Vasopressin-induced differential stimulation of AQP4 splice variants regulates the in-membrane assembly of orthogonal arrays

Alfred N. Van Hoek, Richard Bouley, YingXian Lu, Claudia Silberstein, Dennis Brown, Martin B. Wax, Rajkumar V. Patil

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Aquaporin-4 (AQP4) is a basolateral water channel in collecting duct principal cells and assembles into orthogonal array particles (OAPs), the size of which appears to depend on relative expression levels of AQP4 splice variants. Because the higher-order organization of AQP4 was perturbed by vasopressin in Brattleboro rats and phosphorylation sites have been identified on AQP4, we investigated whether vasopressin and forskolin (Fk) affect AQP4 assembly and/or expression in LLC-PK1 cells stably transfected with the AQP4 splice variant M23, which is responsible for formation of OAPs, and/or the splice variant M1, which does not form OAPs. Our data show that [lys 8]-vasopressin (LVP) and Fk treatment led to differential increases in expression levels of M23-AQP4 and M1-AQP4 that varied as a function of incubation time. At early time points (day 1) expression of M1 was significantly stimulated (4.5-fold), over that of M23 (1.6-fold), but after 3 days the expression of M23 became predominant (4.1-fold) over that of M1 (1.9-fold). This pattern of stimulation was dependent on an intact AQP4 residue serine 111 and required protein synthesis. In cells expressing both M1 and M23 (M1/M23 ∼ 1), with small sized OAPs at the membrane, the LVP/Fk-induced stimulation of M23 was modified and mimicked that of M1 when expressed alone, suggesting a dominant role for M1. In Brattleboro kidney inner medulla, an 8-day chronic exposure to the vasopressin agonist (dDAVP) led to reduction in M1 and a significant increase in M23 immunoblot staining (M1/M23 = 2/3→1/4). These results indicate that AQP4 organization and expression are regulated by vasopressin in vivo and in vitro and demonstrate that the dominant role for M1 is restricted to a one-to-one interaction between AQP4 splice variants that regulates the membrane expression of OAPs.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Renal Physiology
Volume296
Issue number6
DOIs
StatePublished - Jun 2009

Fingerprint

Aquaporin 4
Vasopressins
Membranes
Colforsin
Lypressin
Aquaporin 2
Brattleboro Rats
Kidney Medulla
LLC-PK1 Cells
Particle Size
Serine

Keywords

  • Hydrophobic plasma membrane protein
  • Osmotic water transport

ASJC Scopus subject areas

  • Physiology
  • Urology

Cite this

Vasopressin-induced differential stimulation of AQP4 splice variants regulates the in-membrane assembly of orthogonal arrays. / Van Hoek, Alfred N.; Bouley, Richard; Lu, YingXian; Silberstein, Claudia; Brown, Dennis; Wax, Martin B.; Patil, Rajkumar V.

In: American Journal of Physiology - Renal Physiology, Vol. 296, No. 6, 06.2009.

Research output: Contribution to journalArticle

Van Hoek, Alfred N. ; Bouley, Richard ; Lu, YingXian ; Silberstein, Claudia ; Brown, Dennis ; Wax, Martin B. ; Patil, Rajkumar V. / Vasopressin-induced differential stimulation of AQP4 splice variants regulates the in-membrane assembly of orthogonal arrays. In: American Journal of Physiology - Renal Physiology. 2009 ; Vol. 296, No. 6.
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AU - Bouley, Richard

AU - Lu, YingXian

AU - Silberstein, Claudia

AU - Brown, Dennis

AU - Wax, Martin B.

AU - Patil, Rajkumar V.

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AB - Aquaporin-4 (AQP4) is a basolateral water channel in collecting duct principal cells and assembles into orthogonal array particles (OAPs), the size of which appears to depend on relative expression levels of AQP4 splice variants. Because the higher-order organization of AQP4 was perturbed by vasopressin in Brattleboro rats and phosphorylation sites have been identified on AQP4, we investigated whether vasopressin and forskolin (Fk) affect AQP4 assembly and/or expression in LLC-PK1 cells stably transfected with the AQP4 splice variant M23, which is responsible for formation of OAPs, and/or the splice variant M1, which does not form OAPs. Our data show that [lys 8]-vasopressin (LVP) and Fk treatment led to differential increases in expression levels of M23-AQP4 and M1-AQP4 that varied as a function of incubation time. At early time points (day 1) expression of M1 was significantly stimulated (4.5-fold), over that of M23 (1.6-fold), but after 3 days the expression of M23 became predominant (4.1-fold) over that of M1 (1.9-fold). This pattern of stimulation was dependent on an intact AQP4 residue serine 111 and required protein synthesis. In cells expressing both M1 and M23 (M1/M23 ∼ 1), with small sized OAPs at the membrane, the LVP/Fk-induced stimulation of M23 was modified and mimicked that of M1 when expressed alone, suggesting a dominant role for M1. In Brattleboro kidney inner medulla, an 8-day chronic exposure to the vasopressin agonist (dDAVP) led to reduction in M1 and a significant increase in M23 immunoblot staining (M1/M23 = 2/3→1/4). These results indicate that AQP4 organization and expression are regulated by vasopressin in vivo and in vitro and demonstrate that the dominant role for M1 is restricted to a one-to-one interaction between AQP4 splice variants that regulates the membrane expression of OAPs.

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