1Kidney, Co-transporters Kidney anatomy

Role of Kidneys: filtration of blood & reabsorption of salts & water

Co-transporters:

Structure & function

How do co-transporters work?

Outline:

Kidneys filter the blood, regulate water, electrolyte & pH balance.Waste products that are not reabsorbed into the blood are excreted in the urine.

Drugs that alter renal function, the diuretics, are important for managing cardiovascular disease

Diuretics act on kidney to increase excretion of salts and H2O, thus altering blood volume, and indirectly blood pressure

Used in treatment of: Hypertension Cardiac insufficiency (heart failure) Pulmonary edema Renal failure

2Nephrons are organized in the kidney with the glomerulus in the cortex and the loop of Henle in the medulla

Nephrons are the structural & functional units within the kidney.

~1 million nephrons in each kidney

25 moles!

180 liters = 50 gal !

How much fluid & solute is filtered each day by the kidneys?

Glomerulus:An ultrafiltrate of the blood is created at the start of the nephron.

What enters the renal tubule:salts?water?glucose?protein? Glomerulus

Bowmans Capsule

Proximal Tubule

Incoming blood The ultrafiltrate of the

blood passes through many segments of the nephron as the salts, water, amino acids, & glucose are progressively reabsorbed across the tubule into the blood.

3Nephron with its associated vascular system

A network of blood vessels surrounds the nephrons, & takes up the water and solutes that are reabsorbed across the renal tubule.

Apical Membrane

Basolateral Membrane

( )

Tight Junction

Renal epithelial cells have distinct apical and basolateral membranes. Basolateral membrane contains Na/K ATPase. Utilizes ATP to transport Na+ out of renal epithelial cell to be reabsorbed by blood & transports K+ into cell.

Apical membrane contains cotransporters. Utilize ion gradient (usually Na+) to cotransport solutes out of tubular lumen into epithelial cell.

Ion Channels: Pore Dissipative of gradient Fast transport, near

diffusion limit

Cotransporters:

Secondary Active Transport

Use gradient for one substance to transport another substance

Conformational change Cotransport (symport) or

exchange (antiport) Slow transport

Cotransporters >100 families of secondary active transporters, >40

families in humans Can concentrate solutes across membrane by factor

of 106

Transport ions, neurotransmitters, peptides, sugars, nutrients

Use gradient of one substance (usually Na+ or H+) to cotransport another substance

Examples: In prokaryotes involved in uptake of nutrients Nutrient update in intestine Transport of Na+ and Cl- in kidney (target of diuretics) Uptake of neurotransmitters (target for SSRI

antidepressants (selective serotonin reuptake inhibitors), cocaine, amphetamines)

Uptake of Iodide into thyroid

4Science. 2003 Aug 1; 301(5633): 610-5. & 616-620

AcrB: H+/multidrug resistance transporter

Major Facilitator family of cotransporters: LacY: H+/lactose cotransporter GlpT: Glycerol-6-phosphate/Phosphate

antiporter

Na+-Coupled cotransporters: GltPh: Na+/aspartate cotransporter NhaA: Na+/H+ antiporter LeuT: Na+/Leucine cotransporter (of the

neurotransporter family) sGLT: Na+/sugar cotransporter BetP: Na+/amino acid cotransporter Mhp1: cation/nucleobase cotransporter

X-ray Crystal Structures Solved for Bacterial Cotransporters

Lactose Permease: a H+/Lactose cotransporter:

Typically 10-14 transmembrane segments

Prolines and glycines in TM regionswhat are their function?

Symmetric structure with 2 groups of helicies

Side View Top ViewLactose Permease (LacY)

Wide hydrophilic entrance for substrate

substrate

Core 5 + Architecture of Cotransporters

5

Common theme in cotransporter structure, even though transporters have little amino acid sequence identity and different # of TM segments

Core structure of 10 TM segments TM1-5 related to TM6-10 Odd # of TM segments so

repeated structure is inverted in membrane

vSGLT: Na+/galactose cotransporter

LeuT: bacterial Na+/leucine cotransporterAbrahamson (2009), Krishnamurthy (2009)

5Na/Galactose cotransportervSGLT (14 TM segments)

TM 1-5 TM6-10 TM1-5 (blue) + inverted TM6-10To show structural homology

Inverted 5 + Structural Repeat

5

Abrahamson (2009)

Model of Structural Changes between Inward- and Outward-facing ConformationsDuring Transport

Lactose Permase Glycerol-3-Phosphate Transporter

Alternating Access Model of Transport Alternating Access Mechanism: Crystal Structure of Transport Intermediates

of LeuT, a Na+/Leucine cotransporter

Krishnamurthy (2012)

Internal aqueous cavity

Blue = aqueous cavities in cotransporter protein

External aqueouscavity

6Apical Membrane

Basolateral Membrane

( )

Tight Junction

Proximal Tubule

60-70% of Na+ absorbed 60-70% of H2O absorbed leaky epithelium, permeable to H2O Most diuretics act later in tubule Osmotic diuretics (mannitol):

Are filtered in the glomerulus but cannot be reabsorbed in nephron

Reduce the passive reabsorption of H2O

Site of action is Proximal Tubule & descending limb Loop of Henle

Used in acute renal failure to keep fluid flowing in nephrons, and for emergency treatment of intracranial or intraocular pressure (not kidney effects)

Not useful for control of blood pressure

Proximal Tubule: Absorption of Bicarbonate

Proximal Tubule: 90% of bicarbonate reabsorbed

Carbonic Anhydrase Inhibitors:(Acetazolamide)

Not now used as diuretics Used in treatment of

glaucoma Deplete extracellular

bicarbonate Cause metabolic acidosis

7Summary Each kidney contains ~106 nephrons

Kidneys filter 180 liters (50 gal) of H2O per day & 25 moles of Na+

>99% is reabsorbed!!

The blood is filtered at the glomerulus, & the ultrafiltrate enters the renal tubule

Salts & water are transported across the renal epithelium & reabsorbed into the blood as the fluid passes through the nephron

Co-transporters & Na+/K+ ATPase are the key proteins responsible for transport of salts across the renal epithelium

Co-transporter Summary Co-transporters throughout nephron are involved in ion

reabsorption

Co-transporters use a solute gradient to drive the translocation of other substrates

X-ray crystal structure of co-transporters: Symmetric core of protein with 5 + 5 inverted structural

architecture of transmembrane helicies

Transmembrane helicies often bent at proline or glycine residues suggesting conformational flexibility

Entrance/cavity from one side for substrate

Substrate binding site in center of protein at center of membrane

Alternating Access Model of Transport: conformational change causes alternate exposure of substrate binding site to each side of membrane