Identification and characterization of a cell membrane nucleic acid channel

Abstract

We have identified a 45-kDa protein purified from rat renal brush border membrane that binds short single-stranded nucleic acid sequences. This activity was purified, reconstituted in proteoliposomes, and then fused with model planar lipid bilayers. In voltage-clamp experiments, the reconstituted 45-kDa protein functioned as a gated channel that allows the passage of nucleic acids. Channel activity was observed immediately after addition of oligonucleotide. Channel activity was not observed in the absence of purified protein or of oligonucleotide or when protein was heat-inactivated prior to forming proteoliposomes. In the presence of symmetrical buffered solution and oligonucleotide, current passed linearly over the range of holding potentials tested. Conductance was 10.4 ؎ 0.4 picosiemens (pS) and reversal potential was 0.2 ؎ 1.7 mV. There was no difference in channel conductance or reversal potential between phosphodiester and phosphorothioate oligonucleotides. Ionsubstitution experiments documented a shift in reversal potential only when a concentration gradient for oligonucleotide was established, indicating that movement of oligonucleotide alone was responsible for current. Movement of oligonucleotide across the bilayer was confirmed by using 32 P-labeled oligonucleotides. Channel open probability decreased significantly in the presence of heparan sulfate. These studies provide evidence for a cell surface channel that conducts nucleic acids.

Uptake and trafficking of DNA in keratinocytes: evidence for DNA-binding proteins

“…For comparison, a number of ODN-binding membrane proteins has been identified on kidney epithelial cells, and keratinocyte cell lines (HaCaT and A431 cells) that have a molecular weight of 35 and 61-63 kDa. 49,51 Since the proteins were also detected to a lesser extent in nuclear extracts, the authors hypothesized that these proteins might be involved in the uptake and nuclear transport of DNA. 49 To our knowledge, ezrin and moesin have not been described to bind DNA, neither has their involvement in intracellular trafficking of DNA been reported.…”

ATP as a mediator of macula densa cell signalling

“…This has some appeal, since it would presumably favour a faster on/off transient and could provide a greater degree of precision regarding the amount of ATP that was released from the cell per unit time. Several ATP conductive candidate channels have been proposed, including CFTR, a nucleic acid channel, connexin hemichannels, and the maxi-anion channel [104][105][106]. It is also possible that more than one anion conductive pathway could be permeable to ATP.…”

“…However, it is possible that some VDAC-like protein, which has yet to be identified, could be involved in macula densa release of ATP. It is also possible that proteins such as connexin hemichannels or the nucleic acid channel [104,116,117] could be involved in ATP release. Thus, the unanswered question at this time is the molecular identity of this channel and what controls the opening and closure of this ATP pathway.…”

Pore-blockade times for field-driven polymer translocation

“…DNA, RNA and proteins are such naturally occurring long molecules [1][2][3][4][5] in a variety of biological processes. Translocation is also used in gene therapy [6,7], and in delivery of drug molecules to their activation sites [8]. Consequently, the study of translocation is an active field of research: as a cornerstone of many biological processes, and also due to its relevance for practical applications.…”