No abstract

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.

The voltage-sensitive sodium channel Na v 1.5 (encoded by SCN5A) is expressed in electromechanical organs and is mechanosensitive. This study aimed to determine the mechanosensitive transitions of Na v 1.5 at the molecular level. Na v 1.5 was expressed in HEK 293 cells and mechanosensitivity was studied in cell-attached patches. Patch pressure up to −50 mmHg produced increases in current and large hyperpolarizing shifts of voltage dependence with graded shifts of half-activation and half-inactivation voltages ( V 1/2 ) by ∼0.7 mV mmHg −1 . Voltage dependence shifts affected channel kinetics by a single constant. This suggested that stretch accelerated only one of the activation transitions. Stretch accelerated voltage sensor movement, but not rate constants for gate opening and fast inactivation. Stretch also appeared to stabilize the inactivated states, since recovery from inactivation was slowed with stretch. Unitary conductance and maximum open probability were unaffected by stretch, but peak current was increased due to an increased number of active channels. Stretch effects were partially reversible, but recovery following a single stretch cycle required minutes. These data suggest that mechanical activation of Na v 1.5 results in dose-dependent voltage dependence shifts of activation and inactivation due to mechanical modulation of the voltage sensors.

The SCN5A-encoded Na(v)1.5 Na(+) channel is expressed in interstitial cells of Cajal and smooth muscle in the circular layer of the human intestine. Patients with mutations in SCN5A are more likely to report gastrointestinal symptoms, especially abdominal pain. Twin and family studies of irritable bowel syndrome (IBS) suggest a genetic basis for IBS, but no genes have been identified to date. Therefore, our aims were to evaluate SCN5A as a candidate gene involved in the pathogenesis of IBS and to determine physiological consequences of identified mutations. Mutational analysis was performed on genomic DNA obtained from 49 subjects diagnosed with IBS who reported at least moderately severe abdominal pain. One patient hosted a loss-of-function missense mutation, G298S, that was not observed in >3,000 reference alleles derived from 1,500 healthy control subjects. Na(+) currents were recorded from the four common human SCN5A transcripts in transfected HEK-293 cells. Comparing Na(v)1.5 with G298S-SCN5A versus wild type in HEK cells, Na(+) current density was significantly less by 49-77%, and channel activation time was delayed in backgrounds that also contained the common H558R polymorphism. Single-channel measurements showed no change in Na(v)1.5 conductance. Mechanosensitivity was reduced in the H558/Q1077del transcript but not in the other three backgrounds. In conclusion, the G298S-SCN5A missense mutation caused a marked reduction of whole cell Na(+) current and loss of function of Na(v)1.5, suggesting SCN5A as a candidate gene in the pathophysiology of IBS.

Quartz has a dissipation factor of approximately 10(-4), which is an order of magnitude less than that of the best glasses previously used to fabricate patch pipettes; it's dielectric constant of 3.8 is also lower than that of other glasses. On the basis of these electrical characteristics it is expected that patch pipettes pulled from quartz tubing will produce significantly less noise than pipettes made from other glasses. Our work confirms these expectations and we describe theoretical and practical aspects of the use of quartz pipettes for single channel patch voltage clamp measurements. Methods for pulling quartz pipettes with a laser-based puller and coating them with low-loss elastomers are discussed, as are precautions that are necessary to achieve low noise recordings. We have shown that quartz pipettes can be pulled from tubing with outer diameter to inner diameter ratios as large as 3 and a method of applying heavy elastomer coatings all the way to the tip of pipettes is presented. Noise sources arising from the pipette and its holder are described theoretically, and it is shown that measured noise is in good agreement with such predictions. With low noise capacitive feedback electronics, small geometry holders, and thick-walled quartz pipettes coated with low-loss elastomers we have been routinely able to achieve noise of 100 fA rms or less in a 5-kHz bandwidth with real cell patches and a pipette immersion depth of approximately 2 mm. On occasion we have achieved noise as low as 60 fA rms in this bandwidth.