Increased immunofluorescence sensitivity using 532 nm laser excitation.

OBJECTIVE: We evaluated the use of a high power, diode pulsed solid-state laser emitting 532 nm light for immunofluorescence applications. We compared the sensitivity and utility of this laser with the standard 488 nm excitation. METHODS: A flow cytometer was equipped with both a 488 nm and a 532 nm laser; fluorescence emissions from each laser were collected using the same filters and the same detector system. Cells or compensation beads (e.g. latex beads coated with anti-kappa antibodies) were stained with monoclonal antibodies conjugated to phycoerythrin (PE) as well as the PE tandem dyes TRPE, Cy5PE, Cy5.5PE, and Cy7PE. The sensitivity of detection of these reagents as well as those in heavily compensated channels was quantified by measuring the spreading error for a primary detector into a secondary detector. RESULTS: Measurement of the fluorescence emission of PE and PE-tandem dyes was considerably more sensitive when using 532 nm excitation (150 mW) as compared with 488 nm excitation (20 mW). In addition, as the absolute number of photoelectrons collected was greater, there was less measurement-error-induced spread into the compensated channels. As an example, when comparing the spreading error of PE labeled cells into the TRPE detector, the green laser was found to be 15-fold more sensitive as compared with the blue laser. In addition, the blue laser produced more autofluoresent signal from cells as compared with the green laser. Together, these advantages of the 532 nm excitation line provides for a significantly improved detection of immunofluorescence staining.     

Comparative electron microscopic study of the stomach of Orchestia cavimana and Arcitalitrus sylvaticus (crustacea: Amphipoda)

The functional morphology of stomachs of the European semiterrestrial amphipod Orchestia cavimana and of the Australian terrestrial species Arcitalitrus sylvaticus was studied by electron microscopy. The stomach of the two amphipod species is divided longitudinally into a spacious dorsal food channel and two ventral filtration channels. Additionally, a prominent helically oriented circulation channel is situated on each lateral side of the stomach, forming a semicircular channel separated from the food channel by spines. The food channel conveys coarse food particles directly into the midgut through a funnel. The filtration channels receive fine material filtered through primary and secondary filters. Material forced through the secondary filters by the pressure of the laterally located inferolateralia eventually reaches the openings of the midgut glands. Washing of filters and soaking of ingested food items with enzymes probably is achieved by a forward stream of digestive juice from the midgut glands and conveyed through the circulatory channels. The specializations of the stomach of the two species of Amphipoda investigated are described and compared to the pertinent structures of Mysidacea and Isopoda.     

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine β-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties.     

Anion channels and hormone signalling in plant cells

Current evidences support a central role in signal transduction and turgor regulation for plasma membrane anion channels. The present review focuses on these channels as putative targets for plant hormones. Various approaches have been developed to investigate the contribution of anion channels to hormone responses at the level of integrated responses of intact cells or organs, or to study directly the hormonal regulation of anion channels at the membrane level. These approaches are mainly discussed for two biological models, stomatal guard cells and hypocotyl or coleoptile cells, both cell types being equipped with several types of anion channels. Membrane potential and anion flux measurements, together with pharmacological studies using anion channel inhibitors, reveal that anion permeabilities are involved in the responses of guard cells or hypocotyl cells to abscisic acid and/or auxin. In a few instances, a modulation of anion channel activity can be detected in voltage-clamp or patch-clamp experiments. From these data and other studies, anion channel activation seems to constitute a very early step in many transduction cascades within response pathways to endogenous hormonal signals, but also to abiotic and biotic environmental signals such as light or molecules involved in plant-pathogen interactions. This points to plasma membrane anion channels as major actors in plant signalling networks.     

Electrophysiological and Fluorescence Microscopy Studies with HERG Channel/EGFP Fusion Proteins

HERG (human ether-a-go-go-related gene) encodes the Kv11.1 protein α-subunit that underlies the rapidly activating delayed rectifier K⁺ current (I _Kr) in the heart. Alterations in the functional properties or membrane incorporation of HERG channels, either by genetic mutations or by administration of drugs, play major roles in the development of life-threatening torsades de pointes cardiac arrhythmias. Visualization of ion channel localization is facilitated by enhanced green fluorescent protein (EGFP) tagging, but this process can alter their properties. The aim of the present study was to characterize the electrophysiological properties and the cellular localization of HERG channels in which EGFP was tagged either to the C terminus (HERG/EGFP) or to the N terminus (EGFP/HERG). These fusion constructs were transiently expressed in human embryonic kidney (HEK) 293 cells, and the whole-cell patch-clamp configuration and a confocal laser scanning microscope with primary anti-HERG antibodies and fluorescently labeled secondary antibodies were used. For EGFP/HERG channels the deactivation kinetics were faster and the peak tail current density was reduced when compared to both wild-type HERG channels and HERG/EGFP channels. Laser scanning microscopic studies showed that both fusion proteins were localized in the cytoplasm and on discrete microdomains in the plasma membrane. The extent of labeling with anti-HERG antibodies of HEK 293 cells expressing EGFP/HERG channels was less when compared to HERG/EGFP channels. In conclusion, both electrophysiological and immunocytochemical studies showed that EGFP/HERG channels themselves have a protein trafficking defect. HERG/EGFP channels have similar properties as untagged HERG channels and, thus, might be especially useful for fluorescence microscopy studies.     

A tale of two tails: cytosolic termini and K(+) channel function

The enormous variety of neuronal action potential waveforms can be ascribed, in large part, to the sculpting of their falling phases by currents through voltage-gated potassium channels. These proteins play several additional roles in other tissues such as the regulation of heartbeat and of insulin release from pancreatic cells as well as auditory signal processing in the cochlea. The functional channel is a tetramer with either six or two transmembrane segments per monomer. Selectivity filters, voltage sensors and gating elements have been mapped to residues within the transmembrane region. Cytoplasmic residues, which are accessible targets for signal transduction cascades and provide attractive means of regulation of channel activity, are now seen to be capable of modulating various aspects of channel function. Here we review structural studies on segments of the cytoplasmic tails of K⁺ channels, as well as the range of modulatory activities of these tails.     

Nanopore cheminformatics

A cheminformatics method is described for classification, and biophysical examination, of individual molecules. A novel molecular detector is used--one based on current blockade measurements through a nanometer-scale ion channel (alpha-hemolysin). Classification results are described for blockades caused by DNA molecules in the alpha-hemolysin nanopore detector, with signal analysis and pattern recognition performed using a combination of methods from bioinformatics and machine learning. Due to the size of the alpha-hemolysin protein channel, the blockade events report on one DNA molecule at a time, which enables a variety of reproducible, single-molecule biophysical experiments. To capture the full sensitivity of the nanopore detector's blockade signal, Hidden Markov Models (HMMs) were used with Expectation/Maximization for denoising and for associating a feature vector with the ionic current blockade of each captured DNA molecule. Support Vector Machines (SVMs) that employ novel kernel designs were then used as discriminators. With SVM training performed off-line, and economical HMM processing on-line, blockade classification was possible during capture. HMMs were also used in conjunction with a time-domain finite state automaton (off-line) for feature discovery and kinetics analysis. Analysis of the DNA data indicates a variety of binding (DNA-protein), fraying, and conformational shifts that are consistent with data obtained from thermodynamic analyses (melting curves), X-ray crystallography, and NMR studies. The software tools are designed for analysis of generic blockades in ionic channels, including those in other biological pore-forming toxins, other biological channels in general, and semiconductor-based channels.     

TRPC6 contributes to the Ca(2+) leak of human erythrocytes.

Human erythrocytes express cation channels which contribute to the background leak of Ca(2+), Na(+) and K(+). Excessive activation of these channels upon energy depletion, osmotic shock, Cl(-) depletion, or oxidative stress triggers suicidal death of erythrocytes (eryptosis), characterized by cell-shrinkage and exposure of phosphatidylserine at the cell surface. Eryptotic cells are supposed to be cleared from circulating blood. The present study aimed to identify the cation channels. RT-PCR revealed mRNA encoding the non-selective cation channel TRPC6 in erythroid progenitor cells. Western blotting indicated expression of TRPC6 protein in erythrocytes from man and wildtype mice but not from TRPC6(-/-) mice. According to flow-cytometry, Ca(2+) entry into human ghosts prepared by hemolysis in EGTA-buffered solution containing the Ca(2+) indicator Fluo3/AM was inhibited by the reducing agent dithiothreitol and the erythrocyte cation channel blockers ethylisopropylamiloride and amiloride. Loading of the ghosts with antibodies against TRPC6 or TRPC3/6/7 but neither with antibodies against TRPM2 or TRPC3 nor antibodies pre-adsorbed with the immunizing peptides inhibited ghost Ca(2+) entry. Moreover, free Ca(2+) concentration, cell-shrinkage, and phospholipid scrambling were significantly lower in Cl(-)-depleted TRPC6(-/-) erythrocytes than in wildtype mouse erythrocytes. In conclusion, human and mouse erythrocytes express TRPC6 cation channels which participate in cation leak and Ca(2+)-induced suicidal death.     

Dual-color time-integrated fluorescence cumulant analysis

We introduce dual-color time-integrated fluorescence cumulant analysis (TIFCA) to analyze fluorescence fluctuation spectroscopy data. Dual-color TIFCA utilizes the bivariate cumulants of the integrated fluorescent intensity from two detection channels to extract the brightness in each channel, the occupation number, and the diffusion time of fluorophores simultaneously. Detecting the fluorescence in two detector channels introduces the possibility of differentiating fluorophores based on their fluorescence spectrum. We derive an analytical expression for the bivariate factorial cumulants of photon counts for arbitrary sampling times. The statistical accuracy of each cumulant is described by its variance, which we calculate by the moments-of-moments technique. A method that takes nonideal detector effects such as dead-time and afterpulsing into account is developed and experimentally verified. We perform dual-color TIFCA analysis on simple dye solutions and a mixture of dyes to characterize the performance and accuracy of our theory. We demonstrate the robustness of dual-color TIFCA by measuring fluorescent proteins over a wide concentration range inside cells. Finally we demonstrate the sensitivity of dual-color TIFCA by resolving EGFP/EYFP binary mixtures in living cells with a single measurement.     

Inhibition of matrix metalloproteinase MMP-2 activates chloride current in human airway epithelial cells.

Matrix metalloproteinases (MMPs) are involved in the remodeling and degradation of the extracellular matrix. Recently, it has been found that MMPs also contribute to processes not directly related to tissue remodeling, such as platelet aggregation or degranulation of airway gland cells. Since mucus secretion is closely related to ion channel function, we investigated whether MMPs could also be involved in the regulation of ion channels. We used human airway submucosal cell line Calu-3 to study the effects of MMPs on whole-cell current and transepithelial short-circuit current (I(sc)). Phenanthroline, a specific inhibitor of MMPs, increased whole-cell current with the half-maximally effective dose of 5.2 microM, and reversibly activated I(sc) in transepithelial measurements. Current stimulated by phenanthroline displayed linear current-voltage relationships and had inhibitor pharmacology and ion selectivity consistent with cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity. Zymography and Western blot showed significant expression of MMP-2 in Calu-3 cells. Moreover, anti-MMP-2 antibodies (1 microg/mL) increased whole-cell current and I(sc), whereas human recombinant MMP-2 (10 ng/mL) reduced it. We also studied the expression of MMPs and the effects of phenanthroline on whole-cell current in A549 cells, which are derived from airway surface epithelium and do not express CFTR Cl- channels. While these cells also showed significant expression of MMP-2, inhibition of this enzyme with phenanthroline exerted no significant effect on whole-cell current. It is concluded that MMP-2 is involved in the regulation of CFTR Cl- channels in human airways.     

Dual-color photon-counting histogram

We report on the development of dual-color photon-counting histogram (PCH) analysis. Dual-color PCH is an extension of regular PCH and considers the photon counts received in two detection channels instead of one. Because each detection channel records a different color, dual-color PCH distinguishes fluorescent species not only by differences in their brightness, but also according to their color. The additional discrimination by color increases the sensitivity of PCH in resolving a mixture of species considerably. Most dual-color fluorescence fluctuation experiments are performed on fluorophores with overlapping emission spectra. This overlap results in spectral cross talk between the detector channels, which reduces resolvability. Here, we demonstrate that dual-color PCH is able to resolve binary dye mixtures in the presence of cross talk from a single measurement without any additional information about the sample. We discuss the effect of sampling time on the fit parameters of dual-color PCH. Differences between dual-color fluorescence correlation spectroscopy and dual-color PCH will also be addressed. We quantitatively resolve a mixture of the two fluorescent proteins CFP and YFP, which is challenging because of the strong spectral overlap of their emission spectra. Dichroic mirrors are needed to direct the light into the two detection channels. We quantify the influence of these filters on dual-color PCH analysis and determine the optimal transition wavelength of the dichroic mirror for the CFP-YFP pair.     

A generalized machine for automated flow cytology system design.

A general-purpose multiparameter flow cytophotometry system has been developed for use in the desgin of flow cytophotometers to perform specific tasks in automated cytology. Five separate measurement stations spaced along the axis of a capillary tube can be used to make up to eight optical measurements of individual cells flowing through the capillary. The system uses a broad-band arc source and can measure light scattered at various angles, light absorption by cell constituents and/or dyes and fluorescence of cell constituents and/or fluorochromes, excited directly and/or by energy transfer from neighboring molecules. High numerical aperture optics are used to maximize light-gathering capacity and minimize the effects of cell orientation and eccentricity of position in the fluid stream on measurements. A hard-wired preprocessor is used to detect the presence of cells and adjust sampling timing for changes in cell velocity; the electronic system also controls the gain of the detector photomultiplier tubes to compensate for background variations. Data acquistion and analysis are controled by a small general-purpose digital computer. The system has been used to develop a method and apparatus for blood cell counting and classification.     

The highly conserved Gln49 and Ser50 of mammalian connexin43 are present in chick connexin43 and essential for functional gap junction channels

In an attempt to compare the regulation of chick connexin43 channels to those of mammalian connexin43, we found that the nucleotide sequence reported for chick connexin43 differs from that of the chick connexin gene by two codons that had been entered as histidine49 (H49) and valine50 (V50) (accession no. M29003), but are in fact glutamine49 (Q49) and serine50 (S50). Neuro2A cells were transfected with corrected wild-type (Q49/S50) chick connexin43 (accession no. AF233738), the double-replacement Q49H/S50V connexin43, or the single replacement of Q49H or S50V. All clones had gap junctions in membrane based on immunocytochemistry and immunoblots of the triton-resistant membrane fraction. Wild-type transfectants had three conductance states with a predominant channel conductance of 85 ±5 pS. Cells producing the Q49H-Cx43 or the double-replacement Q49H/S50V-Cx43 protein had no detectable connexin43 channels. In contrast, cells expressing S50V-Cx43 gap junctions had channels with reduced conductances (75 ±8 pS) compared to wild-type controls. Low or high pH of the bathing solution had no effect on the Q49H-Cx43 channels. We conclude that glutamine49 is important for channel function, and replacement of this residue with histidine most likely distorts secondary structure of the first extracellular loop, possibly by changing the orientation of conserved cysteines, and this inhibits channel function. The S50V substitution may also cause similar but less severe structural changes.     

Scanning and transmission electron microscopic observations on the stomach of three mysid species (Crustacea)

Epithelial and cuticular linings of the stomach investigated in three species representing different genera of the Mysidacea are elaborated into a set of structural specializations dividing the stomach longitudinally into one dorsal and two ventral channels. The dorsal, or food, channel contains ingested food and retains coarse particles, which eventually are transported into the midgut through a funnel. The ventral, or filtration, channels, which are separated by an anterior and a posterior median ridge (anteromedianum, inferomedianum), contain fine particles and soluble materials extracted from the dorsal channel through two filter systems: primary filters, which lie anteriorly on either side of the anteromedianum, and posterior secondary filters, which are located on the inferomedianum. The final filtrate is transported into the ventral caeca or midgut glands. The ultrastructure of the cuticle lining the lumen of the stomach shows several specializations, the most prominent of which are stout spines and delicate filter devices. The epithelium is multilayered in circumscribed areas (the lateralia). The basement lamina is extremely developed in the inferomedianum. Detailed knowledge of the microscopic anatomy and the ultrastructure of the stomach allows identification of several homologous gastric structures among different peracaridean groups and in Decapoda.     

A solid-state,portable instrument for measurement of chlorophyll luminescence induction in plants

A newly developed compact instrument is described for the measurement of chlorophyll luminescence induction in plants. The instrument operates with a pulsed light emitting diode (LED) as light source and a photodiode as luminescence detector. A special emitter-detector geometry provides for high irradiance of the sample and efficient collection of luminescence by the detector. With insertion of appropriate filters the same probe is also suited for measuring prompt chlorophyll fluorescence. The instrument shows considerable flexibility with respect to pulse frequency, relative lengths of light/dark intervals and luminescence sampling periods. Due to a selective amplifier system only that part of luminescence is processed which is induced by the individual excitation pulses. By this approach, the problem of slow phase accumulation, encountered with conventional phosphoroscopes, is eliminated. Some examples are given for system operation, demonstrating satisfactory performance in measurements with intact leaves and isolated chloroplasts.     

IK1 channel activity contributes to cisplatin sensitivity of human epidermoid cancer cells

Cisplatin, a platinum-based drug, is an important weapon against many types of cancer. It induces apoptosis by forming adducts with DNA, although many aspects of its mechanism of action remain to be clarified. Previously, we found a role for the volume-sensitive, outwardly rectifying Cl(-) channel in cisplatin-induced apoptosis. To investigate the possibility that cation channels also have a role in the cellular response to cisplatin, we examined the activity of cation channels in cisplatin-sensitive KB-3-1 (KB) epidermoid cancer cells by the whole cell patch-clamp method. A cation channel in KB cells, activated by hypotonic stress, was identified as the Ca2+-activated, intermediate-conductance K+ (IK1) channel on the basis of its requirement for intracellular Ca2+, its blockage by the blockers clotrimazole and triarylmethane-34, and its suppression by a dominant-negative construct. Activity of this channel was not observed in KCP-4 cells, a cisplatin-resistant cell line derived from KB cells, and its molecular expression, observed by semiquantitative RT-PCR and immunostaining, appeared much reduced. Cell volume measurements confirmed a physiological role for the IK1 channel as a component of the volume-regulatory machinery in KB cells. A possible role of the IK1 channel in cisplatin-induced apoptosis was investigated. It was found that clotrimazole and triarylmethane-34 inhibited a cisplatin-induced decrease in cell viability and increase in caspase-3/7 activity, whereas 1-ethyl-2-benzimidazolinone, an activator of the channel, had the opposite effect. Thus IK1 channel activity appears to mediate, at least in part, the response of KB cells to cisplatin treatment.     

Spectral bandwidth in automated leukocyte classification

Investigators have repeatedly pointed out the importance of spectral information in the automated classification of white blood cells. In general, monochromatic images recorded through two or three color filters are used to extract this information. Although it has generally been thought that the use of narrow band filters provides "cleaner" color information than is obtainable through wide band filters, the choice has not been fully investigated and the question is far from being settled. The use of wide band filters has the clear practical advantage of increased light levels at the detector, resulting in higher signal-to-noise ratio with less demand on light source design. In order to investigate this issue, a series of 681 leukocytes of the most frequently occurring types were digitized by the use of both narrow (10 nm) and wide (90 nm) band filters. Parameters were extracted independently from both sets of images. These parameters were then used to develop a classifier for each set of images. The choice of features and classifier results indicate that there are no major performance differences between the two types of filters.     

A light and electron microscope study of the gills of larval lampreys (Geotria australis) with particular reference to the water-blood pathway.

The gills of ammocoetes of the Southern Hemisphere lamprey Geotria australis have been studied using light and electron microscopy. Emphasis has been placed on describing the structures and vessels involved in gaseous exchange, and on providing quantitative data for the water-blood barrier, including diffusion distance, diffusing capacity and the relative volumes of the component tissues. Although lamprey gills lie inside rather than outside the branchial skeleton as in gnathostomatous fishes. the morphology and ultrastructure of the gill filaments and secondary lamellae of G. australis larvae are very similar to those of teleost fishes. The extensive blood spaces within the secondary lamellae are enclosed by pillar cell bodies and pillar cell flanges which support two layers of epithelial cells. The outer surfaces of the epithelial cells are ridged and covered in a flocculent material which probably represents mucus. Differences were observed in the components of the water-blood barrier at the distal edges and at the surface of the secondary lamellae. At the distal edge, the lining of the marginal channel consisted of an endothelial cell rather than the pillar cell flanges which line the blood spaces of other regions. Based on light micrograph measurements, these differences result in a reduction in the arithmetic mean thickness of the water-blood barrier from 3.62 μm over the pillar cells to 2.22 μm over the marginal channel. Using values for the water-blood barrier obtained from light micrographs, the arithmetic and harmonic mean diffusing capacities were calculated as 1.1046 and 1.7589 ml O₂min/mm Hg/Kg.     

Amiloride-sensitive apical membrane sodium channels of everted Ambystoma collecting tubule

Patch clamp methods were used to characterize sodium channels on the apical membrane of Ambystoma distal nephron. The apical membranes were exposed by everting and perfusing initial collecting tubules in vitro. In cell-attached patches, we observed channels whose mean inward unitary current averaged 0.39±0.05 pA (9 patches). The conductance of these channels was 4.3±0.2 pS. The unitary current approached zero at a pipette voltage of –92 mV. When clamped at the membrane potential the channel expressed a relatively high open probability (0.46). These characteristics, together with observation that doses of 0.5 to 2 m amiloride reversibly inhibited the channel activity, are consistent with the presence of the high amiloride affinity, high sodium selectivity channel reported for rat cortical collecting tubule and cultured epithelial cell lines.We used antisodium channel antibodies to identify biochemically the epithelial sodium channels in the distal nephron of Ambystoma. Polyclonal antisodium channel antibodies generated against purified bovine renal, high amiloride affinity epithelial sodium channel specifically recognized 110, 57, and 55 kDa polypeptides in Ambystoma and localized the channels to the apical membrane of the distal nephron. A polyclonal antibody generated against a synthetic peptide corresponding to the C-terminus of Apx, a protein associated with the high amiloride affinity epithelial sodium channel expressed in A6 cells, specifically recognized a 170 kDa polypeptide. These data corroborate that the apically restricted sodium channels in Ambystoma are similar to the high amiloride affinity, sodium selective channels expressed in both A6 cells and the mammalian kidney.This work was supported by American Heart Association, New York Affiliate Grant 91007G (LCS) and National Institute of Diabetes and Digestive and Kidney Disease Grants DK-37206 (DJB) and DK46705 (PRS).     

Gating deficiency in a familial hemiplegic migraine type 1 mutant P/Q-type calcium channel

Familial hemiplegic migraine type 1 (FHM1) arises from missense mutations in the gene encoding alpha1A, the pore-forming subunit of P/Q-type calcium channels. The nature of the channel disorder is fundamental to the disease, yet is not well understood. We studied how the most prevalent FHM1 mutation, a threonine to methionine substitution at position 666 (TM), affects both ionic current and gating current associated with channel activation, a previously unexplored feature of P/Q channels. Whole-cell currents were measured in HEK293 cells expressing channels containing either wild-type (WT) or TM alpha1A. Calcium currents were significantly smaller in cells expressing TM channels, consistent with previous reports. In contrast, surface expression of TM channels, measured by immunostaining against an extracellular epitope, was not decreased, and Western blots demonstrated that TM alpha1A subunits were expressed as full-length proteins. WT and TM gating currents were isolated by replacing Ca2+ with the nonpermeant cation La3+. The gating currents generated by the mutant channels were one-third that of WT, a deficiency sufficient to account for the observed attenuation in calcium current; the remaining gating current was no different in kinetics or voltage dependence. Thus, the decreased calcium influx seen with TM channels can be attributed to a reduced number of channels available to undergo the voltage-dependent conformational changes needed for channel opening, not to fewer channel proteins expressed on the cell surface. This identification of an intrinsic defect in FHM1 mutant channels helps explain their impact on neurotransmission when they occupy type-specific slots for P/Q channels at central nerve terminals.