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

Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1

Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34(+) cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cell

Sodium selectivity of semicircular canal duct epithelial cells

<p>Abstract</p> <p>Background</p> <p>Sodium absorption by semicircular canal duct (SCCD) epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na⁺under control of a glucocorticoid hormone (dexamethasone) and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC), comprised of the three subunits α-, β- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na⁺or also K⁺through an amiloride-sensitive pathway. Parasensory K⁺absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. <it>Hear.Res</it>. doi:10.1016/j.heares.2011.05.003, 2011].</p> <p>Results</p> <p>We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197), whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, β- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na⁺but not K⁺, but the Na⁺selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na⁺.</p> <p>Conclusions</p> <p>These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na⁺-selective channel, likely αβγ-ENaC. These epithelial cells therefore absorb only Na⁺via the amiloride-sensitive pathway and do not provide a parasensory K⁺efflux from the canals via this pathway. The results further provide caution to the culture of epithelial cells on impermeable surfaces.</p