Research work of Karlin group:
The long-term goal of our research is to understand
the function of the nicotinic acetylcholine (ACh) receptors in terms
of their molecular structures (Karlin, 2002). As we originally showed,
the muscle-type ACh receptors have five subunits arranged around
the central channel in the order alpha-gamma-alpha-beta-delta (Karlin
et al., 1983) (Figure 1).
Each subunit has four membrane spanning segments
(M1-M4), and our recent research has been aimed at the structure
and function of these segments. We are attempting to classify every
residue in M1–M4 as water facing, lipid facing, or buried.
Many of the water-facing residues, which include the residues lining
of the channel, have been identified by application of the substituted
cysteine-accessibility method (SCAM), which we developed (Karlin
and Akabas, 1998) (Figure 2).
In SCAM, reactions are quantitated by their perturbation
of receptor function, as measured electrophysiologically. Using
this approach, we have also mapped the resting (Wilson and Karlin,
1998) and desensitization (Wilson and Karlin, 2001) gates (Figure
3), the intrinsic electrostatic potential in the open and resting
channel (Pascual and Karlin, 1998; Wilson et al., 2000) (Figure
4) and the site of binding of open-channel blockers (Pascual and
Karlin, 1998; Yu, Shi, and Karlin, 2003) (Figure 5).
Using a different approach that we developed on
a KcsA, a bacterial potassium channel (Li, Shi, and Karlin, 2003),
we are identifying the lipid facing residues, substituted by cysteines,
by their photoreaction, in the presence and absence of ACh, with
a hydrophobic photolabel that strongly prefers cysteine, quantitating
the reactivity of residues by a cysteine specific gel shift assay.
In addition we are mapping the relative arrangement and paths through
the membrane of M1-M4 by crosslinking. The surface exposure of each
residue and the paths of the segments through the membrane will
be the bases for a detailed model of the 2°, 3° and 4°
structure of the membrane domain. The differences in reaction rates
in the presence and in the absence of ACh will identify structural
elements that move during changes in the functional state of the
1. Yu Y, Shi L, Karlin
A. Structural effects of quinacrine binding in the open channel
of the acetylcholine receptor. Proc Natl Acad Sci U S A. 2003 Apr
2. Li J, Shi L, Karlin A. A photochemical
approach to the lipid accessibility of engineered cysteinyl residues.
Proc Natl Acad Sci U S A. 2003 Feb 4;100(3):886-91.
3. Li J, Xu Q, Cortes DM, Perozo E,
Laskey A, Karlin A. Reactions of cysteines substituted in the amphipathic
N-terminal tail of a bacterial potassium channel with hydrophilic
and hydrophobic maleimides. Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11605-10.
4. Li Y, Karlin A, Loike JD, Silverstein
SC. A critical concentration of neutrophils is required for effective
bacterial killing in suspension. Proc Natl Acad Sci U S A. 2002
5. Karlin A. Emerging structure of
the nicotinic acetylcholine receptors. Nat Rev Neurosci. 2002 Feb;3(2):102-14.
6. Karlin A. The acetylcholine-binding
protein: 'What's in a name?'. Pharmacogenomics J. 2001;1(4):221-3.
7. Karlin A. Of snakes, snails, and
surrogates. Neuron. 2001 Oct 25;32(2):173-4.
8. Hastrup H, Karlin A, Javitch JA.
Symmetrical dimer of the human dopamine transporter revealed by
cross-linking Cys-306 at the extracellular end of the sixth transmembrane
segment. Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10055-60.
9. Karlin A. Scam feels the pinch.
J Gen Physiol. 2001 Mar;117(3):235-8.
10. Wilson G, Karlin A. Acetylcholine
receptor channel structure in the resting, open, and desensitized
states probed with the substituted-cysteine-accessibility method.
Proc Natl Acad Sci U S A. 2001 Jan 30;98(3):1241-8. Epub 2001 Jan
11. Sahin-Toth M, Karlin A, Kaback
HR. Unraveling the mechanism of the lactose permease of Escherichia
coli. Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):10729-32.
12. Wilson GG, Pascual JM, Brooijmans
N, Murray D, Karlin A. The intrinsic electrostatic potential and
the intermediate ring of charge in the acetylcholine receptor channel.
J Gen Physiol. 2000 Feb;115(2):93-106.
13. Pascual JM, Karlin A. Delimiting
the binding site for quaternary ammonium lidocaine derivatives in
the acetylcholine receptor channel. J Gen Physiol. 1998 Nov;112(5):611-21.
14. Karlin A, Akabas MH. Substituted-cysteine
accessibility method. Methods Enzymol. 1998;293:123-45.
15. Wilson GG, Karlin A. The location
of the gate in the acetylcholine receptor channel. Neuron. 1998
16. Pascual JM, Karlin A. State-dependent
accessibility and electrostatic potential in the channel of the
acetylcholine receptor. Inferences from rates of reaction of thiosulfonates
with substituted cysteines in the M2 segment of the alpha subunit.
J Gen Physiol. 1998 Jun;111(6):717-39.
17. Zhang H, Karlin A. Contribution
of the beta subunit M2 segment to the ion-conducting pathway of
the acetylcholine receptor. Biochemistry. 1998 Jun 2;37(22):7952-64.
18. Zhang H, Karlin A. Identification
of acetylcholine receptor channel-lining residues in the M1 segment
of the beta-subunit. Biochemistry. 1997 Dec 16;36(50):15856-64.
19. Martin MD, Karlin A. Functional
effects on the acetylcholine receptor of multiple mutations of gamma
Asp174 and delta Asp180. Biochemistry. 1997 Sep 2;36(35):10742-50.
20. Karlin A. Transport bicycles. Proc
Natl Acad Sci U S A. 1997 May 27;94(11):5508-9. Review.
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