of the body
- Increase osmolality of ECF  draws H2O out of
ICF
- There is increase in water volume in the ECF
even though no water was added to the body
- Total-body content of Na is the major
determinant of ECF volume
Whole body Na content determines ECF volume,
whereas whole-body content determines osmolality
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Effective circulating volume – blood volume necessary
to achieve adequate perfusion
Extracellular Na = total-body content
Increases in ECF volume OR total-body Na content 
renal excretion of Na
- Plasma Na concentration does not regulate
renal excretion of Na
Holding Na constant (major part of total body osmoles)
- Increase in TWB would decrease osmolality
o Net gain or loss of solute-free H2O has
major impact on osmolality and Na of
the ECF
o A gain or loss of solute-free H2O affects
ICF more than ECF
Small decrease in osmolality
- Increase renal water excretion
- Diminished thirst
Emergency states of very low ECF
- Try to conserve Na
- Seek water by triggering thirst
- Conserve water by concentrating urine
TRANSPORT OF SOLUTES AND WATER ACROSS
EPITHELIA
Epithelia – barriers that separate ECF from outside
world
- Uninterrupted sheet of cells joined by junctional
complexes
- Serve as selectively permeable barrier
- Demarcate boundary between apical and
basolateral regions
- Capable of VECTORIAL TRANSPORT
Apical membrane – brush border, mucosal membrane,
or luminal membrane
Basolateral membrane – serosal or peritubular
membrane
The epithelial cell generally has different
electrochemical gradients across its apical and
basolateral membrane
- Transepithelial differences occur – composition
of “outside world” not the same as ECF
o Transepithelial voltage usually not zero
- Electrophysiological methods provide 2 major
types of information:
o Define electrical driving forces
o Electrical measurements define
electrical resistance of epithelium
- Transepithelial voltage – voltage different b/n
solutions on either side of epithelium
- Basolateral cell membrane voltage (Vbl) –
microelectrode into epithelial cell and reference
electrode to blood/interstitial space
15
 - Apical cell membrane voltage (Va) – compare
intracellular electrode with reference electrode
in the lumen
- Va + Vbl = Transepithelial voltage
- From Ohm’s law – possible to calculate the
electrical resistance of entire wall or
transepithelial resistance (Rte)
Tight and Leaky epithelia differ in permeability of their
tight junctions
- Resistance to flow of electrical current – one
measure of how tightly an epithelium separates
o Low electrical resistance – low-
resistance pathway in tight junctions
(leaky)
 More permeant
o Tight – high electrical resistance
- Transcellular pathway – substance crosses
through cell by first passing across apical then
basolateral membranes
- Paracellular pathway – substance passes
through tight junctions and lateral intercellular
spaces
- Leaky epithelia – perform bulk transepithelial
transport; not good with maintaining gradients
o In proximal tubules and small intestine
- Tight epithelia – maintain transepithelial ion
concentration or osmotic gradients
o Distal nephron, large intestines, and
urinary bladder (tightest)
- Na-K pump is located exclusively at basolateral
membrane EXCEPT in CHOROID PLEXUS
- Most of the K recycles back out through K
channel
o K gradient predominantly determines
Vbl
o Usually 50-60 mV inside negative
- Na is lower in epithelial cell than in ECF
o Active extrusion through Na-K pump
o Serves as driving force for Na entry
through apical Na channels and for
secondary active transport of other
solutes
Epithelial cells can absorb or secrete different solutes
by inserting specific channels or transporters at either
the apical or basolateral membrane
Placing different transporters  accomplish net
transepithelial transport of different solutes (absorptive
or secretory direction)
Na absorption
- Basolateral Na-K pump pumps Na out 
generates inward Na electrochemical gradient
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across apical membrane  provides driving
force for Na to enter through ENac Na channels
in apical membrane
- Na-K pump generates current of positive charge
from lumen to interstitium (b/c current is more
positive in interstitium)
o Creates lumen-negative transepithelial
voltage
 Driving force for passive Cl
absorption through paracellular
pathway
K secretion
- K channels at apical membrane allows K from
Na-K pump to be secreted across apical
membrane
o Basis of K secretion
- Amiloride – blocks apical ENaC Na channels 
inhibits K secretion and Na reabsorption
Glucose Absorption
- Absorbed by secondary active cotransport of Na
with organic solutes in SI and proximal tubule
o SGLT for example
- Inward Na gradient drives entry of both Na and
Glucose  accumulates inside cell  passively
exits through carrier mediated transporter
(GLUT)
- Can also drive Cl absorption due to lumen-
negative transepithelial voltage generated
Cl secretion
- Inwardly directed Na drives secondary active Cl
uptake by Na/L/Cl cotransporter NKCC1
- Cl accumulated can exit through CFTR
- Negative charges move across the cell from
interstitium to lumen  lumen-negative
voltage  drives passive Na secretion across
tight junctions
Water transport across epithelia passively follows
solute transport
H2O moves passively in response to osmotic gradients
Finite permeability of bare lipid bilayer and presence of
AQPS ensure rapid equilibriation
Tight junctions provide pathway for H2O
Hydraulic conductivity – epithelial H2O permeability
- Varies widely b/c of:
o Differences in membrane lipid
composition
o Abundance of AQPs (presence either
constitutive or regulated)
Absorption of Hyperosmotic fluid
- Absorbate is hyperosmotic if epithelium
absorbs more salt than water
16
 - In ascending limb of loop of Henle - Passive movement of solutes across tight
o Reabsorbs salt but little water junction can contribute to ‘forward’ or backleak
o Dilute fluid is left behind and renal movement of solute
interstitium is hyperosmotic - Modulate transport by changing permeability of
Absorption of an Isosmotic Fluid pathway
- In renal proximal tubule and small intestine – - Na permeability in proximal tubule increases
no detectable osmotic gradients when ECF volume increases
- Permeability of epithelia for water might be o Lower Na reabsorption b/c of increased
extremely high due to high expression of AQPs backleak of absorbed Na
- Lateral intercellular space – modestly Luminal Supply of Transported Species and Flow Rate
hyperosmotic d/t accumulation of absorbed - Concentration of transported solutes effects
solutes rate of solute transport
o Localized osmotic gradient pull water - Renal proximal tubule – process of glucose
into lateral interspaces absorption depletes glucose  slow further
Absorption of Hypo-osmotic fluid absorption
- Active transport of water does not occur o Increase glucose conc’n on site of
- Water cannot move against an osmotic gradeitn glucose uptake increases rate of glucose
- Occurs but requires osmolality of basolateral absorption
membrane exceed apical compartment - Flow itself (sensed by bending central cilium)
- Medullary collecting duct uses this approach trigger signaling cascades resulting in alteration
o Interstitial fluid in renal medulla is of transporter and channel function
hyperosmotic
o H2O permeability is high d/t AQP2
Epithelia can regulate transport by controlling
transport proteins, tight junctions, and the supply of
all transported substance
Increased Synthesis (or Degradation) of Transport
Proteins
- Change number of transport molecules in the
cell
- Aldosterone directly or indirectly increases
transcription rate of genes that encode Na-K
pump
Recruitment of Transport Proteins to the Cell
Membrane
- Store transporters in intracellular organelle
“pool” then inserting them
- Histamine causes “tubulovescles” with H-K
pumps to fuse with apical membrane of gastric
parietal cell
o Initiates gastric secretion
Post-translational Modification of Pre-Existing
Transport Proteins
- cAMP enhance phosphorylation of apical
membrane Cl channel
o CFTR – Cl channel regulated by
phosphorylation
o Defect in regulation of CFTR – primary
physiological abnormality in cystic
fibrosis
Changes in Paracellular Pathway

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