BIO304 Lecture 6

Sodium channels

activated when Ach binds

Calcium channels

opened when action potential enters nerve terminal

Cytoplasmic calcium

kept at low levels

Transient cytoplasmic signalling molecule

cytoplasmic calcium

Binds oxygen atoms, causes conformational changes in proteins

pros of cytoplasmic calcium

Carboxyl and carbonyl groups on amino acids

oxygen atoms

Good for signalling or activating mechanical processes

conformational changes in proteins

vesicle exocytosis, muscle contraction, activating other ion channels, changes in gene expression, apoptosis, intracellular signalling

6 examples of mechanical processes

Precipitates phosphates which can accumulate and become toxic, can trigger apoptosis, cannot be chemically altered for neutralization

harms of cytoplasmic calcium

10000 fold difference

[Ca2+]in <<< [Ca2+]out

1,500,000 fold

calcium less concentrated inside the cytoplasm than K+

Neuronal excitation, muscle contraction, stroke

[Ca2+]cyt can increase transiently

Positive feedback

stroke

Blood clot

stops flow of blood to a brain region

Thrombolytics

drugs that dissolve blood clots, can restore blood flow

Tissue plasminogen activator

example of thrombolytics

Neurons depolarize and produce many APs

due to lack of oxygen and glucose leading to loss of sodium-potassium pump

Drugs that inhibit voltage gated sodium channels

can reduce the number of APs generated

Glutamate

excitatory NT released by many rapidly firing neurons

Lack of energy in presynaptic neuron

causes glutamate transporters to stop working

Glutamate transporters

remove glutamate from cleft

Drugs that block glutamate receptors

combat excessive stimulation

Post synaptic neurons

bombarded with Glutamate and produce Aps

Calcium and zinc

excessive amounts enter cell

Drugs that block calcium channels

may avert intracellular buildup of calcium

Cell death due to excitotoxicity

excessive calcium and zinc

Cytoplasmic chelators/buffers

bind free calcium to remove it from solution

Pumps and exchangers

extrude calcium from the cytoplasm to the cell exterior or intracellular compartments

Intracellular compartments

sarco/endoplasmic reticulum, mitochondria

Calcium pumps

plasma membrane calcium ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)

Plasma membrane calcium ATPase

PMCA

Sarcoplasmic/endoplasmic reticulum calcium ATPase

SERCA

p-type but do not need a beta subunit

PMCA and SERCA related to the sodium-potassium ATPase

Cycle

hydrolysis of single ATP molecule

PMCA

1 calcium ion pumped out of the cell per cycle

4

human PMCA genes

PMCA Alpha 1

brain/ubiquitous - lethal if mutated

PMCA Alpha 2

brain and muscle - hearing loss and balance

PMCA Alpha 3

brain and muscle

PMCA Alpha 4

broad distribution - male fertility

SERCA

2 calcium ions pumped into the SR/ER per cycle

SERCA alpha 1

muscle contraction

SERCA alpha 2

muscle contraction, neurons

SERCA alpha 3

non-muscle, but expressed in cardiomyocytes (heart)

Sparsely expressed at the cell membrane

PMCA so it is only good at maintaining low cytoplasmic calcium levels when neurons are not highly active

Highly expressed in the SR

SERCA to ensure efficient removal of cytoplasmic calcium and restoration of SR calcium stores

Ion exchangers

remove calcium much more quickly since they do not hydrolyze ATP

Secondary active transport

consume energy from existing ion concentration gradients in exchange for moving desired ions uphill against their concentration gradients

NCX exchanger

sodium calcium exchanger uses the sodium gradient; sodium calcium antiporter

1 calcium out for 3 sodium in

NCX

Sodium and calcium

both want to get into the cell (gradient); whichever ion type experiences the strongest inward pull wins

Pull

charge x driving force

NCX at RMP

3 sodium ions go in and 1 calcium goes out

NCX at depolarized potentials

1 calcium goes in and 3 sodium go out

NCKX

uses sodium and potassium gradients to remove calcium

4 sodium in and 1 potassium out in exchange for 1 calcium out

NCKX

9

NCX transmembrane segments

NCX1

muscle

NCX2 and 3

brain

11

MCKX transmembrane segments

N-terminus of NCKX

cleaved

NCKX1

retina

NCKX2

retina, brain

NCKX3

brain and smooth muscle

NCKX4

brain and smooth muscle

NCKX5

not expressed at the membrane; polymorphism is associated with white skin in individuals from Europe and Asia; might regulate calcium in melanosomes

Immature neurons and almost all other cells

[calcium]in ~ [calcium]out

Mature neurons

active extrude chlorine from the cytoplasm so [calcium]out >> [calcium]in

Kakazu et al

observed developing superior olive neurons of mice

Olive neurons

audition

Glycine

neurotransmitter that activates post-synaptic chlorine channels (glycine receptors)

Postnatal day 0 mice

glycine cause depolarization of membrane voltage

P15 mice

glycine caused pronounced hyperpolarization of membrane voltage

High [Cl-]in

electrically neutral co-transporters use sodium gradient to move Cl- into the cell

1 sodium and 1 chlorine into the cell

NCC

1 sodium and 1 potassium and 2 chlorine into the cell

NKCC

Produce excitable neuron via Cl- channel activation

NCC or NKCC need to produce [Cl-]in ~ [Cl-]out

SLC12A3

NCC genes

SLC12A2 and SLC12A1

NKCC genes

Less [Cl-]in

electrically neutral cotransporters that use the potassium gradient move chlorine out of the cell

1 potassium and 1 chlorine out of the cell

KCC

Inhibit (hyperpolarize) a neuron via chlorine channel activation

need KCC to produce [Cl-]in << [Cl-]out

SLC12A4, SLC12A5, SLC12A6, SLC12A7

KCC genes

Only NKCC transporting Cl- in

immature neurons

KCC transports Cl- out

mature neurons

sodium-dependent Cl-/HCO3- exchange system

uses the sodium gradient to move bicarbonate into the cell and protons out

HCO3-

part of a physiological buffering system crucial in the nervous system, where cells have little tolerance for fluctuations in pH

High cytoplasmic [H+]

promotes H+ efflux and HCO3- influx

High in [HCO3-]

shifts the equation to the left, further neutralizing cytoplasmic pH

Formation of H2CO3

left shift in equation

Neurotransmitters

synthesized in the cytoplasm then actively transported to presynaptic vesicles

Reuptake

after secretion, neurotransmitters are often taken back into cells

Extracellular transmitter clearance

helps stop synaptic signals and replenishes/recycles transmitters

Vesicular transporters

use proton gradients to move transmitters into the lumen of synaptic vesicles

Vacuolar-type hydrogen ATPases

consume ATP to concentrate hydrogen ions in vesicles

Hydrogen gradient

provides energy for transporting NTs

SLC18

monoamines and acetylcholine

Monoamines

norepinephrine, serotonin, histamine, dopamine

SLC32

glycine, GABA

SLC17

glutamate