Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

FIGURE 10.25

<1 µs

10.6 ● Transport Processes Driven by Ion Gradients

311

formational changes (Figure 10.25), but no deprotonation of the Schiff base occurs during the halorhodopsin photocycle. Given the striking similarity of structures for these two proteins, it is intriguing to ask why bacteriorhodopsin pumps H but not Cl and why halorhodopsin pumps Cl but not H . The first question may be answered by the work of H. G. Khorana and his coworkers, who replaced Asp85 and Asp96 in bacteriorhodopsin with asparagine and found that either substitution caused a drastic reduction in H transport. Dieter Oesterhelt and coworkers have shown that Asp85 and Asp96 are important in the deprotonation and reprotonation, respectively, of the Schiff base in bacteriorhodopsin. The absence of these two crucial residues in halorhodopsin may explain why the latter protein can’t reversibly deprotonate the Schiff base and why halorhodopsin doesn’t pump protons.

10.6 ● Transport Processes Driven by Ion Gradients

Amino Acid and Sugar Transport

The gradients of H , Na , and other cations and anions established by ATPases and other energy sources can be used for secondary active transport of various substrates. The best-understood systems use Na or H gradients to transport amino acids and sugars in certain cells. Many of these systems operate as symports, with the ion and the transported amino acid or sugar moving in the same direction (that is, into the cell). In antiport processes, the ion and the other transported species move in opposite directions. (For example, the anion transporter of erythrocytes is an antiport.) Proton symport proteins are used by E. coli and other bacteria to accumulate lactose, arabinose, ribose, and a variety of amino acids. E. coli also possesses Na -symport systems for melibiose as well as for glutamate and other amino acids.

Table 10.2 lists several systems that transport amino acids into mammalian cells. The accumulation of neutral amino acids in the liver by System A rep-

hυ	Cl–	all-trans	hυ
		+
		C = NH
	hR565	hR578	5 ps
	2 ms Cl–
		10 ms
hR630	hR640	hR660	hR632
(77 K)

300 µs

<1 µs

hR500

13-cis

● The photocycle of lightadapted halorhodopsin (hR), shown in the presence and absence of chloride. The superscripts indicate the maxima of the difference spectra between hR and the intermediates.

Table 10.2

Some Mammalian Amino Acid Transport Systems

System		Amino Acids
Designation	Ion Dependence	Transported	Cellular Source
A	Na	Neutral amino acids
ASC	Na	Neutral amino acids
L	Na -independent	Branched-chain and	Ehrlich ascites cells
		aromatic amino acids	Chinese hamster
			ovary cells
			Hepatocytes
N	Na	Nitrogen-containing
		side chains
		(Gln, Asn, His, etc.)
y	Na -independent	Cationic amino acids
xAG	Na	Aspartate and glutamate	Hepatocytes
P	Na	Proline	Chinese hamster
			ovary cells

Adapted from: Collarini, E. J., and Oxender, D. L., 1987. Mechanisms of transport of amino acids across

membranes. Annual Review of Nutrition 7:75–90.

FIGURE 10.26

EIIIg

Glucose-6-P

Inside

Outside

312 Chapter 10 ● Membrane Transport

PEP Pyruvate

1 Phosphorylation

of EI by PEP

EI

EI ~ P

2 Transfer of phosphoryl group from EI to HPr

HPr ~ P HPr

3 Transfer of phosphoryl group from HPr to EIII

EIIIg ~ P

4 Transfer of phosphoryl group44 from EIII to EII and then to glucose to form glucose-6-P

EIIg

Glucose

● Glucose transport in E. coli is mediated by the PEP-dependent phosphotransferase system. Enzyme I is phosphorylated in the first step by PEP. Successive phosphoryl transfers to HPr and Enzyme III in Steps 2 and 3 are followed by transport and phosphorylation of glucose. Enzyme II is the sugar transport channel.

PEP Pyruvate

O–

–O P O

4 N

3

2

1

5 N

Enzyme I

HPr

resents an important metabolic process. Thus, plasma membrane transport of alanine is the rate-limiting step in hepatic alanine metabolism. This system is normally expressed at low levels in the liver, but substrate deprivation and hormonal activation both stimulate System A expression.

10.7 ● Group Translocation

Certain bacteria possess a novel and versatile system for the inward transport of certain sugars. In this process, the sugar becomes phosphorylated during its transport across the membrane; that is, transport and phosphorylation are tightly coupled. This type of process, in which a chemical modification accompanies transport, has been denoted group translocation. Several such systems are known, but the best understood is the phosphoenolpyruvate:glucose phosphotransferase system, or simply the phosphotransferase system (or PTS), discovered by Saul Roseman of Johns Hopkins University in 1964. The advantage of this system lies in the fact that the sugars, once phosphorylated, are trapped in the cell. Membranes are permeable to simple sugars but impermeable to sugar phosphates, which are negatively charged. The overall reaction for the phosphotransferase is:

Sugaroutside PEPinside 88n sugar-Pinside pyruvateinside

The subscripts illustrate an important point: the phosphoryl transfer occurs entirely on the inside surface of the bacterial membrane.

Several unique features distinguish the phosphotransferase. First, phosphoenolpyruvate is both the phosphoryl donor and the energy source for sugar transport. Second, four different proteins are required for this transport. Two of these proteins (Enzyme I and HPr) are general and are required for the phosphorylation of all PTS-transported sugars. The other two proteins (Enzyme II and Enzyme III) are specific for the particular sugar to be transported.

The first step in the phosphotransferase reaction (Figure 10.26) is the phosphorylation of Enzyme I by PEP to form a reactive phosphohistidine intermediate (Figure 10.27). This is followed by phosphoryl transfer to a histidyl residue of HPr, followed by phosphorylation of Enzyme III. At the same time, the sugar to be transported is bound on the outside surface of the cell by Enzyme II, which constitutes the sugar transport channel. As the sugar is moved to the inside surface of the membrane, the phosphoryl group is transferred from Enzyme III to the sugar, forming the desired sugar phosphate, which is

		O–
	N	–O P O	N	Sugar-P
		N
	N	N	N
	–O P		–O P O	Sugar
		O
	O–	His10	O–
	His15		His175
		Enzyme III

FIGURE 10.27 ● The path of the phosphoryl group through the PTS mechanism. Reactive phosphohistidine intermediates of Enzyme I, HPr, and Enzyme III transfer phosphoryl groups from PEP to the transported sugar.

10.8 ● Specialized Membrane Pores

313

released into the cytoplasm. (In some cases, for example the E. coli mannitol system, no Enzyme III has been identified. In these cases, the C-terminal end of the relevant Enzyme II, which resembles an Enzyme III-type sequence, substitutes for Enzyme III.)

10.8 ● Specialized Membrane Pores

Porins in Gram-Negative Bacterial Membranes

The membrane transport systems described previously (and many others like them) are relatively specific and function to transport either a single substrate or a very limited range of substrates under normal conditions. At the same time, several rather nonspecific systems also carry out transport processes. One such class of nonspecific transport proteins is found in the outer membranes of Gram-negative bacteria and mitochondria. Low-molecular-weight nutrients and certain other molecules, such as some antibiotics, cross this outer membrane, but larger molecules such as proteins cannot. The ability of the outer membrane to act as a molecular sieve is due to proteins called porins (Chapter 9). Alternatively, these molecules have been referred to as peptidoglycan-asso- ciated proteins or simply matrix proteins. General porins form nonspecific pores across the outer membrane and sort molecules according to molecular size, whereas specific porins contain binding sites for particular substrates. Porins from several organisms have been isolated and characterized (Table 10.3). Molecular masses of the porins generally range from 30 kD to 50 kD. Most (but not all) porins are arranged in the outer membrane as trimers of identical subunits. The molecular exclusion limits clearly depend on the size of the pore formed by the porin molecule. The pores formed by E. coli and S. typhimurium porins are relatively small, but porin F from Pseudomonas aeruginosa creates a much larger pore, with an exclusion limit of approximately 6 kD. Specific porins LamB and Tsx of E. coli and porins P and DI of P. aeruginosa possess specific binding sites for maltose and related oligosaccharides (Table 10.4), nucleosides, anions, and glucose, respectively.

Table 10.3

Properties of Some General Porins

Porin and Bacterial Source	Pore Diameter (nm)	Mr Exclusion Limit
E. coli
OmpF	1.2
OmpC	1.1	600
PhoE	1.2
S. typhimurium
Mr 38,000	1.4
Mr 39,000	1.4	700
Mr 40,000	1.4
P. aeruginosa
F	2.2	6000

Source: Adapted from Benz, R., 1984. Structure and selectivity of porin channels. Current Topics in Membrane

Transport 21:199–219; and Benz, R., 1988. Structure and function of porins from Gram-negative bacteria.

Annual Review of Microbiology 42:359–393.

FIGURE 10.28

314 Chapter 10 ● Membrane Transport

Table 10.4

Binding and Permeation Properties of the LamB

Channel for Different Sugars

Sugar	K s (mM)*	P (s 1)†
Maltose	10.000	100.0
Maltotriose	0.400	66.0
Maltoheptaose	0.067	2.5
Lactose	56.000	9.0
Sucrose	15.000	2.5
D-Glucose	110.000	290.0
L-Glucose	46.000	—
D-Galactose	42.000	225.0
D-Fructose	600.000	135.0
D-Mannose	160.000	160.0
Stachyose	50.000	1.0

*Half-saturation constant (concentration for 50% saturation of the transport protein).

†Rate of permeation relative to that of maltose. Data adjusted to 100 s 1 for maltose. The LamB-containing liposomes were added to buffer solutions containing 40 mM of the corresponding test sugars.

Source: Adapted from Benz, R. 1988. Structure and function of porins from Gram-negative bacteria. Annual

Review of Microbiology 42:359–393.

Porins show high degrees of sequence homology and similarity. The most intriguing feature of porin secondary and tertiary structure is this: In contrast to nearly all other membrane proteins that adopt -helical structures in the transmembrane segments, porins show little or no evidence of -helical domains and segments. Instead, the porins and other outer membrane proteins adopt -sheet structures for their membrane-spanning segments. Models of membrane insertion, which involve -strands arranged perpendicular to the membrane plane, have been proposed for several porins (Figure 10.28). The crystal structure of the porin from Rhodobacter capsulatus shows a trimer in which each monomer forms a pore (Figure 10.29). The monomer pore consists of a 16-stranded -barrel that traverses the membrane as a tube. The tube is nar-

Outside

Inside

Asp Ser

Gly

Ser

Gly

Ala

Thr Gly

Ala

Phe

Gln

Leu

Ile

Asn

Ser

Thr

Ala

Glu

Thr

Val

Lys

Asn Gln

Ile

Gly

Gly

Gly

Lys Leu

Asp

Lys

Asp

Pro

Asp

Asp

Gln

Ser

Pro

Phe

Glu

Asp

Asn

Asn

Ser

Asp

Ser

Gln

Phe

Asn

Arg

Lys

Glu

Arg

Thr

Ala

Ile

Glu

Asp

Ile

Met

Gly

Glu

Gln

Met

Phe

Asn

Gln

Asn

Gly

Met

Asn

Asp

Asp

Ser

Asn

Tyr

Asp

Ala

Lys

Asp

Met

Glu

Asn

Thr

Thr

Lys

Lys

Lys

Leu

Asp

Asn

His

Gln

Lys

Thr

Thr

Thr

Asn

Gly

Arg

Gly

Arg

Thr

Gly

Val

Leu

Asp

Met

Ser

Asn

Arg

Trp

Lys

Lys

Asp

Asp

Lys

Thr

Gln

Lys

Asn

Gln

Asp

Ala

Tyr

Gly

Leu

Ala

Arg

Gly

Gly

Ser

Arg

Glu

Asn

Ser

Tyr

Asn

Ile

Lys

Ile

Ala

Ala

Glu

Ala

Gln

Phe

Asn

Ala

Ser

Phe

Leu

Ile

Ile

Val

Val

Arg

Phe

Phe

Val

Ser

Tyr

Gly

Thr

Glu

Tyr

Glu

Val

Asp

Lys

Ala

Lys

Phe

Glu

Ala

Asp

Gly

Gln

Thr

Tyr

Ala

Phe

Ala

Tyr

Val

Tyr

Val

Gly

Gly

Ala

Gly

Tyr

Leu

Leu

Ser

Ala

Trp

Thr

Val

Gly

Gly

Asp

Gly

Tyr

Phe

Glu

Leu

Leu

Ala

Thr

Leu

Gly

Ala

Ala

Ala

Leu

Ala

Val

Met

Val

Lys

Trp

Lys

Ala

Thr

Leu

Thr

Ser

Thr

Leu

Gln

Ser

Thr

Phe

Thr

Asp

Gly

Arg

Tyr

Gly

Tyr

Asn

Tyr

Ile

Gly

Tyr

Tyr

Pro

Tyr

Ala

Tyr

Leu

Glu

Gly

Lys

Leu

Arg

Leu

Asp

Ala

Leu

Ile

Gln

Arg

Tyr

Ser

Gln

Lys

Thr

Tyr

Asp

Asn

Asn

Gly

Phe

Phe

Lys

Asn

Phe

Leu

Phe

Met

Phe

NH3+

Asn

Gln

Gly

Leu

Arg

Thr

Asp

Gly

Asp

Tyr

Asn

Asp

Gly

Asn

Asn

COO–

Gly

Ile

Thr

Gly

Gly

Asp

Ile

Gly Ser

Asp Ala

Phe

Lys

Ala

Asp

Asn

Leu

Ser

Tyr

Phe

Val

Glu

Lys

Asp Gln

Phe Asp

Phe Gly

Ile Tyr

Asn

● A model for the arrangement of the porin PhoE in the outer membrane of E. coli. The transmembrane segments are strands of -sheet.

10.8 ● Specialized Membrane Pores

315

(a)

(b)

rowed near the center by peptide chain segments protruding from the inner wall of the barrel. These chain segments form an “eyelet” about 1 nm long and 0.6 to 1 nm across. The eyelet is postulated to determine the exclusion limit for particles diffusing through the pore.

Porins and the other outer membrane proteins of Gram-negative bacteria appear to be the only known membrane proteins that have chosen the -strand over the -helix. Why might this be? Among other reasons, there is an advantage of genetic economy in the use of -strands to traverse the membrane instead of -helices. An -helix requires 21 to 25 amino acid residues to span a typical biological membrane; a -strand can cross the same membrane with 9 to 11 residues. Therefore, a given amount of genetic information could encode a larger number of membrane-spanning segments using a -strand motif instead of -helical arrays. Further, -strands can present alternating hydrophobic and hydrophilic R groups along their length, with hydrophobic R groups facing the lipid bilayer and hydrophilic R groups facing the waterfilled channel (Chapter 9).

The Pore-Forming Toxins

Many organisms produce lethal molecules known as pore-forming toxins, which insert themselves in a host cell’s plasma membrane to form a channel or pore. Pores formed by such toxins can kill the host cell by collapsing ion gradients or by facilitating the entry of toxic agents into the cell. Produced by a variety of organisms and directed toward a similarly diverse range of target cells, these toxins nonetheless share certain features in common. The structures of these remarkable toxins have provided valuable insights into the mechanisms of their membrane insertion and also into the architecture of membrane proteins.

Colicins are pore-forming proteins, produced by certain strains of E. coli, that kill or inhibit the growth of other, competing bacteria and even other strains of E. coli (a process known as allelopathy). Channel-forming colicins are released as soluble monomers. Upon encountering a host cell, the colicin molecule traverses the bacterial outer membrane and periplasm, then inserts itself

(c)

FIGURE 10.29 ● Three-dimensional reconstruction of porin from Rhodobacter capsulatus.

Drawings of (a) side view of porin monomer showing -sheet structure. (b) Top view and

(c) nearly top view of porin trimer.

FIGURE 10.31

FIGURE 10.30

316 Chapter 10 ● Membrane Transport

N

2	9
	10
1

C

(a)

N

8	9
	10

C

● The structure of colicin Ia. Colicin Ia, with a total length of 210 Å, spans the periplasmic space of a Gram-negative bacterium host, with the R (receptor-bind- ing) domain (blue) anchored to proteins in the outer membrane and the C domain (violet) forming a channel in the inner membrane. The T (translocation) domain is shown in red. The image on the right shows details of the C domain, including helices 8 and 9 (green), which are highly hydrophobic.

		(b)
		Closed state
		N
1	2	7
			6
		3		4
	10
		8

(c)
Open state
N
10
3
7	5
4

(d)

● The umbrella model of membrane channel protein insertion. Hydrophobic helices insert directly into the core of the membrane, with amphipathic helices arrayed on the surface like an open umbrella. A trigger signal (low pH or a voltage gradient) draws some of the amphipathic helices into and across the membrane, causing the pore to open.

into the inner (plasma) membrane. The channel thus formed is monomeric and a single colicin molecule can kill a host cell. The structure of colicin Ia, a 626-residue protein, is shown in Figure 10.30. It consists of three domains, termed the T (translocation) domain, the R (receptor-binding) domain, and the C (channel-forming) domain. The T domain mediates translocation across the outer membrane, the R domain binds to an outer-membrane receptor, and the C-domain creates a voltage-gated channel across the inner membrane. The T, R, and C domains are separated by long (160 Å) -helical segments. The peptide is folded at the R domain, so that the C and T domains are juxtaposed and the two long helices form an underwound antiparallel coiled coil. The protein is unusually elongated—210 Å from end to end—with the T and C domains at one end and R at the other. This unusual design permits colicin Ia to span the periplasmic space (which has an average width of 150 Å) and insert in the inner membrane.

The nature of the channel-forming domain provides clues to the process of channel formation in the inner membrane. The C domain consists of a 10helix bundle, with helices 8 and 9 forming an unusually hydrophobic hairpin structure. The other eight helices are amphipathic and serve to stabilize hydrophobic helices 8 and 9 in solution. When this domain inserts in the inner membrane, helices 8 and 9 inject themselves into the hydrophobic membrane core, leaving the other helices behind on the membrane surface (Figure 10.31). Application of a transmembrane potential (voltage) then triggers the amphipathic helices to insert into the membrane, with their hydrophobic faces facing the hydrophobic bilayer and their polar faces forming the channel surface. This model is hypothetical, but it is supported by studies showing that channel opening involves dramatic structural changes and that helices 2 to 5 move across the membrane during channel opening.

Interestingly, certain other pore-forming toxins possess helix-bundle motifs that may participate in channel formation, in a manner similar to that proposed for colicin Ia. For example, the -endotoxin produced by Bacillus thuringiensis is toxic to Coleoptera insects (beetles) and is composed of three domains, including a seven-helix bundle, a three-sheet domain, and a -sand- wich. In the seven-helix bundle, helix 5 is highly hydrophobic, and the other six helices are amphipathic. In solution (Figure 10.32), the six amphipathic

10.8 ● Specialized Membrane Pores

317

(a)

(b)

FIGURE 10.32 ● The structures of (a) -endotoxin (two views) from Bacillus thuringiensis and (b) diphtheria toxin from Corynebacterium diphtheriae. Each of these toxins possesses a bundle of -helices which is presumed to form the transmembrane channel when the toxin is inserted across the host membrane. In -endotoxin, helix 5 (white) is surrounded by 6 helices (red) in a 7-helix bundle. In diphtheria toxin, three hydrophobic helices (white) lie at the center of the transmembrane domain (red).

helices surround helix 5, with their nonpolar faces apposed to helix 5 and their polar faces directed to the solvent. Membrane insertion and channel formation may involve initial insertion of helix 5, as in Figure 10.31, followed by insertion of the amphipathic helices, so that their nonpolar faces contact the bilayer lipids and their polar faces line the channel.

There are a number of other toxins for which the helical channel model is inappropriate. These include -hemolysin from Staphylococcus aureus, aerolysin from Aeromonas hydrophila, and the anthrax toxin protective antigen from Bacillus anthracis. The membrane-spanning domains of these proteins do not possess long stretches of hydrophobic residues that could form -helical transmembrane segments. They do, however, contain substantial peptide segments of alternating hydrophobic and polar residues. Like the porins, such segments can adopt -strand structures, such that one side of the -strand is hydrophobic and the other side is polar. Oligomeric association of several such segments can produce a -barrel motif, with the inside of the barrel lined with polar residues and the outside of the barrel coated with hydrophobic residues —a motif that can be accommodated readily in a bilayer membrane, creating a polar transmembrane channel.

-Hemolysin, a 33.2-kD monomer protein, forms a mushroom-shaped heptameric pore, 100 Å in length, with a diameter that ranges from 14 Å to 46 Å (Figure 10.33). In this structure, each monomer contributes two -strands 65 Å long, which are connected by a hairpin turn. The interior of the 14stranded -barrel structure is hydrophilic and the hydrophobic outer surface of the barrel is 28 Å wide. Pores formed by -hemolysin in human erythrocytes, platelets, and lymphocytes allow rapid Ca2 influx into these cells with toxic consequences.

Aeromonas hydrophila is a bacterium that causes diarrheal diseases and deep wound infections. These complications arise due to pore formation in sensitive cells by the protein toxin aerolysin. Proteolytic processing of the 52-kD precursor proaerolysin (Figure 10.34) produces the toxic form of the protein, aerolysin. Like -hemolysin, aerolysin monomers associate to form a heptameric transmembrane pore. Michael Parker and coworkers have proposed

FIGURE 10.33 ● The structure of the heptameric channel formed by -hemolysin. Each of the seven subunits contributes a -sheet hairpin to the transmembrane channel.

318 Chapter 10 ● Membrane Transport

FIGURE 10.34 ● The structure of proaerolysin, produced by Aeromonas hydrophila. Proteolysis of this precursor yields the active form, aerolysin, which is responsible for the pathogenic effects of the bacterium in deep wound infections and diarrheal diseases. Like hemolysin, aerolysin monomers associate to form heptameric membrane pores. The three-strands that contribute to the formation of the heptameric pore are shown in red. The N- terminal domain (residues 1–80, yellow) is a small lobe that protrudes from the rest of the protein.

Alamethicin I1:

that each monomer in this aggregate contributes three -strands to the -bar- rel pore. Each of these -strands (residues 277 to 287, 290 to 302, and 410 to 422) consists of alternating hydrophobic and polar residues, so that the pore once again places polar residues toward the water-filled channel and nonpolar residues facing the lipid bilayer.

Whether crossing the membrane with aggregates of amphipathic -helices or -barrels, these pore-forming toxins represent Nature’s accommodation to a structural challenge facing all protein-based transmembrane channels: the need to provide hydrogen-bonding partners for the polypeptide backbone NOH and CPO groups in an environment (the bilayer interior) that lacks hydrogen-bond donors or acceptors. The solution to this problem is found, of course, in the extensive hydrogen-bonding possibilities of -helices and-sheets.

Amphipathic Helices Form Transmembrane Ion Channels

Recently, a variety of natural peptides that form transmembrane channels have been identified and characterized. Melittin (Figure 10.35) is a bee venom toxin peptide of 26 residues. The cecropins are peptides induced in Hyalophora cecropia (Figure 10.36) and other related silkworms when challenged by bacterial infections. These peptides are thought to form -helical aggregates in mem-

Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Phol*

Cecropin A:

Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Glu-Lys-Val-Gly-Gln-Asn-Ile-Arg-Asp-Gly-Ile-Ile-Lys-Ala-Gly-Pro-Ala-Val-Ala-Val-Val-Gly-Gln-Ala-Thr-Gln-Ile-Ala-Lys-NH2

Melittin:

Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH2

Magainin 2 amide:

Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-Met-Asn-Ser-NH2

Polar	(Glu19)		Nonpolar
	Gly
Ser	1	Phe	23-NH2)
8		12
Ala

FIGURE 10.35 ● The amino acid sequences of several amphipathic peptide antibiotics. - Helices formed from these peptides cluster polar residues on one face of the helix, with nonpolar residues at other positions.

	15	5
(Asn22)Lys	4	16 Phe
Lys 11		9	Ala
	Magainin 2 amide
Gly	18	2	Ile(Ile20)
	7	13
His		Gly
	14	6
	Lys	17
	3
		10

	Gly	Val
		Lys
	(Met21)

10.8 ● Specialized Membrane Pores

319

FIGURE 10.36 ● Adult (left) and caterpillar (right) stages of the cecropia moth,

Hyalophora cecropia. (left, Greg Neise/Visuals Unlimited; right, Patti Murray/Animals, Animals)

branes, creating an ion channel in the center of the aggregate. The unifying feature of these helices is their amphipathic character, with polar residues clustered on one face of the helix and nonpolar residues elsewhere. In the membrane, the polar residues face the ion channel, leaving the nonpolar residues elsewhere on the helix to interact with the hydrophobic interior of the lipid bilayer.

A D E E P E R L O O K

Melittin—How to Sting Like a Bee

The stings of many stinging insects, like wasps, hornets, and bumblebees, cause a pain that, although mild at first, increases in intensity over 2 to 30 minutes, with a following period of swelling that may last for several days. The sting of the honey bee (Apis mellifera), on the other hand, elicits a sharp, stabbing pain within 10 seconds. This pain may last for several minutes and is followed by several hours of swelling and itching. The immediate, intense pain is caused by melittin, a 26-residue peptide that constitutes about half of the 50 g (dry weight) of material injected during the “sting” (in a total volume of only 0.5 L). How does this simple peptide cause the intense pain that accompanies a bee sting?

The pain appears to arise from the formation of melittin pores in the membranes of nociceptors, free nerve endings that detect harmful (“noxious”—thus the name) stimuli of violent mechanical stress, high temperatures, and irritant chemicals. The creation of pores by melittin depends on the nociceptor membrane potential. Melittin in water solution is tetrameric. However, melittin interacting with membranes in the absence of a membrane potential is monomeric and shows no evidence of oligomer

formation. When an electrical potential (voltage) is applied across the membrane, melittin tetramers form and the membrane becomes permeable to anions such as chloride. Nociceptor membranes maintain a resting potential of 70 mV (negative inside). When melittin binds to the nociceptor membrane, the flow of chloride ions out of the cell diminishes the transmembrane potential, stimulating the nerve and triggering a pain response and also inducing melittin tetramers to dissociate. When the membrane potential is re-established, melittin tetramers reform and the cycle is repeated over and over, causing a prolonged and painful stimulation of the nociceptors. The pain of the sting eventually lessens, perhaps due to the molecules of melittin diffusing apart, so that tetramers can no longer form.

Although the honey bee’s sting is unpleasant, this tiny creature is crucial to the world’s agricultural economy. Honey bees produce more than $100 million worth of honey each year, and, more importantly, the pollination of numerous plants by honey bees is responsible for the production of $20 billion worth of crops in the United States alone.

320 Chapter 10 ● Membrane Transport

Gap Junctions in Mammalian Cell Membranes

When cells lie adjacent to each other in animal tissues, they are often connected by gap junction structures, which permit the passive flow of small molecules from one cell to the other. Such junctions essentially connect the cells metabolically, providing a means of chemical transfer and communication. In certain tissues, such as heart muscle that is not innervated, gap junctions permit very large numbers of cells to act synchronously. Gap junctions also provide a means for transport of nutrients to cells disconnected from the circulatory system, such as the lens cells of the eye.

Gap junctions are formed from hexameric arrays of a single 32-kD protein. Each subunit of the array is cylindrical, with a length of 7.5 nm and a diameter of 2.5 nm. The subunits of the hexameric array are normally tilted with respect to the sixfold axis running down the center of the hexamer (Figure 10.37). In this conformation, a central pore having a diameter of about 1.8 to 2.0 nm is created, and small molecules (up to masses of 1 kD to 1.2 kD) can pass through unimpeded. Proteins, nucleic acids, and other large structures cannot. A complete gap junction is formed from two such hexameric arrays, one from each cell. A twisting, sliding movement of the subunits narrows the channel and closes the gap junction. This closure is a cooperative process, and a localized conformation change at the cytoplasmic end assists in the closing of the channels. Because the closing of the gap junction does not appear to involve massive conformational changes in the individual subunits, the free energy change for closure is small.

Although gap junctions allow cells to communicate metabolically under normal conditions, the ability to close gap junctions provides the tissue with an important intercellular regulation mechanism. In addition, gap junctions provide a means to protect adjacent cells if one or more cells are damaged or

FIGURE 10.37 ● Gap junctions consist of hexameric arrays of cylindrical protein subunits in the plasma membrane. The subunit cylinders are tilted with respect to the axis running through the center of the gap junction. A gap junction between cells is formed when two hexameric arrays of subunits in separate cells contact each other and form a pore through which cellular contents may pass. Gap junctions close by means of a twisting, sliding motion in which the subunits decrease their tilt with respect to the central axis. Closure of the gap junction is Ca2 -dependent.