Channels

lots of primary literature here.

Here is a web site on channels I recently found

TRANSPARENCY
R. TAYLOR, 1994,

The first page gives history, some of which was covered before, a quite thorough treatment.
1780's Galvani
1834 - Faraday - "ion"
1887 - Arrhenius - salts dissociate
1890 - Nernst
1902 - Bernstein - - membrane's selective permeability to potassium breaks down
1902 - Overton - Sodium needed for action potential
1925 - Michaelis ion selective channels
1936 - Young Loligo
1939 - Cole and Curtis
1949-1952 - voltage clamp and Hodgkin - Huxley papers
1964 - Narahashi - TTX (tetrodotoxin)- sodium
1965 - Armstrong and Binstock - TEA - potassium
some isolations of channel with labeled toxins
Electrophorus electricus - eel mRNA => 1820 a.a. with 4 homologous domains
Noda, Numa et al.
1987 - Jan and Jan (and other groups) Drosophila potassium

Ball and chain inactivation:
TRANSPARENCY
T. Hoshi et al., 1990

O. P. Hamill & D. W. McBride, Jr., 1995

"mechanogated"

Numerous (e.g. 17,000 in palm and fingertips) and widespread (to bacteria and Paramecia, plants, fungi and animals, i.e. all kingdoms)

Classically, for touch, there are "phasic" e.g. Pacinian corpuscle and "tonic" e.g. Merkel cell based on how fast the adaptation and thus whether vibration or steady pressure is optimal

When Erwin Neher and Bert Sakman (Max Planck Inst) developed patch clamp (1097's, Nobel Prize 1991) - this helped a lot.

development of "pressure clamp" - TRANSPARENCY

oocytes of toad have channels, Amiloride blocks channels and fertilization

tension on channel may be important plus viscoelastic elements - intracellular and extracellular, TRANSPARENCY e.g. tip links in stereocilia in hair cells (later)
integrins cell surface link to extracellular like laminin
dystrophin found in studies of Duchenne muscular dystrophy - reverse genetics
link with actin etc.

O. P. Hamill & D. W. McBride, Jr., , 1994,

classic work is on VGNa where Electrophorus contributed
and AChR where Torpedo came in handy

cannot patch clamp bacteria - so make spheroplasts (6 micro m) for patch clamping

find channels called MscL and MscS for Mechanosensitive channel large (conductance - 3000 pS) and small (1000 pS)

use 37 a.a sequence near N terminus to get mscL gene

comparisons with alamethicin 20 a.a. pore forming peptide.

J. Liu et al., 1996,
also D. P. Corey & J. Garcia-Anoveros, , 1996,

advantages of C. elegans for genetics and neurobiology

Caenorhabditis elegans -unc-105 is a new member of channel superfamily
TRANSPARENCY
and let-2 is a collagen

DEGs (degenerins) - ion influx and swelling - mechanosensation

family tree (dendrogram) TRANSPARENCY (Fig. 1) with structures
MEC-4 and MEC-10 -- proteins which contribute to heteromultimers

related to epithelial sodium channels (ENaCs) which are sensitive to amiloride

Discuss hair cells - 30 - 300 stereocilia TRANSPARENCY (Fig. 2)
deflection toward tallest cause nonselective cation channels to open

some mec genes encode tubulins, some secreted proteins, one a collagen

no relation to bacterial

gap junctions

[background] (note there are also plasmodesmata in plants)
classic example is electrical connection between myocardial cells at intercalated disk
pass larger molecules (fluorescent dyes, sugars) as well as electrical current
basically, a hexagon of 6 transmembrane molecules on one cell are in register with the 6 molecules on the other, a patch (macula) of these is at site where membranes look very close in the EM but still pass dye (an electron dense tracer like lanthanum) extracellularly [as opposed to occluding junction where outer membrane dense lines (as seen in the EM) merge
connexons - protein can be very variable
MIP = major intrinsic protein (between lens cells)
big brain mutant Drosophila
conductance high - 120 pS
appropriate to cover this between "channels" & "chemical synapses"
pass lots of molecules
invertebrate and lower vertebrate nervous systems mostly, not in mammalian brain
a review paper on gap junctions
E. Edelson, Gap junctions: conduits for cell-cell communication, Mosaic 21, 1990, 48-56
TRANSPARENCY

J. Bergoffen et al., Connexin mutations in X-linked Charcot-Marie-Tooth disease, Science, 262, 1993, 2039-2042 (also "Connexin connection" - This week in Science, p. 1951)

disease is a degeneration of peripheral nerve
connexin-32 TRANSPARENCY (Fig. 3)
find 7 mutations in 8 families, one frameshift, the others nonconservative substitutions
gap junction channels - expression in myelin

P. M. T. Dean et al., Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine, Science 264, 1994, 92-95.

aquaporin-1 acts in descending thin loop of Henle and proximal tubule - constitutively high water permeability

collecting ducts regulated by antidiuretic hormone arginine vasopressin
There are mutations in receptor which cause Nephrogenic diabetes insipidus
but they had a patient who did not have this
TRANSPARENCY (Fig. 2) - protein and mutations

Cystic fibrosis

J. R. Riordan et al., Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA, Science 245, 1066-1072, 1989

J. L. Marx, The cystic fibrosis gene is found (news and comment) & The CF gene hits the news, Science 245, 923-925, 1989

most common genetic disorder in Caucasians (1/2000)
chromosome 7
many indications that a chloride channel was at fault
needed to do chromosome jumping
cDNA library from sweat glands (CF patients have very salty skin)
big gene (250 kb), 24 exons, TRANSPARENCY (Fig. 7)
cystic fibrosis transmembrane conductance regulator (CFTR)
1480 amino acids (168, 138 daltons), membrane protein probably channel
has sequences resembling consensus nucleotide (ATP)-binding folds (NBF's)R R domain with 69/241 a.a.'s being charged
consensus sequences for phosphorylation by PKA and PKC
sequence like White gene in Drosophila => transport
10/12 membrane spans have charge => channel
like GABA (gamma amino butyric acid) chloride channel
do not secrete chloride - water does not follow, thick mucus
phe at #508 is missing in 70% of cases near binding site for ATP

Drosophila

L. Salkoff & R. Wyman, Ion channels in Drosophila muscle, Trends in Neurosciences 6, 1983, 128-133

Use of Drosophila (see TRANSPARENCY [home drawn and from Salkoff and Wyman])
this work emphasizes fact that in muscle, a fast potassium channel can be
isolated by different developmental times

B. L. Tempel, Potassium channel genes and genetics in flies, mice and man, Seminars in the Neurosciences 2, 197-205, 1990

(this is a nice paper and an interesting one)

2 TRANSPARENCIES (home drawn and from Tempel)
Lots of different potassium channels with different functions
well-known is the delayed rectifier
then there is the transient K+ current or A-current
In pancreatic beta cells, blocked by intracellular ATP or ADP, extracellular glucose
cloning (walking), expression
S4 with charged arginines

L. Salkoff et al., An essential "set" of K+ channels conserved in flies, mice and humans (viewpoint), Trends in Neurosciences May, 1992, 15-5, 161-166.

List of K+ channels: voltage activated, Ca2+-sensitive, ATP-sensitive and others
all kinds of functions
human pancreatic beta cells, cardiac tissue, mammalian lymphocytes

Shaker gene in fly with alternative splicing - still there must be others
low stringency hybridization to find these - Shal, Shaw, and Shab
Shal, A-type K+ current
Shaw, and Shab - delayed rectifier-type K+ current
TRANSPARENCY (Figs 1 and 2)
fast inactivation "inactivation ball"

Evolution - gene duplication -
find lots in mice - TRANSPARENCY
mammals lack introns (no splicing except Shaw) - duplicated gene many times
ancestral K+ channel had already diverged into 4 types by Cambrian radiation

Drosophila Shaw has only 4 (not 6) S4 charges - less low voltage sensitivity
Co-express in Xenopus oocyte - and compare properties with real ones
-probably no heteromultimers

M. C. Trudeau, HERG, a human inward rectifier in the voltage-gated potassium channel family, Science 269, 92-95, 1995

ether-a-go-go (eag) outward rectifying K+ channel in family with Elk (eag-like) and Erg (eag-related)

inward rectifiers carry currents at voltages negative to K+ equilibrium
in heart, at positive voltages they close contributing to action potential plateau

heterologous expression of HERG in frog oocytes

M-EAG is outward, typical data TRANSPARANCY (Figs 1 and 2)
note outward current is up
some modeling based on S4 properties most recent work "has focused on a family of channels sharing a hydrophobicity plot that predicts two transmembrane domains"

KAT-1 in Arabidopsis is inward rectifier with S4 to sense very negative V in plants

HERG is locus for a long QT syndrome (LQT-2)
review electrocardiogram

L. M. Manuzzu et al., Direct physical measure of conformational rearrangement underlying potassium channel gating, Science 271, 213-216, 1996

Shaker site-specific fluorescent labeling

Fig. 6 (Jan and Jan 1997) is a comparison of the voltage gated K+ channel and the inwardly-rectifying potassium channel. Note, the latter has a very different structure, only 2 membrane spans but does have the pore-lining loop

Fig. 8 (Jan and Jan 1997) is a description of how this channel is activated by the muscarinic cholinergic recrptor (through the G-protein cascade).

R. Hedrich & P. Dietrich, Plant K+ channels: similarity and diversity, Bot. Acta. 109, 1-8, 1996
This paper refers to "green" (from plants) and "red" (from animals)
this work is electrophysiological, and application of the patch clamp has contributed greatly
Of course, nutritionally, for plants and herbivores, potassium is very relevant (macronutrient)
several rapid volume change responses
for instance, here is a TRANSPARENCY from this department's introductory biology text to show the involvement of K+ in guard cell
responses, responsible for opening and closing of stomata
an overall outline:
Inward rectifying voltage dependent K+ channels for K+ uptake
Outward rectifying voltage dependent K+ channels for K+ release
Also there is a high affinity K+ transporter (HKT1) about which little is known though it is thought that protons are cotransported. Such a
system would be used to get K+ in from low concentration in soil.
This paper concentrates on uptake channels like those cloned from:
Arabidosis thaliana (KAT1)
Solanum tuberosum (KST1)
channel conductance is 5-30 pS
for a 10 x change in K+ gradient, voltage changes 56-58 mV in accord with the Nernst potential (see earlier this semester, discussion of
Paramecium)
not many insect, scorpion, snake, frog or dinoflagellate toxins which affect animal channels affect plant channels
But external Cs+ and TEA+ do block, but weakly
In contrast with animal channels, KST1 and KAT1 have ATP and cyclic nucleotide cassettes and several channels are ATP dependent
Like shaker in S1-S6 and H5 or P (pore forming) - TRANSPARENCY with 21 conserved amino acids except that plant channel has extra 14
amino acids
No N-terminal ball and chain and no inactivation
there are AKT1 = Arabidosis K+ transporters which have ankyrin binding domains


Paramecium

Y. Naitoh & R. Eckert, Ionic mechanisms controlling behavioral responses of Paramecium to mechanical stimulation, Science 164, 1969, 963-965

R. Eckert, Bioelectric control of ciliary activity, Science 176, 1972, 473-481

C. Kung et al., Genetic dissection of behavior in Paramecium, Science 188, 1975, 898-904.

Jennings (1906) avoidance reaction (literature onsimple behaviors of lower organisms goes back a long way)
TRANSPARENCY (home drawn and from papers)

Eckert - anterior stimulation - depolarization - spike - Calcium
-posterior stimulation hyperpolarization - Potassium
(These are good figures for understanding the Nernst equation)

Genetics - good (as well as size)
TRANSPARENCY
Autogamy - makes homozygous - good for isolating recessives

Get mutants - "pawn" (pw) cannot swim backwards - no spike- eliminate calcium current
fast-2 (f) - no depolarization
paranoiac (Pa) no downstroke of the action potential
and others - (see below)

R. R. Preston et al., Calmodulin mutants and Ca2+-dependent channels in Paramecium, Annual Review of Physiology, 53, 1991, 309-319

TRANSPARENCY Calmodulin (Alberts et al., Fig. 15.34, p. 750)

Intracellular Calcium is usually low, Ca2+/CaM complex if micromolar
expose hydrophobic patches for interaction with >20 enzymes
Paramecium tetraurelia - one CaM gene; true for most, yeasts to eel
ORF 444 bases, no introns
Calcium transient increases concentration from <10-7 to > 10-5
then delayed K+ channels repolarizes calcium action potential
(this is eliminated in pantophobiac, CaM structural gene
change gene name from pntA to cam1)
ser -> phe at 101 TRANSPARENCY (Fig. 1)
shows cam 1, 2, 3 mutants all near C-tterminus

also there is a calcium-dependent (not voltage-dependent!) sodium current
lacking in fast
enhanced in paranoiac
cam 11, 12, 13 changes (sometimes two changes!) near N-terminus

Maybe two sites interact differently
Other mtants affect Ca2+-dependent K+ channels without affecting CaM
tea for TEA-insensitive and rst for restless

Chloride:

T. J. Jentesch, Chloride channels: a molecular perspective, Current opinion in neurobiology, 1996, 6, 303-310

There are 3 types of chloride channel:
(1) postsynaptic receptors (like for GABA = gama amino butyric acid) and glycine
(2) CFTR (traffci ATPase superfamily, though cystic fibrosis channel is passive)
(3) CLC
TRANSPARENCY
(1) note, for neurotransmitter, there are 4 membrane spans, called M1-M4
(2) the CFTR, with its 12 TM spans with R and NBF (nucleotide binding fold) between 6 & 7 and NBF near C terminus was covered earlier, R domain is for phosphorylation
(3) CLC different, perhaps still some uncertainty

(1) inhibitory, dampen membrane excitability (anion, 110/5-15 gradient, out to in)
(3) TRANSPARENCY dendrogram - show expression and defects (like myotonia CLC-1, Kidney stones CLC-5)

Channels

K. Mikoshiba, The InsP3 receptor and intracellular Ca2+ signaling, Current opinion in Neurobiology, 1997, 7, 339-345
TRANSPARENCY quick-freeze deep-etch technique actually shows channel arrangement
cartoon shows cut-away of 2 of the 4 pore subunits each with 6 transmembrane domains
note that, relative to Shaker, inside lumen is topologically like outside of cell

C.C.H.Peterson, Store operated calcium entry, Seminars in the Neurosciences, 1996, 8, 293-300
TRANSPARENCY Fig. 1 shows a diagram which overlaps considerably with what will be shown later (Friel paper, Invertebrate Phototransduction lecture)
Note that there must be a pump (here called SERCA) to concentrate calcium into ER TRANSPARENCY here is a detail of trp channel
note suggestion that ankyrin repeats may see to trp - InsP3R proximity

In this lecture so far, I have short-changed calcium channels, and now I will only summarize what will be repeated in the synaptic transmission lecture:

In presynaptic terminal:Ca2+ in through Q or N type voltage gated channel
(N stands for "neither, as opposed to T=transient or L=long lasting, the N channel is blocked by omega toxin from Conus [snail genus])

In Muscle: t-tubules get excitation to near sarcoplasmic reticulum
dyhydropyridine (blocking drug) receptor in t-tubule
homology to sodium channel - voltage sensitive

In Muscle: ryanodine receptor in sarcoplasmic reticulum same family as IP3 receptor
coupled with t-tubule

G. A. Cottrell, The first peptide-gated ion channel (Review), The Journal of Experimental Biology 200, 1997, 2377-2386
TRANSPARENCY Fig. 3 shows FMRF amide and related agonists and antagonists
TRANSPARENCY Fig. 1 - shows unusual structure with only 2 transmembrane domains which is like the epithelial Na+ channels and the degenerins

W. N. Zagotta and S. A. Siegelbaum, Structure and function of cyclic nucleotide-gated channels, Annual Review of Neuroscience, 1996, 19, 235-263
TRANSPARENCY compares Shaker and CNG for domains
- there is a cGMP binding domain beyond (toward C-terminus) S1-S5, P, S6
Figure also shows the predicted membrane topography and the 4 subunit structure
TRANSPARENCY amino acid structure and comparisons of pores

D. Ren et al., A prokaryotic voltage-gated sodium channel, Science 294, 2372-2375, 2001.
also
W. A. Catterall, A one-domain voltage-gated sodium channel in bacteria (perspective), Science 294, 2306-232308., 2001
Took until 2001 to get this. Bacillus halodurans - salt-loving
274 aa -> homotetramer
for eukaryote, ion selectivity probably determined by a few a.a.'s in 2nd half of pore loop
some discussion of critical amino acids and how bacterial channel differs from eukaryotic
tetrodotoxin does not block but some calcium channels do block
loop too small for S4 to move, so gating is probably different
inactivation is 100x slower
the ball in chain now has the jargon N-type inactivation,
by contrast, C-type inactivation requires all the loops and that is likely here.

References

M. Barinaga, Playing tetherball in the nervous system, Science 250, 506-507,1990

J. Bergoffen et al., Connexin mutations in X-linked Charcot-Marie-Tooth disease, Science, 262, 1993, 2039-2042

also D. P. Corey & J. Garcia-Anoveros, Mechanosensation and the DEG/ENaC Ion channels (Perspectives), Science 273, 19 July, 1996, 323-324

G. A. Cottrell, The first peptide-gated ion channel (Review), The Journal of Experimental Biology 200, 1997, 2377-2386

P. M. T. Dean et al., Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine, Science 264, 1994, 92-95.

R. Eckert, Bioelectric control of ciliary activity, Science 176, 1972, 473-481

E. Edelson, Gap junctions: conduits for cell-cell communication, Mosaic 21, 1990, 48-56

O. P. Hamill & D. W. McBride, Jr., The cloning of a mechano-gated membrane ion channel (Research News) Trends in Neurosciences 17-11, Nov., 1994, 439-443

O. P. Hamill & D. W. McBride, Jr., Mechanoreceptive membrane channels, American Scientist 83, Jan-Feb 1995, 30-37

R. Hedrich & P. Dietrich, Plant K+ channels: similarity and diversity, Bot. Acta. 109, 1-8, 1996

T. Hoshi et al., Biophysical and molecular mechanisms of Shaker potassium channel inactivation, Science 250, 533-538, 1990

Jan, L. Y., and Y. N. Jan, 1997 Voltage-gated and inwardly rectifying potassium channels. J Physiol (Lond). 505: 267-82.

T. J. Jentesch, Chloride channels: a molecular perspective, Current opinion in neurobiology, 1996, 6, 303-310

C. Kung et al., Genetic dissection of behavior in Paramecium, Science 188, 1975, 898-904.

J. Liu et al., Interaction between a putative mechanosensory membrane channel and a collagen, Science, 273, 1996, 361-364.

J. L. Marx, The cystic fibrosis gene is found (news and comment) & The CF gene hits the news, Science 245, 923-925, 1989

L. M. Manuzzu et al., Direct physical measure of conformational rearrangement underlying potassium channel gating, Science 271, 213-216, 1996

K. Mikoshiba, The InsP3 receptor and intracellular Ca2+ signaling, Current opinion in Neurobiology, 1997, 7, 339-345

Y. Naitoh & R. Eckert, Ionic mechanisms controlling behavioral responses of Paramecium to mechanical stimulation, Science 164, 1969, 963-965

C.C.H.Peterson, Store operated calcium entry, Seminars in the Neurosciences, 1996, 8, 293-300

D. Ren et al., A prokaryotic voltage-gated sodium channel, Science 294, 2372-2375, 2001.
also
W. A. Catterall, A one-domain voltage-gated sodium channel in bacteria (perspective), Science 294, 2306-232308., 2001

J. R. Riordan et al., Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA, Science 245, 1066-1072, 1989

L. Salkoff et al., An essential "set" of K+ channels conserved in flies, mice and humans (viewpoint), Trends in Neurosciences May, 1992, 15-5, 161-166.

L. Salkoff & R. Wyman, Ion channels in Drosophila muscle, Trends in Neurosciences 6, 1983, 128-133

R. TAYLOR, Evolutions: The voltage-gated sodium channel, The Journal of NIH Research 6, November, 1994, 100-102 & 111-112.

B. L. Tempel, Potassium channel genes and genetics in flies, mice and man, Seminars in the Neurosciences 2, 197-205, 1990

M. C. Trudeau, HERG, a human inward rectifier in the voltage-gated potassium channel family, Science 269, 92-95, 1995

W. N. Zagotta and S. A. Siegelbaum, Structure and function of cyclic nucleotide-gated channels, Annual Review of Neuroscience, 1996, 19, 235-263

This page was last updated on Jan. 24, 2002

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