Fabrication of patch pipets.

Patch clamping refers to a wide range of electrophysiological measurements, all of which have in common the use of patch pipets and the formation of gigaohm seals. The purpose of this unit is to describe the fabrication of patch pipets. The aspects of the pipet geometry that are important to different applications and the different procedures that have been found to most reliably and simply achieve these results are described. Parameters for glass selection are detailed in the beginning of the unit. Pulling patch and whole-cell pipets, elastomer coating, fire polishing, pipet filling, and pipet testing in an experimental setup are highlighted. Additional support protocols describe alternative ways to optimize pipet geometry and cleaning the glass before pulling. Considerations for noise and dynamic performance are emphasized as these two requirements for single-channel and whole-cell current measurements dictate how the pipets must be fabricated.

SCN5A is expressed in human jejunal circular smooth muscle cells.

Tetrodotoxin-resistant Na+currents are expressed in a variety of muscle cells including human jejunal circular smooth muscle (HJCSM) cells. The aim of this study was to determine the molecular identity of the pore-forming alpha-subunit of the HJCSM Na+ channel. Degenerate primers identified a cDNA fragment of 1.5 kb with 99% nucleotide homology with human cardiac SCN5A. The identified clone was also amplified from single smooth muscle cells by reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot analysis showed expression of full-length SCN5A. Laser capture microdissection was used to obtain highly purified populations of HJCSM cells. RT-PCR on the harvested cells showed that SCN5A was present in circular but not in longitudinal muscle. A similar result was obtained using a pan-Na+ channel antibody. The full-length sequence for SCN5A was obtained by combining standard polymerase chain reaction with 5' and 3' rapid amplification of cDNA end techniques. The intestinal SCN5A was nearly identical to the cardiac SCN5A. The data indicate that SCN5A is more widely distributed than previously thought and encodes the pore-forming alpha-subunit of the tetrodotoxin-resistant Na+ current in HJCSM cells.

Kv3.3 potassium channels in lens epithelium and corneal endothelium.

Human Kv3.3/KCNC3 is a Shaw-type potassium channel that has been mapped to chromosome 19q13.3-13.4. Complete mouse and rat Kv3.3 cDNA coding sequences have been published, yet the human Kv3.3 cDNA has remained incomplete for years. We report here for the first time the amino acid sequence for hKv3.3 and the electrophysiological behavior of the encoded channel in transiently transfected mammalian cells. In addition, we report the occurrence of Kv3.3 message in rabbit corneal endothelial cells and the properties of the currents when the corneal channel is expressed. The hKv3.3 gene is highly GC-rich (69%) and contains numerous GC runs which made DNA sequencing and PCR amplification especially problematic. The full-length sequence contains two possible start codons. The encoded 757 amino acid hKv3. 3 protein is about 93% identical to mouse and rat Kv3.3 in the first 659 amino acids before the C-terminal domains diverge greatly as a result of alternative splicing. The rabbit cornea Kv3.3 is a close sequence match for hKv3.3 even in the C-terminal domain. However, we have not yet found a cornea sequence which contains the first potential start codon from hKv3.3. Electrophysiologically, the hKv3. 3 channel produces an A-current although expression of constructs which lack the 5' region of the first start codon inactivate much more slowly than full-length constructs. This short hKv3.3 construct also shows changes in activation.

Kir2.1 Potassium channels and corneal epithelia.

PURPOSE: To determine the existence of inward rectifier (Kir2.1) sequences in mRNA from corneal epithelium and endothelium. METHODS: cDNA library construction, cloning, PCR, patch clamp. RESULTS. Both the corneal epithelium and endothelium contain mRNA for Kir2.1 inwardly rectifying potassium channels. When the cDNA is transfected into Chinese hamster ovary cells, the channel currents match those expected from Kir2.1 inward rectifiers. CONCLUSIONS: The mRNA for Kir2.1 potassium channels exists in corneal epithelium and endothelium. Therefore, Kir2.1 inwardly rectifying potassium channels probably make some contributions to the resting voltages of cornea epithelium and endothelium. Previous data, however, suggest that they are probably not the dominant contributors in these preparations.

A mechanosensitive calcium channel in human intestinal smooth muscle cells.

BACKGROUND & AIMS: Gastrointestinal smooth muscle strips devoid of enteric nerve cells can contract in response to stretch, suggesting that mechanosensitivity and mechanotransduction can occur at the level of the smooth muscle cell. The aim of this study was to determine whether stretch-activated calcium channels are present in gastrointestinal smooth muscle cells. METHODS: Whole-cell and single-channel calcium currents were measured from human jejunal circular smooth muscle cells in response to increased intracellular pressure, bath perfusion, and membrane stretch. RESULTS: At 10 mm Hg positive pressure, peak calcium current increased from -36 +/- 10 pA to -53 +/- 13 pA. Bath perfusion at 10 mL/min increased calcium current from -97.7 +/- 14 pA to -122 +/- 16 pA. Single-channel open probability increased in response to negative pipette pressure. All increases were blocked by nifedipine. CONCLUSIONS: A stretch-activated, nifedipine-sensitive calcium channel is present in human jejunal circular smooth muscle cells. The channel is activated by both an increase in intracellular pressure and by external shear forces. The presence of a stretch-activated calcium channel in gastrointestinal smooth muscle cells may allow the smooth muscle cells to act directly as mechanotransducers and to participate in the regulation of smooth muscle tone and intestinal motility.

Electrically silent potassium channel subunits from human lens epithelium.

We describe the cloning and characterization of the first human members, hKv9.1 and hKv9.3, of the electrically silent delayed-rectifying-like K+ channel subfamily. Their modulatory effects on the electrically active subfamily member hKv2.1 are also quantified. The hKv9 K+ channels were isolated from a human lens epithelium cDNA library, but both hKv9.1 mRNA and hKv9.3 mRNA were found to coexist with the mRNA for hKv2.1 in a large number of human tissues. The hKv9.1 gene is composed of a minimum of five exons, with at least two alternatively spliced exons in the 5'-untranslated region (UTR). In contrast, the hKv9.3 gene is intronless across the coding region, 3'-UTR, and all of the analyzed 5'-UTR. Radiation hybrid mapping localized the hKv9.1 gene to 20q12 and the hKv9.3 gene to 2p24. Each electrically silent subunit, when coexpressed with hKv2.l, slows deactivation and inactivation compared with hKv2. 1 expressed alone. In addition, each results in an increment in the single channel conductance.

Effects of melatonin on ionic currents in cultured ocular tissues.

The effects of melatonin on ionic conductances in a cultured mouse lens epithelial cell line (alpha-TN4) and in cultured human trabecular meshwork (HTM) cells were measured using the amphotericin perforated patch whole cell voltage-clamp technique. Melatonin stimulated a voltage-dependent Na+-selective current in lens epithelial cells and trabecular meshwork cells. The effects of melatonin were observed at 50 pM and were maximal at 100 microM. Melatonin enhanced activation and inactivation kinetics, but no change was observed in the voltage dependence of activation. The results are consistent with an increase in the total number of ion channels available for activation by membrane depolarization. Melatonin was also found to stimulate a K+-selective current at high doses (1 mM). Melatonin did not affect the inwardly rectifying K+ current or the delayed rectifier type K+ current that has been described in cultured mouse lens epithelial cells. The results show that melatonin specifically stimulated the TTX-insensitive voltage-dependent Na+ current by an apparently novel mechanism.

Ion transporters and receptors in cDNA libraries from lens and cornea epithelia.

PURPOSE: To construct tissue-specific cDNA libraries containing copies of genes of ion transporting and receptor proteins in lens and cornea epithelia. To screen the libraries for the cDNA of these molecules and to deduce the protein sequence from the cDNA sequence. METHOD: Lens and corneal cDNA libraries are not commercially available and are difficult to prepare from microdissected single animal eyes or cadaver human eyes due to the small amount of tissue. To overcome these problems, we refined an approach for preparing high efficiency, non-PCR amplified plasmid cDNA libraries, where only nanogram quantities of poly (A) RNA are required. Plasmids were electrotransformed into DH10B bacteria and routinely yielded >10(6) independent colonies with typically >90% recombinants. Randomly selected colonies were subjected to colony PCR analysis to determine the libraries average insert size (typically approximately 1-kb). Primers to known genes were used in PCR amplification to check for representation of the genes in the cDNA libraries. RESULTS: Using libraries from rabbit cornea epithelium and endothelium, cultured human lens epithelium, and alphaTN4 cells, we have found the libraries to contain the cDNA for 3 common housekeeping proteins expected to be at high copy numbers in all cells. In addition, we identified 22 rare proteins and for many we determined the complete coding regions. These include K+ channels, Cl- channels, Ca++ channels, Na+ channels, three exchangers, the Na+-HCO3- cotransporter, and three kinds of receptors. CONCLUSION: Cornea and lens libraries have been constructed from single cornea or lens preparations. The libraries often contain the cDNA sequences for ion transporting and receptor proteins which are expected to be relatively rare in these cells. Many of these cDNA's have now been sequenced and the encoded proteins identified.

Inwardly rectifying potassium channels in lens epithelium are from the IRK1 (Kir 2.1) family.

Lens epithelial cells from many species contain inwardly rectifying K+ channels. The channels are highly selective for K+ over Na+. They have a conductance of 27-30 pS in symmetrical 150 mM K+ solutions. The conductance to inwardly flowing current depends on the external [K+], being 1/2 maximal at about 50 mM and maximal by 110-150 mM. The amino acid sequences from lens epithelium (eight different species) show at least 98% sequence homology to each other and to the potassium channel known as IRK1 (Kir 2.1). Cloned channels from chick, rabbit, and human lens epithelium all make functional channels when their cDNA is transfected into HEK-293 or tsA-201 cells. Human lens inward rectifiers when engineered as fusion proteins with green fluorescent protein (GFP) also make functional channels. In addition, their localization in the membrane and in intracellular organelles can be demonstrated by fluorescence microscopy.

Molecular biology and electrophysiology of calcium-activated potassium channels from lens epithelium.

PURPOSE: We structurally and functionally characterized the alpha and beta subunits of the human lens epithelium Ca(++)-activated potassium channel (BK). METHODS: The two subunits were sequenced following RT-PCR with multiple primer pairs. The subunits were cloned using a PCR approach and were expressed in tsA-201 cells for patch clamp recording. Green fluorescence protein-channel subunit fusion proteins were characterized by patch clamping and were imaged by fluorescence microscopy. RESULTS: Alpha subunits alone make a large single-channel conductance, potassium-selective channel with modest Ca++ sensitivity. Beta subunits alone make no channel but, when coexpressed with the alpha subunit, make a channel with increased Ca++ sensitivity, although still less than for natural channels of this type. GFP-BK subunit fusion proteins continue to function and result in a fluorescing channel, which can be localized by fluorescence microscopy. The alpha subunit codes for a "minimal" BK channel in that none of its potential alternative splicing sites contains an "extra" exon. CONCLUSIONS: The Ca(++)-activated potassium channel known as BK has the nucleotide sequences of its alpha and beta subunits represented in messenger RNA of cultured human lens epithelium. It is the first identified channel, to date, which imparts internal Ca++ dependence to lens epithelial potassium conductance.

Distribution of heme oxygenase and effects of exogenous carbon monoxide in canine jejunum.

Carbon monoxide (CO) has been postulated to be a messenger in the gastrointestinal tract. The aims of this study were to determine the distribution of heme oxygenase (HO), the source for endogenous CO in the canine jejunum, and to determine the effects of CO on jejunal circular smooth muscle cells. HO-2 isoform was present in a population of myenteric and submucosal neuronal cell bodies, in nerve fibers innervating the muscle layers, and in smooth muscle cells. HO-1 isozyme was not detected in the canine jejunum. Exogenous CO increased whole cell current by 285 +/- 86%, hyperpolarized the membrane potential by 8.5 +/- 2.9 mV, and increased guanosine 3',5'-cyclic monophosphate (cGMP) levels in smooth muscle cells. 8-Bromo-cGMP also increased the whole cell current. The data suggest that endogenous activity of HO-2 may be a source of CO in the canine jejunum and that exogenously applied CO can modulate intestinal smooth muscle electrical activity. It is therefore reasonable to suggest a role for endogenously produced CO as a messenger in the canine jejunum.

Magnetic bead capture of cDNAs from double-stranded plasmid cDNA libraries.

We have developed a cDNA library screening method which allows the simultaneous screening of >10 ( 12 ) double-stranded plasmid cDNA molecules with minimal a priori sequence knowledge. A biotinylated, gene-specific oligonucleotide probe along with abutting 'blocking' oligos is hybridized to the plasmid cDNA library and the target plasmid retrieved with paramagnetic streptavidin beads and transformed into Escherichia coli. Multiple rounds of enrichment with a target plasmid represented at 0.002-0.0001% resulted in over one-third positive clones. Our method will be useful for isolating even the rarest cDNAs starting from ESTs, isolated exons or homologous sequence information.

Sequences and expression of pyruvate dehydrogenase genes from Pseudomonas aeruginosa.

A mutant of Pseudomonas aeruginosa, OT2100, which appeared to be defective in the production of the fluorescent yellow-green siderophore pyoverdine had been isolated previously following transposon mutagenesis (T. R. Merriman and I. L. Lamont, Gene 126:17-23, 1993). DNA from either side of the transposon insertion site was cloned, and the sequence was determined. The mutated gene had strong identity with the dihydrolipoamide acetyltransferase (E2) components of pyruvate dehydrogenase (PDH) from other bacterial species. Enzyme assays revealed that the mutant was defective in the E2 subunit of PDH, preventing assembly of a functional complex. PDH activity in OT2100 cell extracts was restored when extract from an E1 mutant was added. On the basis of this evidence, OT2100 was identified as an aceB or E2 mutant. A second gene, aceA, which is likely to encode the E1 component of PDH, was identified upstream from aceB. Transcriptional analysis revealed that aceA and aceB are expressed as a 5-kb polycistronic transcript from a promoter upstream of aceA. An intergenic region of 146 bp was located between aceA and aceB, and a 2-kb aceB transcript that originated from a promoter in the intergenic region was identified. DNA fragments upstream of aceA and aceB were shown to have promoter activities in P. aeruginosa, although only the aceA promoter was active in Escherichia coli. It is likely that the apparent pyoverdine-deficient phenotype of mutant OT2100 is a consequence of acidification of the growth medium due to accumulation of pyruvic acid in the absence of functional PDH.

Effects of pH on the potassium current in rabbit corneal epithelial cells.

The effects of pH on K+ conductance were measured using the amphotericin perforated-patch whole cell voltage-clamp technique in freshly dispersed rabbit corneal epithelial cells. Bath perfusion with pH 6.00 Ringer solution after standard Ringer solution (pH 7.35) increased outward K+-selective current (I(K)) from 120 +/- 29 to 312 +/- 64 pA during a step depolarization to +50 mV and hyperpolarized the resting membrane potential (Em) from -52 +/- 5 to -62 +/- 3 mV (n = 15, P < or = 0.05). Increasing bath pH to 8.5 decreased I(K) from 183 +/- 40 to 114 +/- 35 pA (n = 6, P < or = 0.05) and depolarized Em from -63 +/- 6 to -53 +/- 5 mV (n = 6, P < or = 0.05). Intracellular acidification using the weak electrolyte (NH4)2SO4 also increased I(K) from 83 +/- 15 to 183 +/- 20 pA (n = 4, P = 0.01) and hyperpolarized Em from -51 +/- 8 to -68 +/- 6 mV (P = 0.002). Intracellular alkalinization reduced I(K) to 66 +/- 10 pA and depolarized Em to -36 +/- 8 mV (P = 0.009). Single channel studies in perforated outside-out vesicles showed that a decrease in bath pH from 7.35 to 6.00 was accompanied by an increase in the single channel open probability (NPo) from 0.43 to 0.64 at an Em of 15 mV. NPo was also increased in cell-attached patches. The unitary conductance, measured from -100 to +100 mV, was not changed. These results indicate that pH modulates I(K) in rabbit corneal epithelial cells by changes in NPo.

Physiological properties of the normal lens.

The lens is an avascular organ suspended between the aqueous and vitreous humors of the eye. The cellular structure is symmetric about an axis passing through its anterior and posterior poles but asymmetric about a plane passing through its equator. Because of its asymmetric structure, the lens has historically been assumed to perform transport between the aqueous and vitreous humors. Indeed, when anterior and posterior surfaces were isolated in an Ussing chamber, a translens current was measured. However, in the eye, the two surfaces are not isolated. The vibrating probe technique showed the current densities at the surface of a free-standing lens were surprisingly large, about an order of magnitude greater than measured in an Ussing chamber, and were not directed across the lens. Rather, they were inward in the region of either anterior or posterior pole and outward at the equator. This circulating current is the most dramatic physiological property of a normal lens. We believe it is essential to maintain clarity; hence, this review focuses on factors likely to drive and direct it. We review properties and spatial distribution of lens Na+/K+ pumps, ion channels, and gap junctions. Based on these data, we propose a model in which the difference in electromotive potential of surface versus interior cell membranes drives the current, whereas the distribution of gap junctions directs the current in the observed pattern. Although this model is clearly too simple, it appears to quantitatively predict observed currents. However, the model also predicts fluid will move in the same pattern as ionic current. We therefore speculate that the physiological role of the current is to create an internal circulatory system for the avascular lens.

Ionic channels in corneal endothelium.

Single-channel patch-clamp techniques as well as standard and perforated-patch whole cell voltage-clamp techniques have been applied to the study of ionic channels in the corneal endothelium of several species. These studies have revealed two major K+ currents. One is due to an anion- and temperature-stimulated channel that is blocked by Cs+ but not by most other K+ channel blockers, and the other is similar to the family of A-currents found in excitable cells. The A-current is transient after a depolarizing voltage step and is blocked by both 4-aminopyridine and quinidine. These two currents are probably responsible for setting the -50 to -60 mV resting voltage reported for these cells. A Ca(2+)-activated ATP-inhibited nonselective cation channel and a tetrodotoxin-blocked Na+ channel are possible Na+ inflow pathways, but, given their gating properties, it is not certain that either channel works under physiological conditions. A large-conductance anion channel has also been identified by single-channel patch-clamp techniques. Single corneal endothelial cells have input resistances of 5-10 G omega and have steady-state K+ currents that are approximately 10 pA at the resting voltage. Pairs or monolayers of cells are electrically coupled and dye coupled through gap junctions.

Dye transfer between cells of the lens.

Dye transfer between lens fiber cells and between lens epithelial cells and underlying fiber cells was studied using a wide dynamic range-cooled CCD camera, H2O immersion objectives and image analysis techniques. Each lens was decapsulated by a new technique which leaves the epithelial cells adherent to the lens fiber mass. Lucifer Yellow CH was injected into either single epithelial cells or single fiber cells using the standard whole cell configuration of the patch voltage clamp technique. The results demonstrate extensive dye communication between fiber cells at the lens posterior surface, anterior surface, and equatorial surface. Dye transfer between deep fiber cells was also observed. Dye transfer between approximately 10% of epithelial cells and their underlying fiber cells was apparent when care was taken to yield wide dynamic range images. This was required because the relatively high concentration of dye in the epithelial cell masks the presence of much lower dye concentrations in the underlying fiber cell. A mathematical model which includes dye concentration, time, and spatial spread suggests that those epithelial cells that are coupled to an underlying fiber cell are about as well dye coupled as the epithelial cells themselves. The relatively low dye concentration in a fiber cell is due to its larger volume and diffusion of the dye along the axis of the fiber away from the fiber/epithelial junction.

Calcium currents in human and canine jejunal circular smooth muscle cells.

BACKGROUND & AIMS: Although calcium plays an essential role in intestinal smooth muscle contractile activity, calcium entry pathways in canine and human small intestine are largely unknown. The goal of this study was to characterize calcium channels, a potential entry pathway for calcium, in isolated circular smooth muscle cells of canine and human jejunum. METHODS: Single freshly dissociated human and canine jejunal circular smooth muscle cells were studied using single-channel and perforated whole-cell patch clamp recordings as well as fluorescence dual wavelength ratio imaging. RESULTS: An inward whole-cell current was identified that was carried by a 17 pS (80 mmol/L Ba2+) dihydropyridine-sensitive, barium-permeable channel. The current was potentiated by BayK 8644 (1 mumol/L; n = 3; 82% +/- 34%), acetylcholine (1 mumol/L; n = 8; 42% +/- 5%), and erythromycin (1 mumol/L; n = 9; 70% +/- 11%) and was completely blocked by nifedipine (1 mumol/L; n = 6) or diltiazem (200 mumol/L; n = 4). Application of BayK 8644 (1 mumol/L), acetylcholine (1 mumol/L), or erythromycin (1 mumol/L) to Fura-2-loaded smooth muscle cells bathed in Krebs' solution containing 2.54 mmol/L calcium increased intracellular calcium levels. CONCLUSIONS: A calcium entry pathway was identified in canine and human jejunal circular smooth muscle cells. The pathway was mediated by a dihydropyridine-sensitive calcium channel. The channel allowed the entry of significant amounts of calcium at physiological extracellular calcium concentration.