Pamela A. Pappone

A Flow-Activated Chloride-Selective Membrane Current in Vascular Endothelial Cells

Shear stress-induced activation of endothelial ion channels, one of the earliest responses to flow, is implicated in mechano-signal transduction that results in the regulation of vascular tone. The effects of laminar flow on endothelial membrane potential were studied in vitro using both fluorescent potentiometric dye measurements and whole-cell patch-clamp recordings. The application of flow stimulated membrane hyperpolarization, which was reversed to depolarization within 35 to 160 seconds. The depolarization was caused by a Cl(-)-selective membrane current activated by flow independently of the K(+) channel-mediated hyperpolarization. Thus, flow activated both K(+) and Cl(-) currents, with the net membrane potential being determined by the balance of the responses. Membrane potential sensitivity to flow was unchanged by flow preconditioning that elongated and aligned the cells.

Mutations in the M4 domain of Torpedo californica acetylcholine receptor dramatically alter ion channel function

Site-directed mutagenesis was used to mutate alpha Cys418 and beta Cys447 in the M4 domain of Torpedo californica acetylcholine receptor expressed in Xenopus laevis oocytes. The M4 region is a transmembrane domain thought to be located at the lipid-protein interface. By whole-cell voltage clamp analysis, mutation of both alpha subunits to alpha Trp418 increased maximal channel activity approximately threefold, increased the desensitization rate compared with wild-type receptor, and shifted the EC50 for acetylcholine from 32 microM to 13 microM. Patch measurements of single-channel currents revealed that the alpha Trp418 increased channel open times approximately 28-fold at 13 degrees C with no effect on channel conductance. All of our measured functional changes in the alpha Trp418 mutant are consistent with a simple kinetic model of the acetylcholine receptor in which only the channel closing rate is altered by the mutation. Our results show that changes in protein structure at the putative lipid-protein interface can dramatically affect receptor function.

Ion channels are linked to differentiation in keratinocytes

SummaryIn vivo and in vitro, keratinocyte differentiation is linked with increased extracellular Ca2+. In order to correlate ion channels with cell differentiation and investigate keratinocyte membrane responses to Ca2+, keratinocyte single channel currents were studied using the patch-clamp technique. The most frequently observed channel was a 14 pS nonspecific cation channel. This channel was permeable to Ca2+ and activated by physiological concentrations of Ca2+. We also found a 35 pS Cl− channel whose open probability increased with depolarization. Finally, a 70 pS K+ channel was seen only in cell-attached or nystatin-permeabilized patches. We correlated channel types with staining for involucrin, an early marker of keratinocyte differentiation. While the nonspecific cation channel and Cl− channel were seen in both involucrin positive and involucrin negative cells, all channels in which the K+ channel activity was present were involucrin positive. Membrane currents through these channels may be one pathway by which signals for keratinocyte proliferation or differentiation are sent.

MUTATIONS IN THE M4 DOMAIN OF THE TORPEDO CALIFORNICA NICOTINIC ACETYLCHOLINE RECEPTOR ALTER CHANNEL OPENING AND CLOSING

Abstract. We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type α418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the β subunit (β447Trp) had similar but smaller effects on mean open time. Coexpression of α418Trp and β447Trp had the largest effect on channel open time, increasing mean open time 58-fold. No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested. However, the coexpression of the α418Trp and β447Trp mutated subunits also produced channels with at least two additional conductance levels. Block by acetylcholine was apparent in the current records from α418Trp mutants. Burst analysis of the α418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the α- and β subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating, and that mutations in multiple subunits act additively to increase channel open time.

Site-specific mutations of nicotinic acetylcholine receptor at the lipid-protein interface dramatically alter ion channel gating

McNamee, 1991). cDNA cloning reveals that each subunit has four homologous hydrophobic segments (Ml, M2, M3, and M4) that are believed to span the lipid bilayer. There is strong evidence that M2 is the ion channel-lining domain, but the specific functions of the other hydrophobic segments are not well characterized. In this report, the role ofthe M4 segment in ion channel function was investigatedby usingsite-directedmutagenesis of a specific cysteine residue (aCys418) in the M4 regionofTorpedo californica a subunit. The M4 region is believed to be located at the lipid-protein interface based primarily on chemical labeling studies (Giraudat et aI., 1985), and we have examined both the labeling and mutation of cysteine residues in these domains (Pradier et aI., 1989; Li et aI., 1990). We show here that the mutation on the a subunit alters ion channel gating in a dramatically different way than previous mutations.

Membrane responses to extracellular ATP in rat isolated white adipocytes

Abstract We used whole-cell and perforated-patch voltage-clamp methods to study the membrane electrical properties of isolated rat epididymal and inguinal white adipocytes. We examined cells from both Sprague-Dawley and Zucker lean and Zucker obese (fa/fa) rats. A delayed-rectifier potassium current was present and similar in unstimulated white fat cells from all these sources. The potassium current activated rapidly with depolarization positive to about –30 mV and showed slow inactivation. Stimulation with extracellular ATP activated both hyperpolarizing and depolarizing conductances. ATP exposure also increased cell membrane capacitance by an average of 16%, suggesting that ATP activates exocytosis. Exposure to norepinephrine had little electrophysiological effect. We conclude that white adipocytes are very similar to brown adipocytes in their resting electrophysiological profile and in their responses to extracellular ATP.

Differential Regulation of Ca 2+ Signaling and Membrane Trafficking by Multiple P2 Receptors in Brown Adipocytes

Extracellular ATP triggers changes in intracellular Ca2+, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors might mediate the Ca2+ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence imaging of Ca2+ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology of the Ca2+ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed mRNA for P2Y2, P2Y6, and P2Y12 metabotropic receptors and P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca2+, while 100 μM suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca2+ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely with 1 μM PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells, and that membrane trafficking is regulated by a P2 receptor showing unusual properties.

Ion Permeation in Cell Membranes

Selective membrane permeability to small ions underlies such important cell activities as the generation of electrical impulses in nerve and muscle cells, the release of neurotransmitters and hormones, sensory transduction, and epithelial transport. Because the energy change involved in moving an ion such as potassium or sodium from the aqueous medium to the nonpolar interior of a bare lipid bilayer is enormous, on the order of 60 kcal/mole,(251,252) cells have developed specialized structures which lower the energy for movement of ions through the cell membrane. One class of these structures, termed ion channels, decreases this energy by providing a polar, aqueous pathway through the lipid. Ion channels act as enzymes in that they lower the transition energy for ion movement from one side of the membrane to the other. Ion channels are proteins and show several other properties characteristic of enzymes: the transport through channels saturates with increasing substrate (ion) concentration, channels are specific for the passage of particular ions, and channels can be competitively inhibited (blocked). Ion channels also show the interesting feature that both catalytic functions (permeation) and regulatory functions (gating) are influenced by interactions with the membrane electric field.

Effects of pH on acetylcholine receptor function

SummaryWe have examined the effects of changing extracellular pH on the function of nicotinic acetylcholine receptors fromTorpedo californica using ion flux and electrophysiological methods. Agonist-induced cation efflux from vesicles containing purified, reconstituted receptors showed a monotonic dependence on external hydrogen ion concentration with maximal fluxes at alkaline pH and no agonist-induced efflux at pHs less than ∼5. A similar pH dependence was measured for the peak agonist-activated membrane currents measured in microelectrode voltage-clampedXenopus oocytes induced to expressTorpedo receptor through mRNA injection. Half-maximal inhibition occurred at a similar pH in both systems, in the range of pH 6.5–7.0. Single-channel currents fromTorpedo ACh receptors measured in patch-clamp recordings were also reduced in amplitude at acid pH with an apparent pKa for block of <5. Measurements of channel kinetics had a more complicated dependence on pH. The mean channel open time determined from patch-clamp measurements was maximal at neutral pH and decreased at both acid and alkaline pHs. Thus, both channel permeability properties and channel gating properties are affected by the extracellular pH.

Demyelination as a test for a mobile Na channel modulator in frog node of Ranvier.

We found previously that the external surface of frog skeletal muscle fibers can be irreversibly modified by treatment with the amino group-specific reagent, trinitrobenzene sulfonic acid (TNBS). Reaction of the muscle membrane with TNBS permanently shifts the potential dependence of the sodium channel inactivation gating process, h infinity, to more hyperpolarized potentials. The experiments presented here show nearly identical effects on the sodium currents of voltage-clamped frog node of Ranvier in the presence of TNBS. In contrast to the results in muscle, in myelinated nerve the voltage dependence of sodium-channel inactivation returns rapidly to control values following a brief exposure to TNBS. We have used partial demyelination to test the hypothesis that recovery of the normal voltage dependence for h infinity following TNBS treatment is due to lateral diffusion of reacted groups away from the sodium channels in the node. We find that increasing the membrane area exposed to TNBS by partial demyelination greatly slows reversal of the TNBS effects. This result suggests that a modifiable membrane component that affects sodium channel gating is mobile in the plane of the membrane and can rapidly diffuse between nodal and internodal regions.