The results described above indicate that deletion of the KCNK7 gene results in a mouse that not only is indistinguishable from wildtype or heterozygous animals but also had unaltered response to the three volatile anesthetics tested compared to wildtypes and heterozygotes. These findings most likely indicate that KCNK7 does not play a dominant role in the mechanism of action of volatile anesthetics. These studies cannot however, rule out the possibility of compensatory changes in ion channel gene expression in these mice to mask the effect of KCNK7 knockout.
KCNK7 is grouped with two other members of the weak inward rectifier K2P
subfamily based on sequence homology and similarity of genomic structure. KCNK7 is located on mouse chromosome 19 and like TWIK-1 and TWIK-2, the coding sequence is contained on three exons. An exon-intron boundary is also conserved among the three genes within the first GYG pore sequence after the first nucleotide of the second codon. This conservation suggests that members of this group arose by duplication and radiation type of mechanism. Overall, the sequence homology among the weak inward rectifier K2P
family is low. displays an alignment of representative regions of KCNK7, TWIK-1 and TWIK-2 (Clustal W method) and shows that while in some conserved regions the percentage of identical amino acid residues may reach 50%, the overall sequence identity is in the range of only 30–40% (TWIK-1 v. TWIK-2 41% identify; TWIK-1 v. KCNK7 35% identity; TWIK-2 v. KCNK7 30% identity). This sequence divergence, which is also found within other K2P
channel subfamilies, may indicate that these channels are rapidly evolving into specialized cellular roles, as has been suggested for the plethora of K2P
channels in the C. elegans
Protein sequence alignment (single letter amino acid code) of regions of four closely related K2P channels. Shaded letters indicate areas of sequence identity among the four sequences.
Of the fifteen mammalian K2P
channels, only three (TASK-5, TALK-1 and KCNK7) do not produce functional channels when expressed in heterologous expression systems such as Xenopus
oocytes or transfected cultured cells. Therefore it has remained unclear whether these are truly non-functional in vivo
, whether they are missing essential co-factors or whether they need to be paired with other subunits to achieve functional expression [20
]. Extensive expression studies by Salinas et al. discovered that KCNK7 is retained intracellularly yet could not identify a retention signal sequence either by site-directed mutagenasis or by fusion protein strategies. The negative results suggest that either the KCNK7 channel is not a significant target for the volatile anesthetics, or the effect is compensated for by other K2P
Nevertheless, based on the preservation of basic gene structure for KCNK7, it is also unlikely that KCNK7 represents a pseudogene. There are two main types of pseudogenes, processed and duplicated. Processed pseudogenes arise by reverse transcription of processed mRNA and thus lack intronic sequences. Duplicated pseudogenes arise from errant genomic DNA replication and thus have similar genomic structure to homologous genes. Even though we believe that KCNK7 arose by gene duplication, based on the similarity of its genomic structure to its K2P homologues, it has maintained an intact open reading frame while diverging significantly in primary sequence from its relatives. This observation implies that it provides some function and has not been released from selection pressure to accumulate random stop codons.
Thus it remains unclear why the KCNK7 knockout mouse displays little difference from the wildtype phenotype. KCNK7 transcript has been detected by RT-PCR in many areas of the human central nervous system including cerebellum, cortex, hippocampus and spinal cord [18
]. It was also identified in mouse brain by the Allen Brain Atlas (http://www.brain-map.org/welcome.do
); KCNK7 transcript can be seen at low to intermediate levels in choroid plexus within the lateral ventricle, third ventricle and lateral recess of the fourth ventricle. It is a formal possibility that low level expression of KCNK7 in these regions may contribute in some way to CSF production to indirectly affect CNS function.
This report adds to the evidence that the contribution of K2P
channel family to the mechanism of action of volatile and gaseous anesthetics may be restricted to a few family members. Previous work in knockout mice found that TASK-2 (KCNK5) does not mediate the action of volatile agents [6
]. Other animal studies using blockers of TASK-1 (KCNK3) and TASK-3 (KCNK9) suggest that they also do not play a major role as well [4
], although TASK-1 and TASK-3 knockout mice display a small resistance to halothane and isoflurane [14
]. The inactivation of one K2P
channel gene, TREK-1, produced a mouse that showed a variable increase in MAC (7%–48%) that depended on the agent [11
]. Studies of mice multiple K2P
channel knockouts or conditional knockouts will be needed to fully understand their role in the mechanism of action of volatile anesthetics.