Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed.

BACKGROUND: Intermediate-conductance, calcium-activated potassium channels (IKs) modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses. METHODS: Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1) expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture. RESULTS: hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D). Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques. CONCLUSION: Human keratinocyte differentiation is stimulated by calcium mobilization and influx, and differentiation stimuli coordinately upregulate mRNA levels of the calcium-activated hIK1 channel. This upregulation is paradoxical in that functional hIK1 channels are not observed in cultured keratinocytes. It appears, therefore, that hIK1 does not contribute to the functional electrophysiology of primary human keratinocytes, nor intact human skin. Further, the results indicate caution is required when interpreting experiments utilizing pharmacological hIK1 modulators in human keratinocytes.

The cost burden of multiple sclerosis in the United States: a systematic review of the literature.

OBJECTIVES: To estimate average annual cost per multiple sclerosis (MS) patient in the US using published estimates from the literature. METHODS: A search was performed of English-language literature published between 2007 and June 2012 in PubMed and Embase using the term 'multiple sclerosis' and the subject heading 'healthcare costs'. Included articles were primary studies with MS cost figures that could be converted to per patient per year values. Costs were inflated to 2011 dollars using the medical component of the Consumer Price Index. RESULTS: Fifteen studies met the inclusion criteria. Eight presented only direct cost calculations; the remaining seven presented estimates of total cost, broken down into direct and indirect costs. Total all-cause healthcare costs for MS as reported by studies that included direct and indirect costs ranged from $8528-$54,244 per patient per year. On average, direct costs comprised 77% (range 64-91%) of total costs. Prescription medications accounted for the majority of direct costs. On average, indirect costs comprised 23% (range 9-36%) of total costs. Compared with direct all-cause medical costs for other chronic conditions reported in the literature, MS ranked second behind congestive heart failure. LIMITATIONS: Data sources in these studies were dated, ranging from 1999-2008, and therefore do not include some of the newer, more costly therapies. In addition, this review does not include either assessment of the decrements in quality-of-life associated with MS or costs associated with increasing levels of disability or early retirement. Furthermore, variations in study designs, populations, methodologies, and cost inputs preclude more precise cost estimates. CONCLUSIONS: MS is a costly chronic disease. Further research is needed to understand: costs by MS type, costs associated with increasing disability and early retirement, and the potential impact of new treatments expected to launch in coming years.

Calcium channel upregulation in response to activation of neurotrophin and surrogate neurotrophin receptor tyrosine kinases.

Modulation of calcium channel expression and function in the context of neurotrophin induced neuronal differentiation remains incompletely understood at a mechanistic level. We addressed this issue in the PC12 model neuronal system using patch clamp electrophysiology combined with ectopic expression of the human beta platelet-derived growth factor (betaPDGF) receptor as a surrogate neurotrophin receptor system. PC12 cells ectopically expressing the human betaPDGF receptor were treated with PDGF or nerve growth factor (NGF) for up to 7 days, and Ca2+ channel subtype expression was analyzed using selective pharmacological agents in both whole-cell and cell-attached single channel patch clamp configurations. PDGF-induced upregulation of N- and P/Q-type Ca2+ channel currents completely mimicked upregulation of these currents caused by NGF stimulation of the endogenous TrkA receptor tyrosine kinase (RTK). Neither PDGF nor NGF significantly altered L- or R-type currents. Single channel recordings together with immunocytochemistry implied that growth factor-induced increases in whole-cell Ca2+ currents were a result of synthesis of new channels, and that whereas increased N channel density was apparent in the soma, additional P/Q channels distributed preferentially to extrasomal locations, most likely the proximal neurites. Finally, specific signaling-deficient mutant forms of the betaPDGF receptor were used to show that activation of Src, PI3-kinase, RasGAP, PLCgamma or SHP-2 (some of which are implicated in certain other aspects of PC12 cell differentiation) by RTKs is not required for growth factor-induced Ca2+ channel upregulation. In contrast, activation of the Ras-related G-protein Rap1 was found critical to this process.

G proteins as regulators of ion channel function.

Virtually unknown just a decade ago, GTP-binding proteins (G proteins) have become a major focus of current research. This family of closely related proteins transduce extracellular signals (such as hormones, neurotransmitters and sensory stimuli) into effector responses (1,2). It is now evident that ion channel permeability is one such effector response. In fact, the striking increase in the frequency of reports that demonstrate G protein-regulated ion channel function suggests that channels whose permeability mechanism can be altered by a G protein-mediated process may be more the rule than the exception. It is well-known that the cAMP-dependent modulation of ion channels is under the control of G proteins that regulate adenylate cyclase activity(3,4). However recent studies demonstrate that G proteins also transduce agonist-induced changes in channel activity that do not involve adenylate cyclase. It is on this aspect of G protein signal transduction that this review will focus.

Effect of basal lamina of ovarian follicle on T- and L-type Ca(2+) currents in differentiated granulosa cells.

Patch clamp experiments were conducted to study the effects of basal lamina (basement membrane) of chicken ovarian follicle on membrane Ca(2+) currents in differentiated chicken granulosa cells in a homologous system. The whole cell patch clamp technique was used to simultaneously monitor membrane capacitance (an indirect measure of total cell surface area) and currents flowing through voltage-dependent Ca(2+) channels (using Ba(2+) as the charge carrier). Membrane capacitance was smaller in cells incubated on intact basal lamina than in control cells (incubated on tissue culture-treated plastic substratum). Granulosa cells expressed both T- and L-type Ca(2+) currents, and the amplitudes of the currents in cells incubated on intact basal lamina were significantly lower than those of control cells. Also, granulosa cells incubated on intact basal lamina were found to have significantly lower T- or L-type Ca(2+) current densities than control cells. Intact basal lamina that had been stored for 12 mo produced effects on T- and L-type Ca(2+) currents similar to those caused by freshly isolated basal lamina. The basal lamina was solubilized completely in one step and used to coat glass coverslips (uncoated glass coverslips served as controls). Granulosa cells incubated on coverslips precoated with solubilized basal lamina assumed spherical shape similar to those incubated on intact basal lamina. Similar to the observations made for intact basal lamina, the solubilized basal lamina suppressed T- and L-type Ca(2+) currents in the differentiated granulosa cells. Moreover, fibronectin, laminin, and type IV collagen, obtained from commercial sources, attenuated T- and L-type Ca(2+) currents in the differentiated granulosa cells. This interplay between basal lamina and Ca(2+) currents may be one mechanism that subserves the effects of the matrix material on metabolic functions of granulosa cells.

Basal lamina of ovarian follicle regulates an inward Cl(-) current in differentiated granulosa cells.

Patch-clamp experiments were conducted to study the effects of basal lamina (basement membrane) of preovulatory chicken ovarian follicle on membrane currents in differentiated chicken granulosa cells in a homologous system. The membrane capacitance (measure of total membrane area) was smaller in cells cultured on intact basal lamina than that of control cells. The granulosa cells expressed outward and two inward currents. A small fraction of the cells (3%) expressed only a transient fast-activating and -inactivating inward current carried by Ca(2+). The majority of the cells, however, expressed a slowly activating and inactivating inward current (carried by Cl(-)) that was superimposed on the transient Ca(2+) current. All cells expressed an outward current characteristic of the delayed-rectifier K(+) current. The removal of extracellular Ca(2+) led to elimination of the slow inward Cl(-) current, indicating that it is a Ca(2+)-dependent Cl(-) current. Both peak amplitude and current density of the inward Cl(-) current were significantly lower in cells cultured on freshly isolated intact basal lamina (or basal lamina stored at 4 degrees C for 12 mo) than those of control cells; however, basal lamina had no significant effect on the density of the outward current. Similar to the observations made for intact basal lamina, solubilized basal lamina suppressed the inward Cl(-) current in differentiated granulosa cells. These data show that homologous basal lamina modulates a Ca(2+)-dependent Cl(-) current in differentiated granulosa cells. These findings provide a partial explanation for the mechanisms that subserve the reported effects of basal lamina (basement membrane) on the metabolic functions of differentiated granulosa cells.

The virally encoded fungal toxin KP4 specifically blocks L-type voltage-gated calcium channels.

KP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. Previous studies demonstrated that this toxin inhibits growth of the target fungal cells by blocking calcium uptake rather than forming channels, as had been suggested previously. Unexpectedly, this toxin was also shown to inhibit voltage-gated calcium channel activity in mammalian cells. We used whole-cell patch-clamp techniques to further characterize this activity against mammalian cells. KP4 is shown to specifically block L-type calcium channels with weak voltage dependence to the block. Because KP4 activity is abrogated by calcium, KP4 probably binds competitively with calcium to the channel exterior. Finally, it is shown that chemical reagents that modify lysine residues reduce KP4 activity in both patch-clamp experiments on mammalian cells and in fungal killing assays. Because the only lysine residue is K42, this residue seems to be crucial for both mammalian and fungal channel activity. Our results defining the type of mammalian channel affected by this fungal toxin further support our contention that KP4 inhibits fungal growth by blocking transmembrane calcium flux through fungal calcium channels, and imply a high degree of structural homology between these fungal and mammalian calcium channels.