The Kim Lab
<p>
KIM, Peter, Membrane Transporters, June 1, 2015
Prof Peter Kim is from Dept of Biochem.
This is at noon in Clark S361 on Monday and is a "preseminar" , a tutorial on
transporters (in prep for Olga Boudker's talk here on Thursday at noon)
Peter Kim's lecture style is wonderful: v deliberate with very high signal to noise and
excellent slides. This is outstanding basic material.
he begins... shows a cartoon membrane:</p>
<p>
there are either pores or carriers.</p>
<p>
<b>a pore</b> looks like a cylinder with a hole in the middle
(thru which ions can move in either direction and they move down their
concentration gradients.)
<b>Gramicidin </b>was one of the first to be studied. he shows structure.</p>
<p>
<b>Valinomycin</b>... was one of the first carriers to be studied.</p>
<p>
pix from Lubert Stryer Biochem. text.</p>
<p>
shows K+ in the middle chelated by hydrocarbons on the periphery.</p>
<p>
a <b>carrier</b> <b>binds an ion</b> and then just randomly diffuses to the interior to dump its cargo.</p>
<p>
channels, pumps, and <b>symporters</b>.
(3 types )</p>
<p>
<b>Channels or pores</b> are like Gramicidin..
passive transport and ions can flow in either direction.
can drive flow in either direction... it's spontaneous
but these are often gated.
eg ACh (acetyl choline) receptor, voltage-gated
Na comes in... following its gradient
and K goes out... but it only open whens ACh binds to it.
and is voltage gated.</p>
<p>
<b>but the FLOW is Passive.</b></p>
<p>
</p>
<p>
<b>PUMPS unlike channels involve active transport</b>
(can go against concentration gradient)
so must be coupled to a source of free energy
ATP hydrolysis is one of most common... as in ATPase.
or light can be a source...e g bacteriorhodopsin to pump protons uphill.
(the Channel rhodopsins for optogenetic switches to pump ions)</p>
<p>
3rd category*
<b>SYMPORTERS</b>
one molecule flows uphill
but coupled with another molecule flowing downhill.</p>
<p>
these couple the flow...
Uphill flow of one molecule that is coupled to downhill flow of another molecule
(but both are moving in the same direction)
but there are also
<b>Antiporters</b> that couple flow in the opposite direction
</p>
<p>
History: this was from <b>Oleg Jardetzky in 1966 in Nature</b>.</p>
<p>
Oleg showed a <b>simple allosteric model for membrane pumps</b>...</p>
<p>
need a cavity and two conformations..</p>
<p>
first conformation say is open to the outside and grabs eg Na+ but then
gets phosphorylated and that opens it to the inside where the Na gets released inside the cell.</p>
<p>
simple picture tells you how the job gets done.</p>
<p>
*
and now... on to GLUTAMATE (Olga will talk abt aspartate (which has similar mechanism)</p>
<p>
Glutamate is the major excitatory neurotransmitter in the brain.</p>
<p>
<b>NMDA and AMPA receptors... both are ionotropic (flow of ions) </b> and
then there are the mGluR and mGluR I (these are GCPRs)
that involve 2nd messengers like cAMP, IP3, DAG and Ca++.</p>
<p>
but once glutamate is released it must be taken up (by glial cells) to get recycled)
<b>they can pump against a concentration gradient of a million fold</b>.</p>
<p>
Glutamine can be transsformed into glutamate...</p>
<p>
</p>
<p>
Olga will talk abt EAATs Excitatory amino acid transporters.</p>
<p>
they are responsible for uptake of glutamate from synapses
there are diseases assoc with deficits of transporters.</p>
<p>
Defects of EAATs in the brain: those cause
hyper excitation: and lead to neuronal and glial cell death;
this underlies brain damage following stroke and ischemia
and progressive cell death in several neurodegen diseases.</p>
<p>
these drive glutamate transport against a v v steep concentration gradient.</p>
<p>
this coupling of uptake : for each mole of glutamate to symport ... it uses three Na ions
and one H+ and counter transport of one K+.</p>
<p>
he draws diagram: recall symport is in the SAME direction.</p>
<p>
so, glutamate goes from outside to inside the glial cell
and it is symprt coupled (same direc to 3 Na and one H+ and
antiport (ie countertransport) of K+ inside to outside
so, note it takes a lot of ions moving down the concentration gradient in order
to move one glutamate against a steep gradient from outside to inside.</p>
<p>
but it also permits passive bidirectional ANION (Chloride) flux
in response to Na and glutamate binding.</p>
<p>
in response to a question: (The diseases are all due to loss of function).</p>
<p>
we will hear abt <b>Glt(Ph) pronounced glit...</b>from Olga B.</p>
<p>
Archea are comfy at 100 degr C.</p>
<p>
these are archeal homologues of EAATs (that Olga will discuss)</p>
<p>
and drives aspartate transport against steep conc gradients.</p>
<p>
these couple each molec of aspartate to symport of three Na ions...</p>
<p>
(this molecule is a homologue of mammalian glutamate symport)...</p>
<p>
review article in Science in 2005 Gouaux and Rod MacKinnon (Nobelist)</p>
<p>
shows architectures of ion channel and ion pumps.</p>
<p>
first time we saw (from MacKinnon) how ions are transported across membranes
(in 1998) and he got Nobel in 2003 (xray crystalographyy)</p>
<p>
K+ channel is a tetramer...
forms a pore and how it is gated.</p>
<p>
and shows Ca channel ATPase... v complex.</p>
<p>
by the way... the glutamate symporter is more like an elevator.
Olga discovered this in 2009 (one piece goes up and the other goes down)
one domain gets transported.</p>
<p>
Olga Boudker in Nature in 2009 transport mech of bacter homolog of glutamate transproters</p>
<p>
and 2015 paper in Nautre
Akyuz et al in 2015 in Nature 518
transport domain unlocking sets the uptake rate of an aspartate transporter.</p>
<p>
I ask abt selectivity (how do they pull it off?)</p>
<p>
A: consider K+ is hydrated...it brings a hydration shell with it..
McKinnon figured this out...</p>
<p>
there are dipole moments in carbonyls of the channel that help..</p>
<p>
but Na is smaller...it is a
selectivity filter: that uses size, hydration...
for the pores</p>
<p>
but with transporters... can have any configuration (just like proteins and enzymes)</p>
<p>
the symporters are huge molecules...(unlike mere pores)</p>
<p>
Q: do the elevators all involve transport of something like an AA like glutamate
A prob YES... metabolites... these are important in kidney eg...and are
not used by simple ions.</p>
<p>
ABC class of transporters... eg pumps like Efflux pumps that pump junk OUT of the
blood brain barrier (that happen to sneak in)</p>
<p>
BBB blood brain barrier is formed by tight junctions across endothelial cells.</p>
<p>
these pumps will pump drugs out of the brain. (so, must disable them if you want to turn that off)
</p>