Lecture 6

Sodium channels

activated when Ach binds

Calcium channels

opened when action potential enters nerve terminal

Action potential

invades nerve terminals, causing opening of calcium channels

Cytoplasmic calcium

binds oxygen atoms (carboxyl and carbonyl groups on amino acids); causes conformational changes in proteins (good for signalling or activating mechanical processes)

Mechanical processes

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

Cytoplasmic calcium harms

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

Transient cytoplasmic signalling molecule

cytoplasmic calcium is kept at very low levels

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

10000 fold difference; 1,500,000 fold less concentrated inside the cytoplasm than K+

Neuronal excitation and muscle contraction

[Ca2+]cyt can increase transiently

Stroke

positive feedback triggers increase in [Ca2+]cyt

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)

PMCA and SERCA

related to the sodium-potassium ATPase; p-type but do not need a beta subunit; sluggish at removing calcium

PMCA

1 calcium ion pumped out of the cell per cycle (hydrolysis of a single ATP molecule); sparsely expressed at the cell membrane, so it is only good at maintaining low cytoplasmic calcium levels when neurons are not highly active

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 (hydrolysis of a single ATP molecule); highly expressed in the SR to ensure efficient removal of cytoplasmic calcium and restoration of SR calcium stores

SERCA alpha 1

muscle contraction

SERCA alpha 2

muscle contraction, neurons

SERCA alpha 3

non-muscle, but expressed in cardiomyocytes (heart)

Ion exchangers

remove calcium much more quickly since they do not hydrolyze ATP as energy source for moving ions against their gradients, consume energy from existing ion concentration gradients in exchange for moving desired ions uphill against their concentration gradients; referred to as secondary active transport

NCX exchanger

sodium calcium exchanger uses the sodium gradient; aka sodium calcium antiporter; 1 calcium out for 3 sodium in (can depolarize membrane voltage); most widely distributed sodium-calcium exchanger

NCX can be made to operate in reverse

since not electrically neutral

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

RMP

3 sodium ions go in and 1 calcium goes out

Depolarized potentials

1 calcium goes in and 3 sodium go out

NCKX

better at removing cytosolic calcium; uses sodium and potassium gradients to remove calcium; 4 sodium in and 1 potassium out in exchange for 1 calcium out

9

NCX transmembrane segments

NCX1-3

mammalian genes, 1 in muscle, 2 and 3 in 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

NCC

transports 1 sodium and 1 chlorine into the cell

NKCC

transports 1 sodium and 1 potassium and 2 chlorine into the cell

Produce excitable neuron via Cl- channel activation

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

NCC gene

solute carrier 12 A3 (SLC12A3)

NKCC genes

SLC12A2 and SLC12A1

Less [Cl-]in

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

KCC

transports 1 potassium and 1 chlorine out of the cell

Inhibit (hyperpolarize) a neuron via chlorine channel activation

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

KCC genes

SLC12A4, SLC12A4, SLC12A5, SLC12A6, SLC12A7

Na+-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

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

Human V-ATPase

structure solved via cryo-electron microscopy; large complex with many subunits; used the toxin SidK which binds and inhibits the pump to isolate the protein from cell extracts for structural analysis

Membrane reuptake transporters

use sodium, potassium, and other gradients to transport transmitters from the extracellular environment into the cytoplasm