Membrane e trasporti transmembrana

TYPES OF ATP POWERED PUMPS

• V-type ATPases - Proton pump across lysosomal vacuolar membranes to maintain a high acidity of lysosomes
• F-type ATPases - Proton pump involved in generating ATP for the cell, i.e., inner mitochondrial membrane and thylakoid membrane of chloroplast
• P-type transport ATPases - Na-K pump, Ca pump, H pump
• ABC Superfamily - Large diverse family of pumps named for their characteristic ATP binding motif or cassette. Consist of two transmembrane (TM) domains and two cytosolic ATP binding (ABC) domains. The two TM domains (each with 6 membrane α-helices) spanning thought to form pathway for substrate transport through membranes.

ATPases - Many proteins use the energy of ATP hydrolysis to fuel transport.

1. P-type ATPase - Ca2+-ATPase is important in muscle activation, K+-H+-ATPase is important in acid secretion in stomach

ATPases - Many proteins use the energy of ATP hydrolysis to fuel transport.

2. V-type ATPases - Proton pump across various membranes to maintain pH gradients

ATPase

Multimeric transporters often work in the reversedirection (ATP synthesis)

F_o ATPase is the mitochondrial ATP synthase

1 +H -ATPase of lysosomes acidify the organelle

ATPases(3) ABC Transporters

Many proteins have an “ATP-Binding Cassette” and move large molecules across membranes

ATPases(3) ABC Transporters-Multi-drug resistant protein can pump drugs out of cells. Some cancers become resistant to cancer therapy by increasing expression of MDR, either by gene duplication or increased transcription Na ,K -ATPase

Enzyme Structure

α– -Subunit of about 100 kDa

ATPase activity

Contains all known substrate and inhibitor binding sites

β– -Subunit, a small glycopeptide of 30-40 kDa

Necessary for activity but role unknown

Necessary for proper membrane insertion during synthesis.Na ,K -ATPase: Mechanism

Na and K bind to separate sites.

Inhibition of K binding by drugs is associated with relief of congestive heart

failure (dropsy).

Na ,K -ATPase: Mechanism

• 3 Na bind
• ATPphosphorylatesactive site
• Conformationalchange releases+Na
• 2 K bind 2+Mg2+
• Mg catalyzesdephosphorylationof enzyme
• Conformational +change releases K

Na+,K+-ATPase: Mechanism

• Digitalis class cardiac glycosides competitively bind to+(extracellular) K site.

Digitalis purpurea (foxglove)F.E. Köhler (1883-1914) Köhler'sMedizinal-Pflanzen in naturgetreuenAbbildungen mit kurz erläuterndemTexte, publ. Gera-Untermhaus.

Ion channels

Ion channels are pores that permit the movement ofspecific ions.

When open, the channels allow ions to move downconcentration gradients

Ion channels

Can "transport" 100,000,000 ions/ sec

A "selectivity filter" restricts movement to specific ions

Movement is controlled by regulating "openness" -the proportion of time spent in the openconfiguration

Ion channels can be regulated by specific conditionsto be

open or closed

If opened for a prolonged period, the channels can become desensitized

Ion channels-

• Anion channel (Cl-)
• K+ channel
• K+ channel
• K+ channel
• K+ channel
• Acetylcholine receptor
• Acetylcholine receptor
• Voltage-gated Na channel protein
• Voltage-gated Na channel protein
• Voltage-gated Na channel protein
• ATPases (3) ABC

Transporters

Many proteins have an "ATP-Binding Cassette" and move large molecules across membranes

• ABC Superfamily
• Large diverse family of pumps named for their characteristic ATP binding motif or cassette.
• Consist of two transmembrane (TM) domains and two cytosolic ATP binding (ABC) domains. The two TM domains (each with 6 α-helices) membrane spanning thought to form pathway for substrate transport through membranes.

Human diseases associated with an ABC Transporter

(ABC1)Progressive familial intrahepatic cholestasis ABCB11 (SPGP), ABCB4 (MDR2)Dubin-Johnson syndrome ABCC2 (MRP2)Pseudoxanthoma elasticum ABCC6 (MRP6)Persistent hypoglycemia of infancy ABCC8 (SUR1), ABCC9 (SUR2)Sideroblastic anemia and ataxia ABCB7 (ABC7)Adrenoleukodystrophy ABCD1 (ALD)Sitosterolemia ABCG5, ABCG8Immune deficiency ABCB2 (Tap1), ABCB3 (Tap2)

What is the Scope of the Problem?

Estimated New Cancer Cases & Deaths2001 **Site New Deaths %CasesAll Sites 1,268,000 553,400 44%Oral Cavity & Pharynx 30,100 7,800 26%Digestive System 235,700 131,300 56%Respiratory System 184,600 162,500 88%11,600 5,800 50%Bones, Joints, & Soft TissuesBreast 193,700 40,600 21%Prostate 198,100 31,500 16%Genital System 88,700 58,500 66%Urinary System 87,500 25,000 29%Endocrine System 21,400 2,300 11%Brain & Nervous System 17,200 13,100 76%Lymphoma & Leukemia 95,100 49,100 52%Other & Unspecified 31,400 36,200**Vast majority of deaths due to chemoresistanceCA Cancer J Clin.51:23,

2001DRUG RESISTANCE: The major obstacle to successful cancer chemotherapy

Chemotherapy Chemotherapy

DRUG ACCUMULATION IS REDUCED IN MITOXANTRONE-RESISTANT CELLS

MCF-7 wt MCF-7 AdVp3000 S1 wt S1-M1-80

Mitoxantrone

Topotecan Resistance Can Develop Simultaneously to Several Unrelated Drugs

This is called MULTIDRUG RESISTANCE

How Drugs Get Into Cells

Diffusion Transport Endocytosis

DD DD DD De.g., vinblastine, e.g., nucleoside e.g., immunotoxins doxorubicin analogs

ABC TRANSPORTERS INVOLVED IN DRUG RESISTANCE