Ion Homeostasis, Channels, and Transporters:

1
Ion Homeostasis, Channels, and Transporters
An Update on Cellular Mechanisms
Experimental Biology 2004 APS Refresher
Course Washington, DC
George R. Dubyak, Ph.D. Dept of Physiology
Biophysics
2
Ion Homeostasis, Channels, and Transporters An
Update on Cellular Mechanisms

• Ion Transport Proteins as Channels versus
  Transporters Not as different as we think
• Interactions of Ion Transport Proteins with
  Adapter Proteins No transporter is an island
• Interactions of Ion Transport Proteins with Local
  Lipids The bilayer as more than a low
  dielectric permeability barrier
• Interactions between Ion Transport Proteins and
  Modulator Proteins Cell-specific context
  explains all

3
Ion Homeostasis, Channels, and Transporters An
Update on Cellular Mechanisms

• Review of Cellular Ion Homeostasis
• Basic Concepts from the Pre-Genomics Era
• Ionic Compartments and Electrochemical Driving
  Forces
• Ionic Permeability Pathways
• Mechanistic Differences between Ion Channels and
  Ion Transporter Proteins (including ATP-Powered
  Pumps)

4
Basic Concept 1 Compartmentation of Ionic Pools
and Electrochemical Driving Forces
Boron and Boulpaep (2002) Medical Physiology
Saunders
5
Basic Concept 2 Categories of Ion Permeability
Pathways
Lodish et al. (2000) Molecular Cell Biology 4th
Edition W.H. Freeman Co.
6
Basic Concept 3 Disparate Mechanisms for Ion
Flux via Channels versus Transporters - The
Channel Story

• Minimal energetic interaction between the
  transported ion and the channel protein
• Ionic flux is limited by opening and closing of a
  single major gate
• Gating is regulated by conformational changes
  extrinsic to the permeability barrier or pore

Gadsby (2004) Nature 427 795-796
Gating is not allosterically coupled to the
movement of ions through the pore of the open
channel
7
Basic Concept 3 Disparate Mechanisms for Ion
Flux via Channels versus Transporters - The
Transporter Story

• There are strong and selective energetic
  interactions between the transported ion(s) and
  the transporter protein
• Ionic flux is limited by the alternating opening
  and closing of two gates
• Both gates can be simultaneously closed to
  produce trapping or occlusion of the transported
  ion(s) within the permeability barrier
• Movement of each gate is regulated by
  conformational changes intrinsic to the
  permeability barrier

8
Basic Concept 3 Disparate Mechanisms for Ion
Flux via Channels versus Transporters - The
Transporter Story
Gadsby (2004) Nature 427 795-796
Gating is allosterically coupled to the movement
of ions through the permeability barrier of the
transporter
9
Basic Concept 3 Disparate Mechanisms for Ion
Flux via Channels versus Transporters
Characterization of channels primarily focuses on
Ionic selectivity and permeability of the pore
Forces and factors that move the gates
Boron and Boulpaep (2002) Medical Physiology
Saunders
Lodish et al. (2000) Molecular Cell Biology 4th
Edition W.H. Freeman Co.
10
Basic Concept 4 Disparate Mechanisms for Ion
Flux via Channels versus Transporters
Characterization of transporters primarily
focuses on
Affinity, selectivity, and stoichiometry of ion
binding
Biochemical or biophysical reactions that are
allosterically coupled to the transport cycle
Gadsby (2004) Nature 427 795-796
11
Ion Transport Proteins as Channels versus
Carriers Not as different as we think

• Concept Both channel-like activity and
  transporter-like activity can be accommodated
  within the basic structures of most transport
  proteins
• Model Example The induction of channel activity
  in the Na,K-ATPase pump upon binding of
  palytoxin, a marine toxin, that stabilizes both
  gates of the Na pump in the open state

12
Ion Transport Proteins as Channels versus
Carriers Not as different as we think
Palytoxin Structure
Boron and Boulpaep (2002) Medical Physiology
Saunders
Hilgemann (2003) PNAS 100 386-388

• Palytoxin is a lethal toxin from a marine
  coelenterate (Palythoa coral)
• Palytoxin binding to the Na pump induces
  appearance of non-selective cation channel
  activity

13
Ion Transport Proteins as Channels versus
Carriers Not as different as we think

• Model guinea pig ventricular myocytes
• Outside-out membrane patch recording in symmetric
  NaCl
• Palytoxin (PTX) induces Na channel activity
  independent of ATP
• However, ATP still acts as a positive allosteric
  regulator

Artigas and Gadsby (2003) PNAS 100 501-505
14
Ion Transport Proteins as Channels versus
Carriers Not as different as we think

• K acts as a negative allosteric regulator of
  PTX-induced Na pump-channels

Artigas and Gadsby (2003) PNAS 100 501-505

• PTX stabilizes opening of both gates of the Na
  pump, i.e., no occluded state
• Permeability/ conformation of the non-occluded
  pore remains allosterically sensitive to ATP
  and K

Boron and Boulpaep (2002) Medical Physiology
Saunders
15
Ion Transport Proteins as Channels versus
Carriers Not as different as we think

• ClC-family proteins comprise a large family of
  structurally related membrane proteins that
  function as Cl- channels in eukaryotic cells
• The resolved crystal of a prokaryotic member -
  ClC-ec1 from E. coli - has provided the
  structural template BUT.

Dutzler, Campbell, Cadene, Chait, and MacKinnon
(2002) Nature 415 287-294
A. Accardi and C. Miller (2004) Nature 427
803-807
16
Interactions of Ion Transport Proteins with
Adapter Proteins No transporter is an island
Concept Many channels and transporters
physically associate with adapter proteins that
regulate the subcellular localization of the
transport protein and/or its direct interaction
within signal transduction complexes that include
receptors, 2nd-messenger effector enzymes, and
protein kinases Model Example The role of
NHERF-family adapter proteins in the localization
and acute regulation of Na-Phosphate
cotransporters within apical signaling complexes
of renal epithelial cells
17
Interactions of Ion Transport Proteins with
Adapter Proteins No transporter is an island
18
Interactions of Ion Transport Proteins with
Adapter Proteins No transporter is an island

• PDZ (PSD-95, discs large, ZO1) domains are
  protein-protein interaction sites found in a
  large number of adapter proteins
• Such adapter proteins act to localize channels or
  transporters within large signaling complexes at
  the sub-membrane cytoskeleton
• Examples include the PSD-95 (post-synaptic
  density) adapter that co-assembles
  neurotransmitter-gated ion channels with
  cytoskeletal elements, kinases, and small GTPases
  into signaling complexes at the neuronal synapses
• NHERFs (Na/H Exchanger Regulatory Factors)
  comprise another family of PDZ-containing
  adapters expressed in many epithelia

19
Interactions of Ion Transport Proteins with
Adapter Proteins No transporter is an island

• Structure of the related NHERF1 and NHERF2
• ERM domains bind to cytoskeletal proteins while
  PDZ domains bind to various transport proteins
  and signaling proteins
• Recent studies have shown that NHERFs can also
  bind the parathyroid hormone receptor (PTH-R) and
  the type 2 Na-Phosphate Cotransporter (NPT2)

PTH-R
NPT2
Shenolikar and Weinman (2001) Am J Physiol -
Renal 280 F389-F395
PLCb1
20
Interactions of Ion Transport Proteins with
Adapter Proteins No transporter is an island

• Phosphate (Pi) reaccumulation from glomerular
  filtrate reflects activity of apical NPT2 protein
  in proximal tubule cells
• During phosphate excess, PTH acts to repress
  NPT2-mediated Pi reuptake
• PTH induces rapid endocytosis of the apical NPT2
  pool by signaling mechanisms that involve both
  phospholipase C (PLC) and adenylyl cyclase (AC)
  pathways

Boron and Boulpaep (2002) Medical Physiology
Saunders
21
Interactions of Ion Transport Proteins with
Adapter Proteins No transporter is an island

• Opossum Kidney (OK) line cells exhibit normal
  PTH-induced repression of NPT2 -mediated Pi
  uptake
• OKH cells (a subline of OK) exhibit weak NPT2
  response to PTH despite similar expression of
  NPT2
• These effects are correlated with high NHERF1
  levels in OK cells and low NHERF1 levels in OKH
  cells
• Transfection of NHERF1 into OKH cells restores
  PTH-induced repression of NPT2 activity

OKH mut NHERF
OKH
OK-wt
OKH NHERF
Mahon, Cole, Lederer, and Segre (2003) Mol
Endocrin 17 2355-2364
22
Interactions of Ion Transport Proteins with
Adapter Proteins No transporter is an island

• In absence of PTH, NPT2 is apically expressed in
  both OKH cells and OKH transfected with NHERF1
  (OKH-N1)
• PTH induces rapid NPT2 (aka Na-Pi-4)
  internalization in OKH-N1 cells but not OKH cells

Mahon, Cole, Lederer, and Segre (2003) Mol
Endocrin 17 2355-2364
23
Interactions of Ion Transport Proteins with
Adapter Proteins No transporter is an island

• Knockout of NHERF1 in mice induces reduced
  steady-state levels of NPT2 in brush-border
  membranes from kidney but not reduction in total
  kidney NPT2 content
• Proximal tubule cells from NHERF1 -/- mice show
  increased internal pools of NPT2 at steady-state
• NHERF1 -/- mice exhibit exhibit significant
  phosphate wasting into urine despite normal serum
  Pi levels
• Most NHERF1 -/- females die within 35 days post
  birth and show multiple bone fractures

Shenolikar, Voltz, Minkoff, Wade, and Weinman
(2002) PNAS 99 11470-11475
24
Interactions of Ion Transport Proteins with
Adapter Proteins No transporter is an island
NHERF association with PTH receptor
directs coupling to distal G proteins and
effector enzymes
PTH-R alone
PTH-R complexed to NHERF1/2
Phospho- lipase C
Adenylyl Cyclase
PTH-R
PTH-R
Gs
Gi/q
25
Interactions of Ion Transport Proteins with
Adapter Proteins No transporter is an island
Adenylyl Cyclase

• NHERF 1/2 bind to a novel PDZ-interaction site at
  the PTH-R C-terminus
• Expression of wildtype PTH-R in absence of NHERF
  induces default coupling to Gs/ AC
• Coexpression of wildtype PTH-R with NHERF
  redirects coupling to Gi/Gq/ PLC

PTH-R
Gs
PTH-R / NHERF
Phospho- lipase C
Gi/q
Mahon, Donowitz, and Segre (2002) Nature 417
858-861
26
Interactions of Ion Transport Proteins with Local
Lipids The bilayer as more than a low
dielectric permeability barrier
Concept The function of many channels and
transporters is directly modulated by the
specific binding of phosphatidylinositol
4,5-bisphosphate (PIP2) this facilitates rapid
modulation of transport protein activity by
highly localized changes in PIP2 synthesis or
degradation Model Example The
hypersensitization of nociceptive vanillanoid
receptor (VR1) channel activity by inflammatory
mediators that activate phospholipase C
(PLC)-dependent PIP2 breakdown
27
Interactions of Ion Transport Proteins with Local
Lipids The bilayer as more than a low
dielectric permeability barrier
PIP2 can positively or negatively modulate
activity of a wide range of ion channels and ion
transporters
These effects usually reflect direct binding of
PIP2 to specific domains of the transport
proteins with consequent allosteric modulation
Hilgemann, Feng, and Nasuhoglu (2001) Science-STKE
111-RE19 1-8
28
Interactions of Ion Transport Proteins with Local
Lipids The bilayer as more than a low
dielectric permeability barrier
29
Interactions of Ion Transport Proteins with Local
Lipids The bilayer as more than a low
dielectric permeability barrier

• Vanillanoid receptors are non-selective cation
  channels of sensory nerve endings that are gated
  by diverse nociceptive stimuli produced at
  damaged tissue
• The major physiological stimuli are acidic pH
  and increased temperature
• Sensitivity of VR1 to these stimuli can be
  greatly enhanced by diverse hydrophobic ligands,
  e.g. capsaicins from peppers

Caterina and Julius (2001) Annu Rev Neurosci 24
487-517
30
Interactions of Ion Transport Proteins with Local
Lipids The bilayer as more than a low
dielectric permeability barrier

• Model VR1 channels heterologously expressed in
  HEK293 cells
• VR1 channels gated by threshold levels of primary
  stimuli (acid or capsaicin)
• Activation of native bradykinin receptors
  (PLC-coupled) greatly potentiates gating by
  primary stimuli

Chuang, Prescott, Kong, Shields, Jordl, Basbaum,
Chao, and Julius (2001) Nature 411 957-962
31
Interactions of Ion Transport Proteins with Local
Lipids The bilayer as more than a low
dielectric permeability barrier

• VR1 channels belong to TRP superfamily
• VR1 C-terminus contains sequences conserved in
  PIP2-interaction domains of other PIP2-sensitive
  channels, e.g. inward rectifier K channels

ONeill and Brown (2003) News Physiol Sci 18
226-231
32
Interactions of Ion Transport Proteins with Local
Lipids The bilayer as more than a low
dielectric permeability barrier
Mutation of this VR1 C-terminal domain increases
gating by primary nociceptive stimuli but reduces
potentiation of gating by PLC-activating
secondary stimuli
VR1 C-terminal variants
Prescott and Julius (2003) Science 300 1284-1288
33
Interactions among Ion Transport Proteins and
Modulator Proteins Cell-specific context
explains all
Concept An ion transport protein can exhibit
tissue-specific differences in function that
reflect its direct association with modulator
proteins that are expressed in a tissue-specific
or stimulus-specific manner. Model Example
The role of FXYD-family membrane proteins in the
tissue-specific modulation of Na,K-ATPase pump
activity
34
Interactions among Ion Transport Proteins and
Modulator Proteins Cell-specific context
explains all
35
Interactions among Ion Transport Proteins and
Modulator Proteins Cell-specific context
explains all
FXYD proteins as tissue-specific modulators of
the Na,K-ATPase
Crambert and Geering (2003) Science-STKE
166-RE1 1-9
36
Interactions among Ion Transport Proteins and
Modulator Proteins Cell-specific context
explains all

• FXYD proteins comprise a family of structurally
  related type-1 membrane proteins that are
  expressed in tissue-specific patterns
• Includes previously identified, but poorly
  understood proteins, e.g., phospholemman (PLM) in
  heart and corticosteroid hormone-induced factor
  (CHIF) in kidney

Crambert and Geering (2003 Science-STKE 166-RE1
1-9
37
Interactions among Ion Transport Proteins and
Modulator Proteins Cell-specific context
explains all
FXYD proteins as tissue-specific modulators of
the Na,K-ATPase Different effects of different
FXYDs
Crambert and Geering (2003 Science-STKE 166-RE1
1-9
38
Interactions among Ion Transport Proteins and
Modulator Proteins Cell-specific context
explains all
Treatment of collecting duct with aldosterone
coordinately increases CHIF and ENaC expression
CHIF increases Na affinity of the Na pump to
increase transcellular Na flux while decreasing
cytosolic Na
Crambert and Geering (2003 Science-STKE 166-RE1
1-9
39
Take-Home Lessons

• Precise homeostasis of the major inorganic
  cations (Na, K, H) and anions (Cl-, PO43-,
  HCO3-) is fundamental to all cells
• However, cell-specific expression of different
  membrane transport proteins and regulatory
  factors permits wide variations in the absolute
  rates of transmembrane flux of these ions
• These cell-specific differences in ionic flux are
  exploited for tissue-specific differences in
  function such as solute flow (e.g.
  transepithelial movements of metabolites) or
  information transfer

40
Take-Home Lessons

• These tissue-specific differences in ionic flux
  are regulated at multiple levels
• via increased/ decreased expression of membrane
  transport protein genes
• via changes in the steady-state trafficking of
  membrane transport protein to and from the plasma
  membrane
• via direct post-translational modification (e.g.
  phosphorylation) of the membrane transport
  proteins
• via direct association with tissue-specific
  adapter or modulator proteins
• via the local lipid composition of the membrane
  bilayer

41
References Original Research Papers

• Accardi and Miller (2004) Nature 427 803-807
• Artigas and Gadsby (2003) PNAS 100 501-505
• Chuang, Prescott, Kong, Shields, Jordl, Basbaum,
  Chao, and Julius (2001) Nature 411 957-962
• Dutzler, Campbell, Cadene, Chait, and MacKinnon
  (2002) Nature 415 287-294
• Mahon, Cole, Lederer, and Segre (2003) Mol
  Endocrin 17 2355-2364
• Mahon, Donowitz, and Segre (2002) Nature 417
  858-861
• Prescott and Julius (2003) Science 300 1284-1288
• Shenolikar, Voltz, Minkoff, Wade, and Weinman
  (2002) PNAS 99 11470-11475

42
References Reviews and Commentaries

• Channel versus Transporter Mechanisms
• Hilgemann (2003) PNAS 100 386-388
• Gadsby (2004) Nature 427 795-796
• Adapter/ PDZ Proteins and Channel/ Transporter
  Regulation
• Shenolikar and Weinman (2001) Am J Physiol -
  Renal 280 F389-F395
• Noury, Grant, and Borg (2003) Science-STKE
  179-RE7 1-12
• PIP2 and Channel/ Transporter Regulation
• ONeill and Brown (2003) News Physiol Sci 18
  226-231
• Hilgemann, Feng, and Nasuhoglu (2001)
  Science-STKE 111-RE19 1-8
• Caterina and Julius (2001) Annu Rev Neurosci 24
  487-517
• Modulator/ FXYD Proteins and Channel/ Transporter
  Regulation
• Cornelius and Mahmmoud (2003) News Physiol Sci
  18 119-124
• Crambert and Geering (2003) Science-STKE 166-RE1
  1-9

43
References Textbooks

• Boron and Boulpaep (2002) Medical Physiology
  Saunders
• Alberts et al. (2001) Molecular Biology of the
  Cell, 4th Edition Garland
• Lodish et al. (2000) Molecular Cell Biology, 4th
  Edition W.H. Freeman Co.