Download Second-Messenger Gated Ion Channels

Second-Messenger Gated
Ion Channels
Membrane Biophysics, 2014
Ion Channel Presentation
Vehpi Yildirim and Joe McKenna
Overview
Stimulus triggers iIntracelluar signal that
modulates channel activity
●
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Examples
–G-protein
coupled channels
–IP3-regulated
–Adenine
channels
nucleotide-sensitive channels
Examples
G-protein coupled inward
rectifying K+ channel
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IP3-regulated Ca2+
release from ER
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Review Article
ATP-sensitive
+
K
Channels
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Link cellular energetics and excitability
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Gate efflux of K+
–Inward
rectifier
–Shallow
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voltage-dependence
Inhibited by ATP, activated by Mg2+
KATP Architecture
Functional Octamer
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Kir 6.2: 4 sub-units
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–Channel
pore
–Site
of ATP
inhibition
Sulphonylurea
Receptor (SUR): 4
sub-units
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of Mg2+
activation
–Site
Kir 6.2/SUR Model
Extrapolated from
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–Bacteria
K+ channel crystal
–Prokaryotic
–Targeted
Kir
mutation
ATP binds at interface of SUR
NBF1 & 2 (b. green)
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Mechanism of Gating
Fast ligand-independent gating
by ion selectivity filter
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Ligand-dependent gating by
hinged motion of M2
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–Inhibited
by ATP
–Activated
by PIP2, MgADP
Gating Kinetic Model
Fast ligandindependent gating and
slow ligand-dependent
gating
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One subunit in closed
configuration → channel
closed
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Two ways to achieve
same half-maximal
inhibition
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KATP-related disease
Pancreatic beta-cells
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–Loss
of function mutation
→Hyperglycemia/diabetes
–Gain
of function mutation →
Hyperinsulinemia
Coronoary cells
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–Loss
of function mutation →
spontaneous contraction, early
death
Identification and Properties of
+
an ATP-Sensitive K Current
in Rabbit Sino-Atrial Node
Pacemaker Cells
X. Han, P. E. Light, W. R. Giles and R. J. French
Journal of Physiology (1996), 490.2, pp.337-350
INTRODUCTION
K(ATP) channles have been
identified in many cell
types.
Most studies use myocytes
from atrium.
Here they use cells from
sino-atrium node.
Questions to be Answered
Are K(ATP) channels present in SA node and, if so, what
are their single channel properties?
Can physiological, pharmacological and pathological
conditions which are known to activate K(ATP) channels
alter SA-node activity?
METHODS
Isolated single cells from SA node of rabbit heart are
studied by measuring spontaneous activity.
Both whole cell and single channel currents are
measured.
Pharmacological blockers or openers are used.
Ventricular myocytes also isolated to compare
results from different regions of heart.
Perforated patch technique for Whole cell.
Inside-out configuration for single channel.
RESULTS
Glibenclamide: K(ATP) channel
blocker.
Acts on SUR subunit.
Cromakalim and Pinacidil: K(ATP)
channel openers.
Act on SUR subunit.
Effects of
glibenclamide on
electrical activity and
ion curents.
Effects of KATP channel openers on the Current
Effect of metabolic
inhibition by NaCN
NaCN (Sodium
Cyanide) : inhibits ATP
production.
Properties of Single KATP Channels
Effects of drugs on single
channel activity.
Effects with high ATP
concentration.
Open and Close Times
Neonatal Diabetes (NDM) Overview
Presents within first 3 months of life, requires
insulin treatment
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Insulin response to sulphonylureas but not
glucose or glucagon
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May result from Kir 6.2 gain of function mutations
in pancreatic beta-cells
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KATP Channels and NDM
Glucose → ATP → channel closure → Ca2+ influx → Insulin
secretion
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NDM Patient Screening
Patients with known
diabetes-related mutations
excluded
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Physical exam including
insulin, sulphonylurea
challenges
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Kir 6.2 gene sequenced
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Identified 6 novel
mutations
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NDM seen only in patients
with Kir6.2 mutations
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Kir6.2 Affected Residues
Highly conserved regions →
functional role
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Near ATP-binding site or
slide helix
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Patient Response to Secretagogues
3 patients with mutations in
ATP binding site (ABS)
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–No
secretion from glucose
–Secretion
opener
from KATP channel
KATP Channels in Oocytes
Channels with mutated
ABS residues
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–Larger
current in steady
[ATP]
–Current
increased by
sulphonylurea
–Weakly
inhibition by ATP
KATP Channels in Oocytes
NDM pathology more
severe in homozygote
mutants
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–Significant
difference in
half-maximal activation
by ATP
Conclusion
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Activating mutations in Kir6.2 causes NDM
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Found in 34% of patients with NDM
Accompanying complications point to vital role of
KATP channels in brain and muscle
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Potential therapy: channel blocker acting on SUR
receptors
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and Enhanced Insulin Action
in KATP Channel Deficient
Mice
Takashi Miki, Kazuaki Nagashima, Fumi Tashiro, Kazumi Kotake,
Hideyuki Yoshitomi, Atsuko Tamamoto, Tohru Gonoi, Toshihiko
Iwanaga, Jun-ichi Miyazaki, And Susumu Seino
PNAS Vol. 95, pp. 10402-10406, September 1998, Biochemistry
INTRODUCTION
KATP Channels in pancreatic Beta Cells comprise
Kir6.2 and SUR1 subunits.
KATP Channels are ATP and ADP sensors and play a
very important role in insulin secretion.
Mutations in regulatory genes cause hypoglycemia.
Here they use Kir6.2-/- mice to study the role of KATP
channels in insulin secretion.
Kir6.2+/+ and Kir6.2-/- cells are dialyzed with ATP-free pipette solution.
Glucose or Tolbutamide does not effect [Ca] in Kir6.2-\- cells.
AcetylCholine and High K+ does effect [Ca] like in wild type cells. Showing voltage gated
Ca channels and IP3 sensitive Ca stores are functioning normally in Kir6.2-\- cells.
A rapid rise in Ca concentration is needed for glucose induced insulin
secretion, rather than a continuous elevated [Ca].
In Kir6.2-/- mice, only a small first phase and no second phase secretion
observed. (In Vitro)
Glucose induced insulin secretion is reduced in knock-out mice.
But surprisingly glucose lowering effect of insulin is significantly increased in knock-out
mice.
Beta
Kir6.2+/+
Kir6.2-/-
Alpha
CONCLUSION
KATP channels play a significant role in insulin
secretion.
Glucose metabolism itself is insufficient for glucoseinduced and sulfonylurea-induced insulin secretion,
both of which require the rapid rise in [Ca2] caused by
closure of the KATP channels.